library(tidyverse)
library(knitr)Content Source: Slides 4, 7, 17, 18, 22, 29
Expanded Explanation:
Metabolism refers to the entire network of biochemical reactions in a cell, which can be broadly categorized into catabolism (breaking down molecules to extract energy) and anabolism (building complex molecules from simpler ones).
These reactions are tightly interconnected; for example, carbohydrates, fats, and proteins can all be funneled into central metabolic pathways like glycolysis, β-oxidation, and the Krebs cycle.
Fuels like glucose and fatty acids are broken into 2-carbon units (acetyl-CoA), which enter the Krebs cycle for further oxidation and ATP production.
The intermediates in these pathways are shared, making them crucial metabolic “crossroads.”
🔗 Interactive biochemical map: http://biochemical-pathways.com/#/map/1
Content Source: Slides 8, 11–13
Expanded Explanation:
Enzymes are biological catalysts that lower the activation energy required for a reaction, thereby increasing the rate.
Enzyme types:
Kinases: catalyze phosphorylation (add phosphate, often from ATP)
Phosphatases: remove phosphate groups
Phosphorylases: uses phosphate grpup to break things apart
Dehydrogenases: perform oxidation-reduction reactions using NAD⁺ or FAD
\[{pyruvate + NADH ⇌ lactate + NAD^+}\]
NADH is the reduced form of NAD+
NADH oxidise (lose e-) → NAD+
NAD+ reduced (gain e-) → NADH
opposite for other substrate in equation:
If NADH oxidise → pyruvate reduced to lactate
If NAD+ reduce → lactate oxidise to pyruvate
📷 Diagram of enzyme action: Wikimedia Commons - Enzyme Mechanism
Content Source: Slides 10, 32, 33
Expanded Explanation:
Regulation ensures metabolic homeostasis and efficiency, adapting metabolism to energy demand.
Major mechanisms:
Allosteric regulation: Binding of regulators at non-active sites (e.g., ATP, ADP levels).
Covalent modification: Phosphorylation/dephosphorylation.
Transcriptional control: Regulation of enzyme synthesis.
Energy sensors like AMP-activated protein kinase (AMPK) respond to cellular energy status.
🧪 Real-world link: How AMPK regulates metabolism (Nature Reviews)
Content Source: Slides 14, 15, 19–21
Expanded Explanation:
In catabolism, electrons (as hydrogen atoms, H/e⁻) are stripped from fuels and transferred to carriers:
NAD⁺ → NADH: oxidizes alcohol groups
FAD → FADH₂: oxidizes alkyl chains (C-C double bond)
These reduced cofactors then feed electrons into the Electron Transport Chain (ETC), driving proton pumping to generate a proton gradient for ATP production.
📘 Supplementary visual and explanation:
NAD⁺ and FAD in metabolism – YouTube (5 min)
Content Source: Slides 24–28
Expanded Explanation:
Carbohydrates:
Basic unit: monosaccharides (e.g., glucose).
Hydrophilic; stored as glycogen (300g).
Broken down by glycolysis to pyruvate.
Energy yield: ~16 kJ/g (wet weight).
Lipids:
Basic unit: fatty acids.
Hydrophobic; stored as triglycerides (many kg).
Oxidized via β-oxidation to acetyl-CoA.
Energy-dense: ~37 kJ/g.
Proteins:
Made from amino acids.
Catabolized when needed, funnel into various points (pyruvate, acetyl-CoA).
No true “storage” – breakdown means losing function.
Energy: ~17 kJ/g.
📷 Image for basic biomolecule structures: Biomolecule Basics - Wikimedia
Content Source: Slides 17–23, 30–31
Expanded Explanation:
Fuel Oxidation (Stage 1)
Fuels → acetyl-CoA via glycolysis, β-oxidation, or amino acid breakdown.
H/e⁻ stripped by NAD⁺/FAD.
Krebs Cycle (Stage 2)
Acetyl-CoA + oxaloacetate → CO₂ + NADH + FADH₂
2-carbon chunks don’t just float around, they are always carried on a CoA → forming acetyl-CoA
No direct ATP, but reduced carriers are made.
Electron Transport Chain (Stage 3)
NADH/FADH₂ donate electrons to ETC (inner mitochondrial membrane).
H⁺ pumped into intermembrane space → proton gradient.
movement of H/e- down the chain provides energy to pump out protons towards cytoplasm
ATP Synthase uses H⁺ flow to convert ADP + Pi → ATP.
THE 7 BIG CONCEPTS:
The H/e- carriers are in short supply
ADP is in short supply
Why? Proton gradient is created when H+ pumped out of matrix into intermembrane space. Protons can re-enter the matrix via a channel of ATP synthase. When the H+ re-enter the subunit rotates and spins which converts ADP + Pi to ATP. So no ATP production = No rotation = No proton flow
So, if ATP synthase inhibited → proton gradient builds up → makes it harder for ETC to pump protons out to cytoplasm → ETC slows/stops
The proton pumps don’t work if the proton gradient is very high
No proton pumping, no H/e- movement down ETC
🌀 Animation of ATP Synthase in action: Harvard’s Molecular Biology Visual – ATP Synthase
Content Source: Slides 14–16
Explanation:
During exercise, muscle cells increase glucose uptake via GLUT-4 transporters, reducing blood glucose levels.
Even gentle activity triggers these transporters to move to the cell surface.
Since glucose is essential for brain function, the body must maintain blood glucose at ~5 mM.
Hormonal response:
Insulin levels fall: Less stimulation of glucose uptake by liver and adipose tissue.
Glucagon levels rise: Stimulates glycogen breakdown in liver and lipolysis in adipose tissue to release glucose and fatty acids respectively.
These actions together stabilise blood glucose levels despite muscle usage.
Summary Table:
| Hormone | Response to Exercise / Muscle Uptake | Effect |
|---|---|---|
| Insulin ↓ | Suppressed by sympathetic activation | Prevents further glucose uptake by liver/fat |
| Glucagon ↑ | Stimulated by low glucose and catecholamines | Increases hepatic glucose production |
| Adrenaline/Noradrenaline ↑ | Sympathetic response | Stimulates glycogenolysis (liver + muscle), lipolysis |
| Cortisol ↑ (longer term) | Supports gluconeogenesis, protein catabolism | Increases substrate availability |
📘 More on hormonal regulation of glucose: Video: Hormonal Regulation of Blood Glucose (Armando Hasudungan)
Content Source: Slide 16
Explanation:
First, What Are Insulin and Glucagon?
Insulin is like a storage hormone. It tells your body to put away fuel (like glucose and fat) when you’ve eaten.
Glucagon is like a release hormone. It tells your body to get fuel out of storage when you’re fasting or exercising
Now Imagine You Haven’t Eaten for a While
Your insulin levels go down, and your glucagon levels go up. Here’s what happens:
In the Muscle:
Muscles can’t take in glucose easily without insulin.
So they don’t get much glucose from the blood.
*Instead, they start burning fat for energy.
In the Liver:
Glucagon tells the liver to:
Break down glycogen (glycogenolysis) → glucose
Make new glucose (gluconeogenesis)
Send that glucose into the blood
So the liver saves the brain by making and releasing glucose.
In Fat (Adipose Tissue):
Low insulin + high glucagon = fat is broken down
This releases fatty acids into the blood
Muscles and other organs burn the fat instead of glucose
In Blood:
| Fuel | What Happens |
|---|---|
| Glucose | Stays steady or may go up (from liver) |
| Fatty acids | Go way up (from fat breakdown) |
| Ketones | Go up later (especially during starvation) |
Low insulin:
↓ Glucose uptake in adipose/muscle (except contracting muscle).
↓ Glycogen synthesis.
High glucagon:
↑ Glycogenolysis in liver → ↑ blood glucose.
↑ Lipolysis in adipose tissue → ↑ fatty acids.
Result: Blood fuel shifts from glucose to fatty acids for most tissues (glucose is conserved for brain).
🧬 Visual guide to hormone effects on metabolism: Glucagon vs Insulin infographic (Kenhub)
Content Source: Slide 17, 20
Explanation:
Glucose stores (glycogen) are limited; brain requires steady glucose supply.
Fatty acids cannot be converted into glucose (no net gluconeogenesis from acetyl-CoA).
So glucose must be:
Recycled via the Cori cycle: Muscle produces lactate → liver converts it to glucose via gluconeogenesis.
Spared: Use of fatty acids for ATP spares glucose for essential tissues.
This prevents rapid depletion of glucose reserves and avoids hypoglycemia.
📘 Cori Cycle Visual Explanation (Osmosis)
Content Source: Slides 12, 14, 20
Explanation:
In low-intensity exercise:
Initial energy comes from glucose (glycolysis).
After minutes, fatty acids (via β-oxidation) become the main fuel.
The increase in ADP activates oxidative phosphorylation.
Fatty acids are mobilised from white adipose tissue and oxidised in mitochondria to acetyl-CoA → Krebs → ETC.
🧪 Fatty Acid β-Oxidation Pathway – Video
Content Source: Slides 12, 14, 20
Explanation:
ATP demand increases slightly → ↑ ADP → stimulates ATP synthase and ETC.
Initial reliance on blood glucose, but transitions to:
Energy is generated primarily aerobically.
📘 Fuel use over time – graph example (OER Commons)
Content Source: Slides 21–24
Explanation:
As pace increases:
🧬 Comparison of fuel usage by intensity (ResearchGate figure)
Content Source: Slide 24
Explanation:
Gentle: Mostly fatty acid oxidation, some glucose → lactate.
Moderate:
Net effect: Mixed oxidation, increased reliance on glucose, faster glycogen depletion.
📘 Carbohydrate and fat usage in exercise – Lecture image
Content Source: Slides 24–26
Explanation:
🧪 Study: Fat and carbohydrate metabolism during exercise (J Appl Physiol)
Content Source: Slides 25–27, 29
Explanation:
Glycogen is mobilised when:
Glycogen → G6P → glycolysis → ATP/lactate.
Glycogen is stored within the muscle and is readily accessible.
📘 Why glycogen is critical to athletes (Examine.com)
Content Source: Slides 25–27
Explanation:
🎥 Anaerobic vs aerobic metabolism (Armando)
Content Source: Slide 29, 33
Explanation:
Glycogen is the only fuel fast enough for high-intensity efforts like sprinting.
Once depleted:
Adequate glycogen stores:
📘 Why do athletes carbo-load? (Scientific American)
Think of the body like a hybrid car that runs on different fuels (glucose, fatty acids, amino acids), depending on what’s available and what you’re doing (resting, exercising, fasting, etc.).
Goal = Make ATP
ATP is your body’s “battery”.
All fuel oxidation pathways exist to make ATP to power cells.
Fuel Types:
| Fuel | When Used | Features |
|---|---|---|
| Glucose | After eating, fast bursts of exercise | Fast, anaerobic possible |
| Fatty acids | Fasting, rest, prolonged exercise | Slow, high yield |
| Amino acids | Starvation, muscle breakdown | Backup fuel |
Oxidation Happens in Stages:
Glycolysis / β-oxidation: Break fuel into 2-carbon units (acetyl-CoA)
Krebs Cycle: Burn acetyl-CoA → release electrons
ETC + Oxidative Phosphorylation: Use electrons to make ATP
Flexibility is key:
Insulin promotes storage and glucose use.
Glucagon promotes release and fat use.
Glucose is like petrol — easy to use, burns fast.
Fat is like diesel — lasts longer but burns slowly.
Protein is like burning the car’s seats — you only use it when you’re desperate.
Even though understanding is most important, there are certain steps and enzymes you just have to memorise.
Happens in cytoplasm
No oxygen needed
Key enzymes:
Hexokinase / Glucokinase
Phosphofructokinase (PFK-1) → rate-limiting step
Pyruvate kinase
🧪 ATP Yield:
2 ATP net per glucose (plus 2 NADH)
Happens in mitochondria
Requires carnitine shuttle for entry
Cycles remove 2 carbon units as acetyl-CoA
Generates lots of NADH, FADH₂
🧪 ATP Yield:
~106 ATP from one palmitate (16C fatty acid)
Happens in mitochondrial matrix
Each turn:
3 NADH
1 FADH₂
1 GTP (≈ ATP)
Enzymes to learn:
Citrate synthase
Isocitrate dehydrogenase
α-ketoglutarate dehydrogenase
Happens in inner mitochondrial membrane
NADH/FADH₂ donate electrons → pump protons
ATP Synthase uses proton gradient to make ATP
🧪 ATP Yield:
2.5 ATP per NADH
1.5 ATP per FADH₂
Glucose → Glycolysis → Pyruvate → Acetyl-CoA → Krebs → ETC → ATP
Fatty Acid → β-Oxidation → Acetyl-CoA → Krebs → ETC → ATP
Amino Acid → Transamination → Keto Acids → Acetyl-CoA or TCA Intermediates → ATP | Condition | Preferred Fuel | Hormone | Notes |
|---|---|---|---|
| Fed | Glucose | ↑ Insulin | Store fat, make ATP |
| Fasting | Fatty acids | ↑ Glucagon | Gluconeogenesis for brain |
| Exercise (short, intense) | Glucose (muscle glycogen) | ↓ Insulin | Anaerobic possible |
| Exercise (prolonged) | Fatty acids | ↓ Insulin, ↑ Glucagon | Conserves glucose |
Coupling means that two things are linked — if one happens, the other happens too.
In the mitochondria:
Fuel oxidation (burning glucose or fat) is coupled to ATP synthesis.
So:
👉 If the cell needs more ATP, it will also burn more fuel.
👉 If no ATP is needed, fuel oxidation slows down or stops.
It’s all about ADP levels and the proton gradient in the mitochondria:
ATP Synthase makes ATP by letting protons (H⁺) flow back into the mitochondrial matrix.
This proton flow only happens when there is ADP + Pi to turn into ATP.
If there’s no ADP, protons stop flowing, and the proton gradient builds up.
This stops the electron transport chain, which stops fuel oxidation.
Lots of ADP = lots of ATP being made = fuel gets burned
Little or no ADP = no ATP being made = fuel oxidation slows or stops
This is chemiosmotic coupling — ATP synthesis is linked to fuel oxidation through the proton gradient.
Imagine a hydroelectric dam (the mitochondria):
- The water behind the dam = proton gradient
- The turbine = ATP synthase
- Electricity = ATP
The water (H⁺) only flows through the turbine if you’re using electricity (need ATP).
If you’re not using electricity (no ADP), the water stops flowing, and the generators (ETC) shut down.
| Component | Function |
|---|---|
| Fuel (glucose, fat) | Oxidised to provide electrons (NADH/FADH₂) |
| ETC (Complex I–IV) | Pumps protons into intermembrane space |
| Proton Gradient (PMF) | Stores energy like a battery |
| ATP Synthase (Complex V) | Uses proton flow to make ATP |
| ADP availability | Acts like a switch – turns ATP synthesis (and fuel use) on or off |
| Condition | ADP Level | Proton Flow | ATP Made | Fuel Oxidised |
|---|---|---|---|---|
| Rest | Low | Slow | Little | Little |
| Exercise | High | Fast | Lots | Lots |
| ATP synthase blocked (e.g. oligomycin) | Irrelevant | Blocked | None | None |
| Uncoupled (e.g. UCP-1) | Normal | Flow allowed | Less | Lots (but wasted as heat) |
Coupling prevents waste.
The body only uses fuel when ATP is actually needed.
This keeps metabolism efficient, like a smart energy-saving system.
Content Source: Slide 5, presenter notes
Explanation:
Fatty acids are long-chain hydrocarbons with a carboxyl group (-COOH) at one end.
Numbering systems:
📘 Fatty acid nomenclature tutorial (Lumen Learning)
Content Source: Slides 7–10, 11, presenter notes
Explanation:
Transport: In blood, fatty acids are bound to albumin due to their hydrophobicity.
Trapping: In the cytoplasm, fatty acids are activated to fatty acyl-CoA by acyl-CoA synthetase, which:
Requires ATP → AMP + PPi (equivalent to 2 ATP).
Makes the reaction irreversible.
Mitochondrial import:
FA-CoA cannot cross the inner mitochondrial membrane.
It is transferred to carnitine by CAT-1, forming FA-carnitine.
Translocase shuttles it across.
Inside matrix, CAT-2 converts it back to FA-CoA.
📺 Beta-oxidation animation (YouTube)
Content Source: Slide 11
Explanation:
Coenzyme A (CoA) has a reactive thiol group (-SH) that forms high-energy thioester bonds with acyl groups (e.g., acetyl-CoA).
It’s a large, charged molecule, preventing free diffusion across membranes—this helps “trap” metabolites inside cells.
Acts as a carrier, transferring acyl groups in metabolism (e.g., fatty acid oxidation, Krebs).
It costs 2 ATP molecules to join CoA to FA
📘 Coenzyme A structure and role (Khan Academy)
Content Source: Slides 12–14
Explanation:
In β-oxidation, two key redox steps:
FAD oxidises the β–α bond, forming a trans double bond (→ FADH₂).
NAD⁺ oxidises the resulting β-hydroxy group to a β-keto group (→ NADH).
Both feed into the ETC to produce ATP:
FADH₂ → 1.5 ATP
NADH → 2.5 ATP
📘 FAD/NAD redox roles explained (LibreTexts)
Content Source: Slides 13–16
Explanation:
Each cycle of β-oxidation removes 2 carbons as acetyl-CoA and generates 1 NADH and 1 FADH₂:
Oxidation by FAD: FA-CoA → enoyl-CoA (trans double bond formed).
Hydration: Water adds across the double bond → hydroxyacyl-CoA.
Oxidation by NAD⁺: → ketoacyl-CoA.
Thiolysis: CoA cleaves acetyl-CoA off → shortened FA-CoA.
Example: Palmitate (C16) → 7 cycles → 8 acetyl-CoA, 7 FADH₂, 7 NADH
📘 Full β-oxidation pathway – Slide + summary
Content Source: Slide 20, 21, 29
Explanation:
Glycolysis is a cytosolic pathway that breaks down glucose (6C) into 2 pyruvate (3C) molecules.
Two main phases:
Investment: 2 ATP consumed.
Payoff: 4 ATP + 2 NADH generated.
Anaerobic: No oxygen required.
📘 Glycolysis Overview – Harvard Animation
Content Source: Slide 21
Explanation:
Glucose enters via GLUT transporters:
Once inside, glucose is phosphorylated by hexokinase/glucokinase → glucose-6-phosphate (G6P).
This traps glucose in the cell, as G6P cannot exit through GLUTs.
📘 GLUT transporters illustrated (NCBI)
Content Source: Slides 22–25
Explanation:
Investment Phase:
2 ATP used:
Glucose → G6P (via hexokinase)
F6P → F-1,6-BP (via PFK) - this adds another phosphate to glucose
Payoff Phase:
4 ATP formed (net gain = 2 ATP).
2 NAD⁺ → 2 NADH.
End products: 2 pyruvate + 2 NADH + 2 ATP (net)
👉 You spend ATP to get things going
This is like putting money in to set up a lemonade stand.
The cell uses 2 ATP to “activate” glucose and turn it into something ready to break apart.
| Step | Enzyme | What Happens |
|---|---|---|
| Step 1 | Hexokinase | Glucose → Glucose-6-phosphate (uses 1 ATP) |
| Step 3 | Phosphofructokinase-1 (PFK-1) | Fructose-6-phosphate → Fructose-1,6-bisphosphate (uses 1 ATP) |
🧪 Total ATP used: 2
👉 You get ATP and NADH back
Now the cell breaks the sugar in half and makes energy and electrons:
For each glucose, you get:
| Step | Enzyme | What’s Made |
|---|---|---|
| Step 6 | Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) | Makes NADH |
| Step 7 | Phosphoglycerate kinase | Makes ATP |
| Step 10 | Pyruvate kinase | Makes ATP and pyruvate |
🧪 Total made: 4 ATP, 2 NADH
| Molecule | Total Made | Total Used | Net Gain |
|---|---|---|---|
| ATP | 4 | 2 | ✅ +2 ATP |
| NADH | 2 | 0 | ✅ +2 NADH |
| Pyruvate | 2 | – | ✅ 2 Pyruvate |
The cell spends energy first, then makes more back.
This is “priming the pump” — like pushing a swing before it comes back higher.
Glycolysis is fast, anaerobic, and the first step in all metabolism, whether or not oxygen is available.
🔵 Glucose
↓ [–1 ATP: Hexokinase]
🔵 Glucose-6-P
↓
🔵 Fructose-6-P
↓ [–1 ATP: PFK-1]
🔵 Fructose-1,6-BP
↓
🔵 2 G3P
↓ [GAPDH → +2 NADH]
↓ [PGK, PK → +4 ATP]
🔵 2 PyruvateContent Source: Slides 27, 29
Explanation:
Aerobic conditions:
Pyruvate enters mitochondria.
Converted to acetyl-CoA by PDH → enters Krebs cycle.
Anaerobic conditions:
Alternative fates: In yeast → ethanol; in liver → gluconeogenesis.
When glucose is broken down in glycolysis, the final product is pyruvate.
Now the cell has to decide:
👉 What do I do with this pyruvate?
Think of pyruvate like a train at a station with multiple tracks.
Which track it takes depends on oxygen, energy needs, and cell type.
🔹 Aerobic conditions (with oxygen)
🔹 Happens in mitochondria
| Enzyme | Pyruvate dehydrogenase (PDH) |
|---|
This is the main pathway in cells when oxygen is available.
🧬 Acetyl-CoA goes into the Krebs cycle → then to ETC → lots of ATP!
✅ Used when:
Oxygen is present
Cell wants to make lots of ATP
Tissues like muscle, brain
🔹 Anaerobic conditions (no oxygen)
🔹 Cytoplasm
| Enzyme | Lactate dehydrogenase (LDH) |
|---|
This happens in:
Working muscles during intense exercise
Red blood cells (no mitochondria)
🧪 Why? To regenerate NAD⁺ so glycolysis can keep going!
✅ Used when:
Oxygen is low
You need ATP fast
For short-term emergency energy
🧪 Example: Sprinting → burning legs → lactate buildup
🔹 Amino acid metabolism
| Enzyme | Alanine aminotransferase (ALT) |
This happens in muscle when amino groups are transferred.
Alanine then travels to the liver, gets turned back into pyruvate, and used for gluconeogenesis.
✅ Used when:
Muscle is breaking down protein
You need to send fuel to the liver
Part of the glucose-alanine cycle
🔹 For gluconeogenesis (making new glucose)
🔹 Also replenishes Krebs cycle intermediates
| Enzyme | Pyruvate carboxylase (needs biotin + ATP) |
🧬 Oxaloacetate is the entry point into gluconeogenesis → helps maintain blood glucose during fasting.
✅ Used when:
You’re fasting
The body needs to make glucose (e.g. for brain)
Or to recharge the Krebs cycle (anaplerosis)
| Pathway | Product | Enzyme | When/Why |
|---|---|---|---|
| Oxidation | Acetyl-CoA | Pyruvate dehydrogenase | With oxygen, for ATP |
| Anaerobic | Lactate | Lactate dehydrogenase | No oxygen, fast ATP |
| Transamination | Alanine | ALT | Transport nitrogen + fuel |
| Carboxylation | Oxaloacetate | Pyruvate carboxylase | Gluconeogenesis or anaplerosis |
Pyruvate is like a 3-way fork in the road:
🛣️ Go to power plant (mitochondria) → Acetyl-CoA
🛻 Shortcut to keep running (lactate) if there’s no oxygen
🚚 Delivery route to liver (alanine or oxaloacetate)
Content Source: Slides 31–33
Explanation:
Central hub of metabolism.
Acetyl-CoA from glycolysis, β-oxidation, or amino acids enters cycle.
Occurs in mitochondrial matrix.
Oxidises acetyl groups to 2 CO₂, while generating NADH, FADH₂, and GTP.
📘 Krebs Cycle as metabolic hub (Nature Education)
Content Source: Slide 33
Explanation:
Acetyl-CoA (2C) + Oxaloacetate (4C) → Citrate (6C)
Two decarboxylations → 2 CO₂
Yields: 3 NADH, 1 FADH₂, 1 GTP per cycle
Oxaloacetate regenerated = cycle.
NADH/FADH₂ go to ETC for ATP production.
📘 Krebs cycle summary (Sackville HS)
Content Source: Slides 34–35 Explanation:
Key intermediates: Citrate, α-ketoglutarate, succinate, malate, oxaloacetate
Regulated enzymes:
Regulation by:
📘 Regulation summary (Biology LibreTexts)
Explanation:
Your body burns fuel (like glucose and fat) to make ATP, just like a fire burns wood to make heat.
But to burn fuel, your body needs oxygen — and when it burns it, it releases carbon dioxide.
So:
🧪 The more oxygen you use, the more energy you’re burning.
💨 The more carbon dioxide you produce, the more fuel you’ve used.
Scientists use a machine called a metabolic cart to measure:
VO₂ = Oxygen consumption (how much O₂ you breathe in and use)
VCO₂ = Carbon dioxide production (how much CO₂ you breathe out)
These numbers tell us how much energy your body is using.
When your body uses oxygen to break down fuel:
Glucose uses O₂ and makes CO₂ (1:1 ratio)
Fat uses more O₂ and makes less CO₂
Each litre of oxygen consumed = about 20.1 kJ of energy burned (on average)
Imagine you’re watching a campfire 🔥
- The more wood (fuel) you burn, the more oxygen is sucked in, and more smoke (CO₂) comes out.
- So by measuring the air going in and out, you can tell how big the fire is — that’s what your body is doing with food!
| Term | What It Means |
|---|---|
| VO₂ | Oxygen consumption (used to oxidise fuel) |
| VCO₂ | CO₂ produced when fuel is broken down |
| Respiratory Quotient (RQ) | VCO₂ / VO₂ → tells which fuel you’re using |
| Energy Expenditure | How much energy (kJ or kcal) you’re burning |
If someone uses 2 L of oxygen per minute, they are burning:
\[ 2 \, \text{L/min} \times 20.1 \, \text{kJ/L} = 40.2 \, \text{kJ/min} \]
That’s how scientists calculate how much energy your whole body is using — just from the air you breathe.
Explanation:
Coupling: ETC activity is limited by ATP demand. Without ATP synthesis, the proton gradient becomes too steep, halting ETC.
Uncoupling = Protons return to the matrix without passing through ATP synthase, dissipating the gradient:
DNP (2,4-dinitrophenol): synthetic uncoupler that carries H⁺ across membrane → uncontrolled, dangerous.
UCP-1 (thermogenin): a regulated proton channel in brown adipose tissue, activated by noradrenaline → generates heat (thermogenesis).
Uncoupling increases O₂ use and fuel oxidation but prevents ATP synthesis.
📘 Image: Mechanism of DNP & UCP-1 – Biology LibreTexts
Explanation:
NAD⁺/NADH: Hydrophilic, mobile electron carrier; donates e⁻ to Complex I.
FAD/FADH₂: Bound tightly to Complex II, accepts 2H⁺ and 2e⁻ from β-oxidation and Krebs.
Ubiquinone (UQ):
Hydrophobic, mobile within the inner membrane.
Accepts electrons from Complex I, II, β-oxidation, and G3P shuttle → transfers them to Complex III.
Complexes I–IV contain iron-sulphur clusters and cytochromes that pass e⁻ down the chain.
🎥 NADH, FADH₂, and UQ roles – YouTube Animation
Explanation:
Carriers that accept both e⁻ and H⁺ (like UQH₂) can transfer only the e⁻ to the next carrier.
The H⁺ is released into the intermembrane space → this is proton pumping.
Complexes I, III, IV pump protons:
I: ~4 H⁺
III: ~4 H⁺
IV: ~2 H⁺
FADH₂ → Complex II → UQ → bypasses proton pumping at Complex I.
📘 Mechanism: Proton pumping – Nature Education
Explanation:
The proton-motive force (PMF) is made of:
This electrochemical gradient drives H⁺ back into the matrix via ATP synthase or other transporters.
📘 Diagram: Proton Motive Force
Explanation:
Glycolysis produces NADH in the cytosol, but the mitochondrial inner membrane is impermeable to NADH.
Two shuttles transfer H⁺/e⁻ into mitochondria:
📘 Shuttle Comparison Chart – Lumen Learning
Explanation:
Ubiquinone (Q) receives electrons from:
Complex I (via NADH)
Complex II (succinate dehydrogenase, via FADH₂)
G3P shuttle (via G3P → FADH₂)
β-oxidation (via electron-transferring flavoprotein → ETF:UQ oxidoreductase)
📘 Figure: 4 routes to Q – Lehninger Fig. 19.8
Explanation:
📘 ROS and mitochondrial antioxidant systems – ScienceDirect
Explanation:
ATP synthase (F₀F₁-ATPase):
F₀: membrane-embedded proton channel; protons cause rotation of γ-subunit.
F₁: catalytic headpiece; β-subunits cycle through 3 conformations:
Bind ADP + Pi
Form ATP
Release ATP
3 H⁺ = 1 ATP (approx.)
Rotation-driven conformational changes result in ATP formation without direct chemical input.
ATP Synthase Animation I – Harvard
ATP Synthase Animation II – Harvard
Explanation:
Besides ATP production, the proton gradient powers:
Also used in:
Gradient is essential for multiple mitochondrial functions.
📘 Transport roles of PMF – Molecular Cell Biology
Explanation:
Assumptions include:
These values may vary based on:
Therefore, these are theoretical estimates, not absolute.
📘 ATP yield breakdown – BioNinja
Explanation:
If ATP demand falls → ADP falls → ATP synthase slows → proton gradient builds → ETC stalls → oxidation slows.
If ATP synthase is blocked (e.g. oligomycin):
Gradient becomes too steep
ETC halts
NADH accumulates → oxidation stops
Uncoupling (e.g. DNP):
Gradient collapses
ETC speeds up (unrestrained)
But no ATP produced
Inhibitors:
Rotenone: blocks Complex I → NADH can’t be oxidised
Cyanide: blocks Complex IV → ETC backup
Methylene blue: bypasses block, acts as e⁻ acceptor
📘 Coupling logic – LearnBiochem
Explanation:
Major energy stores:
Fat (white adipose tissue): ~12–15 kg, high energy density (37 kJ/g).
Liver glycogen: ~100 g, maintains blood glucose.
Muscle glycogen: ~400 g, used locally in muscle.
Protein: 5–10 kg, not stored for fuel; catabolised only under extreme conditions (e.g., late starvation).
Fat is the dominant long-term energy store, but the brain cannot use it directly due to the blood-brain barrier.
📘 Fuel store comparison – Lumen Learning
Explanation:
Euglycemia = maintaining blood glucose ~5 mM.
Vital for tissues like brain, kidney medulla, RBCs.
Achieved by:
Glycogenolysis in liver (rapid).
Gluconeogenesis from lactate, glycerol, and amino acids.
Inhibition of glucose oxidation in peripheral tissues.
Hormonal control:
📘 Glucose homeostasis overview – NCBI
Explanation:
Glucose use continues while dietary sources drop → blood glucose starts falling.
Liver rapidly releases glucose via glycogenolysis.
Glucagon secretion increases, insulin drops.
Blood glucose stabilises at ~4 mM due to hormonal compensation.
🧠 Diagram – Early fasting glucose control
Explanation:
Glucagon:
↑ in liver and adipose tissue (not muscle).
Activates cAMP-PKA pathways → ↑ glycogenolysis and lipolysis .
Insulin:
Acts on muscle, liver, and fat.
↓ during fasting → inhibits glycogenesis, lipogenesis, and glucose uptake.
Muscle relies on GLUT1 for baseline glucose uptake (GLUT4 is insulin-dependent).
📘 Infographic: Glucagon vs Insulin effects – Kenhub
Explanation:
0–6 hours post-absorptive:
6–24 hours:
>24 hours:
📘 Timeline – Starvation phases chart (UpToDate)
Source: L6.2 pg 6, 8
Explanation:
Glycogenolysis:
Glycogen → G1P → G6P → Glucose (via G6Pase)
G6P itself can’t get back out to blood so ONLY in the liver there is G6Pase that takes phosphate off and turn it back into glucose to be carried back out into blood
G6Pase is hidden away in endoplasmic reticulum
Exported via GLUT2
Regulated by glucagon:
–> Activates adenylate cyclase → ↑ cAMP
–> cAMP activates PKA, which:
–> Phosphorylates phosphorylase kinase to make it active
–> Phosphorylate glycogen phosphorylase to make it active
–> active & phosphorylated glycogen phosphorylase will break off glucose-1-phosphate off the end of glycogen chain
Debranching enzyme releases ~10% of glucose directly (as free glucose) rather than glucose-1-phosphate
complex cascade of phosphorylation from 1 glucagon can amplify the amount of glucose that’s released into blood glucose rather than binding directly
Flowchart
Glucagon binds to receptor on liver cell surface ↓ Activates adenylate cyclase ↓ Increases cAMP levels ↓ cAMP activates Protein Kinase A (PKA) ↓ PKA phosphorylates phosphorylase kinase → activates it ↓ Active phosphorylase kinase phosphorylates glycogen phosphorylase ↓ Glycogen phosphorylase becomes active ↓ Active glycogen phosphorylase breaks glucose-1-phosphate off the ends of glycogen chains
📘 Pathway image – Glycogenolysis
Explanation:
Muscle lacks glucagon receptors → no hormonal activation of glycogenolysis.
Muscle lacks G6Pase → G6P cannot be converted to free glucose.
Thus, glucose from muscle glycogen is used locally, not released into blood.
~10% glucose can be released via hydrolysis at branch points.
📘 Explanation – Muscle glycogen is local only
Explanation:
🧠 Graph – Liver glycogen over fasting
Explanation:
Low insulin and high glucagon promote fat mobilisation.
In WAT:
Glucagon → ↑ cAMP → ↑ PKA activity
PKA phosphorylates Hormone Sensitive Lipase (HSL) and perilipin
HSL hydrolyses triglycerides → 3 FA + 1 glycerol
Because cytoplasm is hydrophilic and lipid droplet is hydrophobic –> hard to work between these two
So, PERILIPIN unpicks the edges of th lipid vacuole which creates an opening for HSL to come in and start breaking down the fat.
FAs bind albumin in blood; glycerol goes to liver for gluconeogenesis.
glycerol can diffuse into the blood stream and be used as an alternative fuel to glucose
can diffuse into muscle cell as alternative
📘 Animation – Hormone-sensitive lipase activation
Explanation:
Fatty acid oxidation → lots of acetyl-CoA.
Acetyl-CoA activates PDH kinase, which inhibits PDH (pyruvate dehydrogenase).
Inhibiting PDH prevents pyruvate → acetyl-CoA → blocks glucose oxidation.
Pyruvate is instead converted to lactate, sparing glucose for the liver.
Ac-CoA does not inhibit PDH directly
PDH works opposite to other enzymes –> phosphorylation actually INACTIVATES PDH and dephosphorylation ACTIVATES PDH
high levels of Ac-CoA will activate PDH kinase –> phosphorylate PDH –> when PDH phosphorylated it will become inactive
PDH phosphotase will activate it again
📘 Mechanism – Glucose-fatty acid cycle (Randle cycle)
Explanation:
Glucose-Fatty Acid Cycle (Randle Cycle):
FA oxidation → ↑ acetyl-CoA → inhibits PDH → ↓ glucose oxidation.
Muscle switches to fat → glucose spared.
Cori Cycle:
Muscle: glucose → pyruvate → lactate
Lactate → liver → converted back to glucose via gluconeogenesis
Recycled glucose returns to blood → reused by muscle/RBCs.
📘 Cori cycle diagram – Osmosis
Explanation:
Only three substrates contribute to new (de novo) glucose:
Glycerol: from lipolysis; enters as dehydroxyacetone phosphate (DHAP) (30 g/day).
Amino acids: from proteolysis; carbon skeletons become glucose intermediates.
Lactate: from anaerobic glycolysis; however, this is recycling, not de novo.
📘 Overview – Gluconeogenic substrates (Khan Academy)
Explanation:
Lactate → glucose via Cori Cycle is a closed loop:
Glucose → pyruvate → lactate in muscle
Lactate → pyruvate → glucose in liver
Net result: no new glucose added to the pool, only recycled.
Requires ATP in liver but doesn’t expand total glucose availability.
Explanation:
Gluconeogenesis = almost reverse of glycolysis, but:
Bypasses 3 irreversible steps:
Pyruvate kinase → uses pyruvate carboxylase + PEPCK
Phosphofructokinase → bypassed by fructose-1,6-bisphosphatase
Hexokinase → bypassed by G6Pase
Occurs mainly in the liver, partly mitochondrial (pyruvate carboxylase).
Uses ATP and NADH.
📘 Pathway map – Lumen Learning
Explanation:
After proteolysis, amino acids are:
Deaminated using PLP (vitamin B6)
Amine groups, using amino-transferase enzyme, transferred to:
Pyruvate → alanine
α-ketoglutarate → glutamate
OAA → aspartate
Carbon skeletons → enter gluconeogenesis.
Amine groups → sent to urea cycle for excretion.
If an AA backbone can only be made into acetyl-CoA, it CANNOT be made into glucose
If it can be made into pyruvate or Krebs cycle intermediate, it CAN be made into glucose.
📘 Amino group transfer and urea cycle – LibreTexts
Explanation:
Most nitrogen comes from muscle protein degradation.
NH₃ is toxic → converted into urea in the liver:
Inputs: glutamate + aspartate
Urea excreted in urine.
Urea cycle is energy-intensive.
📘 Urea cycle simplified – Osmosis
Explanation:
After 2–3 days: liver oxidises fatty acids → acetyl-CoA accumulates.
Every step of beta-oxidation needs FAD, NAD+ and CoA
So, Acetyl-CoA converted into ketone bodies:
Acetoacetate
β-hydroxybutyrate
Turning Ac-CoA into something that has no CoA on it so we can get our CoAs back to continue to burn fats in the liver despite no demand for ATP
Taken up by brain, converted to acetyl-CoA → Krebs cycle fuel.
Reduces brain glucose demand from 120g → ~30g/day.
📘 Ketone body formation – Nature
Explanation:
Ketone body oxidation itself is efficient, but:
Some ketones are lost in urine or sweat (e.g., acetone).
Spontaneous decarboxylation → metabolic waste.
Net loss of carbon-containing energy substrates.
📘 Inefficiencies in ketone metabolism – PMC
Explanation:
| Tissue | Primary Fuel |
|---|---|
| Brain | Ketone bodies, some glucose |
| Muscle | Fatty acids |
| Liver | Fatty acids (for ATP), amino acids and glycerol for gluconeogenesis |
| Kidney | Ketones, some gluconeogenesis |
| RBCs | Glucose → lactate (Cori cycle) |
By day 5:
Proteolysis drops (from >100g to ~50g/day)
Ketones take over as primary brain fuel
Fat and glycerol support gluconeogenesis
📘 Metabolic shift in starvation – Cahill 2006
Diagram Suggestion: L6 pg 18, 21
Adipose Tissue → FA & Glycerol
↓ ↓
(Liver) (Liver)
β-oxidation → ATP
↓ ↓
Ketogenesis Gluconeogenesis ← Amino Acids (from muscle)
↓
Blood glucose + ketones → Brain, RBCs, kidney📘 [Starvation metabolism overview – BioRender or Lehninger Textbook Fig 27-3]
Explanation:
Glucagon dominates in prolonged fasting:
Maintains blood glucose and ketone levels despite low insulin.
A person has been starving for 12 hours. They are then treated with a compound. Which IMMEDIATE outcome (i.e. in the next hour) matches each the treatment. (NB. Each outcome could result from more than one treatment):
Injection of an inhibitor of hormone sensitive lipase
Injection of an inhibitor of carnitine acyl transferase
Injection of an inhibitor of muscle pyruvate dehydrogenase
| Blood [Glycerol] | Muscle Fatty Acid Oxidation | Brain Glucose Oxidation | Liver Gluconeogenesis | |
|---|---|---|---|---|
| A | DECREASES | DECREASES | SAME | DECREASES |
| B | SAME | SAME | SAME | SAME |
| C | SAME | DECREASES | SAME | DECREASES |
| D | DECREASES | INCREASES | INCREASES | SAME |
| E | SAME | SAME | DECREASES | INCREASES |
ANSWER
1. Inhibitor of Hormone Sensitive Lipase (HSL)
HSL function: HSL breaks down triglycerides in adipose tissue, releasing free fatty acids (FFA) and glycerol into the blood. Inhibition effect:
2. Inhibitor of Carnitine Acyl Transferase (CAT)
CAT (specifically CAT I) is essential for transporting long-chain fatty acids into mitochondria for β-oxidation in muscle. Inhibition effect:
3. Inhibitor of Muscle Pyruvate Dehydrogenase (PDH)
PDH function: Converts pyruvate to acetyl-CoA in muscle. Inhibition effect:
Final Matching Table:
| Treatment | Matched Outcome | Explanation Summary |
|---|---|---|
| 1. Inhibitor of Hormone Sensitive Lipase | A | ↓ Lipolysis → ↓ glycerol, ↓ muscle FA use, ↓ gluconeogenesis (↓ glycerol substrate) |
| 2. Inhibitor of Carnitine Acyl Transferase | C | ↓ FA oxidation in muscle, ↓ gluconeogenesis (↓ acetyl-CoA activation of PC) |
| 3. Inhibitor of Muscle Pyruvate Dehydrogenase | D | Muscle uses more fat, brain uses more glucose (more available), gluconeogenesis unaffected |
Explanation:
ATP contains three phosphate groups, each with high-energy phosphoanhydride bonds.
Hydrolysis of ATP → ADP or AMP releases substantial free energy (~-30.5 kJ/mol).
Phosphoryl group transfer is kinetically stable but thermodynamically favourable.
ATP is soluble, abundant (~5 mM), and universally recognised by enzymes.
📘 ATP structure and energy – Khan Academy
Explanation:
Energy charge reflects cellular energy status:
\(\text{Energy Charge} = \frac{[ATP] + 0.5[ADP]}{[ATP] + [ADP] + [AMP]}\)
Range: 0 (all AMP) → 1 (all ATP).
Cells maintain charge ~0.9; slight ATP changes → large AMP response.
Acts as a metabolic gauge, especially through AMP-activated protein kinase (AMPK).
📘 Energy charge – Biochemistry LibreTexts
Explanation:
Via the action of adenylate kinase:
\(2 ADP ⇌ ATP + AMP\)
Minor ATP depletion → large increase in AMP, which potently activates AMPK and other enzymes.
AMP is the most sensitive indicator of energy stress.
📘 Adenylate kinase role – PubMed
Explanation:
Control is focused on irreversible steps (far from equilibrium).
Often the first committed step in a pathway.
Enzymes working at saturation (low Km, high [S]) are ideal control points.
📘 Control points in glycolysis – Wikipedia
Explanation:
RLS enzymes typically:
Have low Km (high affinity)
don’t have high reaction velocity
Enzymes (that we’re trying to control flux of) are generally working at substrate concentration that are much higher than their Km value.
so, changes in substrate concentration on high end (where plateau is) don’t change the rate of reaction –> already plenty of substrate
Enzyme working at max velocity trying to turn substrates into products
substrates basically just waiting there —> giving more substrate or taking some away doesn’t change rate of reaction
only time that substrate concentration does affect an enzyme to catalyse a reaction is when concentration gets close to Km value.
Km is the substrate concentration at which the enzyme is catalysing the reaction at half of the max (half of max reaction velocity).
Operate near Vmax
Show strong saturation
Are regulated allosterically or by covalent modification
📘 Vmax and Km visualisation – Lumen Learning
Explanation:
Irreversible under physiological conditions.
Control flux through the pathway.
Sites of feedback and feedforward regulation.
Often committed steps unique to the pathway.
📘 RLS in metabolism – ScienceDirect
Source: L3.1 pg 11
Explanation:
Allosteric regulation: ligand binding at non-active site → conformational change (e.g., AMP, citrate).
Covalent modification: phosphorylation/dephosphorylation alters activity (e.g., PDH).
Isoenzyme expression: tissue-specific variants.
Transcriptional control: change enzyme levels.
From lecture:
Change intrinsic activity of the step: make rate limiting enzyme go faster/slower
Switch on/off: turn the rate limiting enzyme on/off or make it work the other way around
Make and destroy gates according to need: increase the rate of transcription/translation of the RLS, or change its rate of degradation
📘 Mechanisms of enzyme regulation – NCERT
Source: L3.1 pg 12
Examples:
| Pathway | RLS Enzyme |
|---|---|
| Glycolysis | Phosphofructokinase-1 (PFK-1) |
| β-Oxidation | Carnitine acyltransferase I |
| Krebs Cycle | Isocitrate dehydrogenase |
| Glycogenolysis | Glycogen phosphorylase |
| Pyruvate Oxidation | Pyruvate dehydrogenase (PDH) |
Possible rate limiting steps:
Fatty acid oxidation - need the availability of CoA in cytoplasm to trap FA in the cell
Beta oxidation - need availability of carnitine to transport FA-CoA from cytoplasm into the mitochondria martrix
Beta oxidation - dehydrogenases working above Km (working at max rate to break FA into 2-carbon chunks)
Beta oxidation - availability of CoA, NAD+, or FAD
Glycolysis - transport of glucose into the cell using GLUT 2 or 4 (especially GLUT-4 which is insulin-sensitive)
Glycolysis - Hexokinase will stop trapping more glucose if we don’t use the phosphorylated glucose, allowing it to flow back out again
glycolysis - Phosphofructokinase (PFK) phosphorylating G6P to Glucose 1,6-bisphosphate
glycolysis - pyruvate kinase
glycolysis - NAD+ for glycolysis to continue. Unlike Fatty acid oxidation, glycolysis does not need FAD
Pyruvate oxidation - PDH
Pyruvate oxidation - CoA and NAD+, for PDH to convert into Ac-CoA it will need a CoA and this step also converts one NAD+ to NADH
glycogenolysis - glycogen phosphorylase which takes glucose off glycogen
📘 [Table – Lehninger Principles of Biochemistry]
Source: L3.1 pg 13 - graph on this page
Explanation:
Allosteric means site away from active site
Allosterically inhibited by:
ATP (substrate and inhibitor) –> i.e. it doesn’t really like ATP
at physiological [ATP], PFK rx rate very low
but if AMP is added (even in tiny amounts) now PFK activity greatly activated
Citrate (signal of Krebs cycle activity)
How inhibition works:
Activated by:
AMP (signals low energy)
Fructose-2,6-bisphosphate (potent liver activator)
How it works:
Classic control point of glycolysis.
Source: L3.1 pg 14
Explanation:
Inhibited by product G6P (negative feedback).
Prevents excess glucose trapping.
Allows glucose to exit cell via GLUT2 if not needed.
Liver enzyme glucokinase is not inhibited by G6P, enabling storage.
📘 Hexokinase vs glucokinase – Osmosis
Source: L3.1 pg 15 - diagram here
Example:
At rest:
glucose converted to G6P by hexokinase but since we’re restinf not much glycolysis is performed
causes build up of G6P which gives negative feedback loop to inhibit hexokinase
we stop trapping glucose as G6P
energy charge is high when resting and lots of ATP but no AMP
energy charge will act on PFK
PFK gets switched off since there’s not much AMP around and it doesn’t like ATP
PK also gets switched off
During exercise, falling ATP and rising AMP:
Using up trapped G6P so inhibition on Hexokinase is relieved
energy charge switched from high ATP to high AMP
Activate PFK-1 allosterically to switch on glycolysis
Inhibit PDH kinase → activates PDH
feedforward fructose 1,6-bisphosphate onto last enzyme in glycolysis Pyruvate kinase (i.e. its telling PK to hurry up)
All work together to upregulate glycolysis and fuel oxidation.
📘 AMPK and exercise – Cell Metabolism
Also pg 16:
The Role of Mitochondrial Fat Oxidation in Cancer Cell Proliferation and Survival - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/The-key-enzymes-determining-the-competition-of-glycolysis-versus-mitochondrial-fatty-acid_fig1_347695455 [accessed 5 May, 2022]
Figure 1. The key enzymes determining the competition of glycolysis versus mitochondrial fatty acid oxidation (FAO) defined by the Randle cycle. (A) Increased extracellular free fatty acids availability (FFA) can increase mitochondrial fatty acid oxidation (FAO), resulting in higher levels of acetyl-CoA, NADH, and ATP inside the mitochondria.
As a result, citrate production by citrate synthase (CS) is increased, but the activity of PDH and TCA cycle dehydrogenases is decreased by the elevation in acetyl CoA/CoA, NADH/NAD+, and ATP/ADP ratios. Thus, FAO leads to an accumulation of mitochondrial citrate, which is exported to the cytosol via the CIC/SLC25A1 exporter.
High citrate in the cytosol can inhibit phosphofructokinase-1 (PFK-1) and pyruvate kinase (PK) activities, decreasing glycolysis, pyruvate synthesis and oxidation. (B) When extracellular glucose increases, the concomitant upregulation in glycolysis provides more pyruvate to the mitochondria. Pyruvate oxidation by PDH and pyruvate carboxylation by pyruvate carboxylase (PC) generate acetyl-CoA and OAA respectively, increasing citrate synthase (CS) activity.
The amount of citrate produced is higher than needed to sustain the carbon pool of TCA cycle intermediates, which causes its export to the cytosol. Under high glucose, cytosolic citrate is hydrolyzed by ATP-citrate lyase (ACLY) into acetylCoA and OAA. Acetyl-CoA is carboxylated by two acetyl-CoA carboxylases, ACC1 and ACC2, to generate malonyl-CoA. ACC2 is localized in the outer mitochondrial membrane, causing malonylCoA to be more accessible to carnitine-palmitoyl transferase 1 (CPT1). Malonyl-CoA inhibits CPT1
Source: L3.1 pg 17
Example: PDH Regulation
PDH kinase phosphorylates PDH → inactivates it.
PDH phosphatase dephosphorylates → reactivates PDH.
Controlled by energy status (acetyl-CoA, NADH levels).
📘 PDH regulation – KEGG Pathway
Explanation:
RLS are context-dependent:
Fasted vs fed state
Rest vs exercise
Oxygen availability
Example: In fasting, carnitine shuttle becomes limiting for β-oxidation.
generally not ‘availability of substrates’ because we are demand driven not supply driven
demand = want/ don’t want, supply = enough/ not enough
normally every cell gets some glucose through GLUT-1 which is on every cell in body. Therefore, will always have SOME supply to a degree so it comes down to whether or not we NEED it
can be ‘transport & trapping’ –> maybe need CoA to trap glucose inside cell. maybe hexokinase need ATP to trap glucose inside cell
can be ‘transport into mitochondria’ –> so to get FA into matrix we need carnitine (which is the RLS)
can be ‘having enough gates (enzymes) at each step’ –> to get all the way through pathway
IN GENERAL: usually dictated by AMP level and availbility of NAD+, FAD, CoA, or whatever cofactors required by these enzymes. NAD+ and FAD are controlled by the cellular demand for ATP. If there’s no demand for ATP –> NADH and FADH2 stuck holding H/e- –> can’t drop off at ETC until some ATP is used.
📘 Dynamic RLS in metabolism – PMC
Explanation:
Opposing pathways are regulated in opposite ways:
E.g., glycolysis vs gluconeogenesis
β-oxidation vs fatty acid synthesis
Prevents futile cycling.
Often mediated by shared molecules (e.g., citrate, fructose 2,6-bisphosphate).
LOOK AT L3.1 pg20 for example with citrate
The regulatory role of citrate in the metabolism. Citrate is synthesized inside the mitochondria by citrate synthase from acetyl-CoA and OAA. It is exported outside the mitochondria by CIC. Citrate inhibits PFK1, PK, PDH, and SDH. Citrate inhibits also PFK2, which produces F2,6P, an allosteric activator of PFK1 in cancer cells. Activation effect is exerted on lipid biosynthesis through ACC, which produces malonyl-CoA, the first product of lipid biosynthesis, which, in turn, inhibits the CPT-1, the first enzyme of β -oxidation process. Through F1,6BPase, citrate stimulates gluconeogenesis. ACC, acetyl-CoA carboxylase; CS, citrate synthase; F1,6BPase, fruc- tose 1,6 bisphosphatase; G6P, glucose-6-phosphate; F6P, fructose- 6-phosphate; HK, hexokinase; PEP, phosphoenolpyruvate. Symbols + and – indicate stimulation and inhibition, respectively.
Citrate - new functions for an old metabolite - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/The-regulatory-role-of-citrate-in-the-metabolism-Citrate-is-synthesized-inside-the_fig1_259825846 [accessed 5 May, 2022]
Source: L3.2 pg 2, 3
Explanation:
Gluconeogenesis: From 3-Carbon to 6-Carbon Intermediates
Gluconeogenesis synthesizes glucose (6-carbons) from smaller carbon units, especially 3-carbon intermediates like pyruvate and lactate.
Here’s how 3-carbon intermediates are converted to 6-carbon sugars:
2 × pyruvate → 2 × oxaloacetate (OAA) via pyruvate carboxylase (mitochondrial)
OAA → 2 × phosphoenolpyruvate (PEP) via PEPCK
After several steps: 2 × triose phosphates → fructose 1,6-bisphosphate → F6P → G6P
The process bypasses irreversible glycolysis steps (hexokinase, PFK-1, pyruvate kinase).
Stepwise Process (with opposing glycolytic enzymes shown in pink)
Step 1: Pyruvate to Phosphoenolpyruvate (PEP)
Enzymes:
Pyruvate carboxylase (in mitochondria):
PEP carboxykinase (in cytosol):
Opposes: Pyruvate kinase (glycolysis, irreversible)
Step 2: PEP to 1,3-Bisphosphoglycerate → Glyceraldehyde-3-phosphate (G3P)
Multiple reversible steps occur here (opposite of glycolysis), involving:
Conversion of PEP → 2-phosphoglycerate → 3-phosphoglycerate → 1,3-bisphosphoglycerate → G3P
NADH + H⁺ used to reduce 1,3-BPG to G3P (reversal of glycolysis)
Step 3: Two 3-carbon G3P molecules → Fructose-1,6-bisphosphate (F1,6BP)
Via aldolase, two G3Ps are combined (via DHAP intermediate) to form:
\[ \text{G3P} + \text{DHAP} \rightarrow \text{Fructose-1,6-bisphosphate} \]
Now we have a 6-carbon intermediate
Step 4: Fructose-1,6-bisphosphate → Fructose-6-phosphate
Enzyme: Fructose-1,6-bisphosphatase
Opposes: Phosphofructokinase-1 (PFK-1) in glycolysis
Step 5: Fructose-6-phosphate → Glucose-6-phosphate → Glucose
Final steps:
Fructose-6-P → Glucose-6-P (reversible isomerization)
Glucose-6-P → Glucose via Glucose-6-phosphatase
Opposes: Hexokinase in glycolysis
Summary Table: Gluconeogenesis vs Glycolysis Key Steps
| Step | Gluconeogenesis (Blue) | Glycolysis (Pink) | Notes |
|---|---|---|---|
| 1 | Pyruvate → OAA → PEP (pyruvate carboxylase, PEPCK) | PEP → Pyruvate (pyruvate kinase) | Bypasses irreversible step |
| 2 | 1,3-BPG → G3P (uses NADH) | G3P → 1,3-BPG (makes NADH) | Reversible |
| 3 | G3P + DHAP → F1,6BP (aldolase) | Same | Reversible |
| 4 | F1,6BP → F6P (F1,6-bisphosphatase) | F6P → F1,6BP (PFK-1) | Bypasses irreversible step |
| 5 | G6P → Glucose (G6-phosphatase) | Glucose → G6P (hexokinase) | Final bypass step |
Key Point:
The conversion of two 3-carbon molecules (G3P/DHAP) into a single 6-carbon intermediate (F1,6BP) is a central milestone in gluconeogenesis, opposing the breakdown of glucose in glycolysis.
Source: L3.2 pg 4 –> ins diagram
Explanation:
To get G6P converted back to glucose –>
G6P enters the ER lumen via a transporter (T1).
Hydrolysed by glucose-6-phosphatase → glucose + Pi.
Glucose exits via T3; released into blood via GLUT2.
Only occurs in liver and pancreas.
📘 [G6Pase system – Lehninger Figure 15-28]
Source: L3.2 pg 7
Explanation:
2-Deoxyglucose (2-DG) lacks a 2’-OH → cannot be isomerised to F6P.
Trapped in cells as 2-DG-6P.
Used in FDG-PET imaging to track glucose uptake (e.g. in cancers).
Accumulation inhibits glycolysis (Warburg effect exploited).
📘 FDG-PET application – Radiopaedia
Explanation:
PFK-1 activity → glycolysis.
F1,6-BPase activity → gluconeogenesis.
Fructose 2,6-bisphosphate regulates both:
📘 Diagram – F26BP control loop (Biochem Text)
Explanation:
| Regulator | Effect on PFK-1 | Effect on F1,6-BPase |
|---|---|---|
| ATP | Inhibits | – |
| AMP | Activates | Inhibits |
| Citrate | Inhibits | – |
| Fructose 2,6-BP | Strong activator | Strong inhibitor |
📘 [Comparison – Stryer Biochemistry, Ch. 16]
Explanation:
F26BP is a key switch:
Activates glycolysis (PFK-1)
Inhibits gluconeogenesis (F1,6-BPase)
Synthesised by PFK-2, degraded by F2,6-BPase
Allows rapid hormonal control (glucagon vs insulin).
📘 [Visual Summary – Figure 15-17, Lehninger]
Source: L3.2 pg 10-16
Explanation:
↑ F26BP:
Lowers Km of PFK-1 for F6P → ↑ glycolysis
Inhibits F1,6-BPase → ↓ gluconeogenesis
↓ F26BP: reverse occurs
Rapidly adjusts metabolism based on fed vs fasting state.
F2,6BP is a powerful regulatory molecule that controls the balance between glycolysis and gluconeogenesis by affecting two opposing enzymes:
PFK-1 function: Converts fructose-6-phosphate → fructose-1,6-bisphosphate
F2,6BP effect: Activates PFK-1
This speeds up glycolysis, helping the cell break down glucose for energy.
| Molecule | Target Enzyme | Effect | Pathway Affected |
|---|---|---|---|
| F2,6BP | PFK-1 | ✅ Activates | ↑ Glycolysis |
| F2,6BP | F1,6BPase | 🚫 Inhibits | ↓ Gluconeogenesis |
F2,6BP acts like a switch:
When energy is needed, it turns glycolysis ON and gluconeogenesis OFF.
This helps the cell avoid doing both pathways at once, which would waste energy.
Source: L3.2 pg 10-16
Explanation:
PFK-2 and F26BPase are parts of the same bifunctional enzyme.
Phosphorylation by PKA (activated by cAMP/glucagon) → activates F26BPase.
Dephosphorylation (insulin) → activates PFK-2.
A classic example of reciprocal regulation.
📘 [Bifunctional enzyme mechanism – Nature Fig. 16-31]
Source: L3.2 pg 10-16
Explanation:
Fed state (insulin):
Fasting (glucagon):
Allows the liver to switch rapidly between pathways.
Explanation:
| Process | Enzymes Involved | Regulation |
|---|---|---|
| Glycolysis | Pyruvate kinase | Inhibited by ATP, alanine |
| Gluconeogenesis | Pyruvate carboxylase (PC) + PEPCK | PC activated by acetyl-CoA |
Involves 2 steps and ATP/GTP expenditure.
Pyruvate –> phosphoenolpyruvate: require 2 enzymes to get back
pyruvate carboxylase - stimulated by Ac-CoA
PEP pyruvate carboxykinase - quickly turns the 4-carbon oxaloacetate (OAA) into a 3-carbon and also adds phosphate group as well
Summary: getting pyruvate back to PEP - going from 3-carbon pyruvate –> pyruvate carboxylase uses CO2 (from bicarbonate) to join onto pyruvate –> taking CO2 back off again (using different enzymes (using GTP) and adds a phosphate.
Explanation:
FA oxidation → ↑ acetyl-CoA → activates pyruvate carboxylase
which is the first enzyme in gluconeogenesis
and forms more OAA which can potentially increase the number of Krebs cycles performed
Also inhibits PDH, limiting glucose oxidation
Promotes gluconeogenesis during fasting
Explanation:
Anaplerosis = replenishment of Krebs cycle intermediates.
OAA pulled out for gluconeogenesis must be replaced.
Pyruvate → OAA (via PC) serves both gluconeogenesis and anaplerosis.
Critical during exercise, starvation, or rapid biosynthesis.
📘 Anaplerosis and metabolic flux – PMC
Explanation:
After a meal (~50g glucose), blood glucose spikes and must be cleared rapidly to prevent toxicity.
The key players:
Insulin (from pancreatic β-cells): promotes glucose uptake and storage.
Liver acts as a “glucose sponge” via GLUT2.
Muscle and WAT take up glucose via insulin-responsive GLUT4 transporters.
Target: return glucose to ~5 mM within 2 hours.
Post-prandial glucose disposal is biphasic: initial hepatic uptake followed by peripheral tissue clearance.
📘 Video: Post-prandial glucose regulation
Explanation:
| Condition | Fasting Glucose | Insulin Response | Clearance Speed |
|---|---|---|---|
| Tolerant | ~5 mM | Normal, dynamic | Fast (returns in ~2 h) |
| Intolerant | Normal | High (ineffective) | Slow |
| Insulin resistant | Normal or high | Very high | Sluggish |
| Type 2 Diabetes | High | Impaired | Severely delayed |
📘 HbA1c and glucose tolerance – CDC
Explanation:
Glycemic Index (GI) = (AUC test food / AUC glucose) × 100
Test: 50g available carbohydrate → measure blood glucose over 2 hours.
High GI: rapid rise/fall (e.g., white bread, amylopectin-rich).
Low GI: slower rise/fall (e.g., legumes, amylose-rich foods).
Clinically useful in managing diabetes, appetite, and long-term risk of metabolic disease.
📘 Glycemic index explained – Harvard School of Public Health
Explanation:
| Transporter | Location | Features |
|---|---|---|
| GLUT1 | All cells | Basal glucose uptake |
| GLUT2 | Liver, β-cells | High capacity, low affinity; bidirectional |
| GLUT4 | Muscle, WAT | Insulin-dependent; translocates to membrane |
Insulin → GLUT4 translocation → increased uptake in muscle and fat.
Liver uses GLUT2, independent of insulin.
📘 GLUT transporters visual – Osmosis
Source L3.3 pg 15, 16 - diagram here
Explanation:
Glycogen = branched polymer of glucose, α-1,4 chains with α-1,6 branches.
we’d love to build up muscle glycogen stores
so you’ve got 2 choices: Glucose comes in and phosphorylated by hexokinase, trapped as G6P, then:
go down glycolysis to give ATP
get converted to glucose 1-phosphate through isomerisation reaction. it then needs to be activated if we want it to turn into glycogen storage. This activation requires UTP
Synthesised from UDP-glucose, which is formed using UTP + G1P.
Glycogen synthase adds glucose units (C1→C4).
Branching enzyme creates new α-1,6 linkages.
This is the story:
got UTP coming in. UTP carries 3 phosphates and we’re working on G1P which already has 1 phosphate. So, together there’s 4 phosphates involved.
Now 2 phosphates are released as PPi (Pyrophosphate). So now we have the 1 phosphate on G1P and also the remaining one that’s now on UMP (becomes monophosphate since it lost 2 phosphates)
Then these join together to give UDP glucose (diphosphate joined to glucose)
So we’re adding UDP to glucose to prepare it to join onto the glycogen chains. And it specifically joins on the ends of the branches
So now, when UDP glucose gets joined onto the glycogen chain it just takes the glucose part and now we’re left with the UDP
The UDP left behind can now go through that process again. But we need some UTP so we use ATP to convert the UDP back into UTP. So it gets converted by taking the phosphate from ATP, so now our ATP becomes ADP.
glycogen needs branching otherwise it will end up as one long linear chain. So the enzyme ‘glycogen-branching enzyme’ will cut the glycogen chain and join it back as a branch
📘 Glycogen structure & synthesis – LibreTexts
Explanation:
Glycogenesis is anabolic: uses ATP → reduces energy charge → ↑ AMP/ADP
This drop activates phosphofructokinase (PFK-1).
Therefore, insulin indirectly stimulates glycolysis by stimulating storage processes that lower ATP.
📘 Coupling of glycogenesis and glycolysis – Nature Education
Glycogenesis (glycogen synthesis) is an energy-consuming anabolic process, and this energy demand indirectly stimulates glycolysis, a catabolic pathway that provides ATP.
Insulin → activates Protein Phosphatase 1 (PPI)
PPI dephosphorylates glycogen synthase → activates GS
Result: ↑ Glycogen synthesis
Forming UDP-glucose and linking glucose to glycogen requires ATP
This lowers ATP, raises ADP and AMP levels
↓ ATP / ↑ AMP = low energy charge
PFK-1 is not directly activated by insulin
But it’s allosterically activated by AMP, which rises due to ATP usage
Result: ↑ Glycolytic flux → more ATP produced
When insulin tells the cell to store glucose as glycogen (an energy-expensive task), the drop in ATP signals the cell to burn some glucose for energy — stimulating glycolysis.
This is a beautiful example of metabolic balance:
Anabolism (storage) pulls on catabolism (energy production)
Insulin, though anabolic, causes some glucose to be burned to fuel the storage process
| Pathway | Triggered by | Effect |
|---|---|---|
| Glycogen synthesis | Insulin → PPI → Active GS | Uses ATP |
| Glycolysis | AMP ↑ → Activates PFK-1 | Makes ATP |
| Coupling effect | Energy demand from glycogen synthesis indirectly stimulates glycolysis |
Explanation:
| Feature | Hexokinase | Glucokinase |
|---|---|---|
| Tissue | All cells | Liver and β-cells |
| Substrate range | Any 6C sugar | Only glucose |
| Km for glucose | Low (~0.1 mM) → high affinity | High (~10 mM) → low affinity |
| Inhibition | Inhibited by G6P | Not inhibited by G6P |
| Saturation | Easily saturated | Active only at high [glucose] |
📘 Glucokinase vs Hexokinase – Osmosis
Explanation:
| Feature | Liver | Muscle |
|---|---|---|
| Uptake transporter | GLUT2 (insulin-independent) | GLUT4 (insulin-dependent) |
| Hexokinase isoform | Glucokinase (high Km, not inhibited by G6P) | Hexokinase (low Km, G6P-inhibited) |
| Regulation style | Push – high [glucose] → passive uptake | Pull – insulin stimulates GLUT4 + GS |
| GS regulation | Stimulated by glucose and G6P | Stimulated by insulin-dependent GS action |
| Function | Maintains blood glucose | Fuel for contraction |
📘 [Glycogenesis in liver vs muscle – Lehninger Ch. 15]
Explanation:
Glucose → G6P → pyruvate via glycolysis
Pyruvate enters mitochondria → converted to acetyl-CoA by PDH
Acetyl-CoA condenses with OAA → citrate
Citrate exported → cleaved by ATP-citrate lyase (ACL) → cytosolic acetyl-CoA
Acetyl-CoA → malonyl-CoA (via ACC) → fatty acid synthesis (FAS)
Requires: NADPH (from PPP), ATP, glycerol-3-phosphate (from glycolysis)
📘 Video: Overview of Lipogenesis
Reaction:
\[ \text{Acetyl-CoA} + \text{HCO₃⁻} + \text{ATP} \xrightarrow{\text{ACC}} \text{Malonyl-CoA} + \text{ADP} + \text{Pi} \] - taking Ac-CoA and adding CO2 (from bicarbonate) to form 3-carbon chain called malonyl-CoA
conversion step requires vitamin called biotin and uses some ATP
conversion also requires cofactor magnesium
basically an activated/primed ‘Ac-CoA’ ready for lipogenesis
Regulation:
Activated by insulin (via dephosphorylation)
insulin activates the phosphotase to perform dephosphorylation on ACC which activates ACC
so insulin is telling cells to convert Ac-CoA to malonyl-CoA for fatty acid synthesis.
Allosterically activated by citrate (forms polymeric filaments)
Inhibited by fatty acyl-CoA (feedback inhibition)
📘 Biochemical pathway – ACC regulation
Source: L4.1 pg 6
Explanation:
Lipogenesis is basically reduction then dehydration then reduction again (to add on 2-carbon chains at a time)
Large multifunctional enzyme complex
Key steps (per 2C addition cycle):
Loading: Acetyl group + Malonyl group attached to ACP (FAS binds to its substrates)
Condensation - malynol will be decarboxylated so the CO2 comes off
Reduction (NADPH) - negative charge will attack at the cabonyk carbon and join itself onto the chain, maing it 4-carbon long
Dehydration
Second reduction (NADPH)
Chain transfer and repeat
Final product: Palmitate (C16) → released when chain reaches 14–18 carbons
📘 Diagram – Fatty Acid Synthase Cycle
Fed state (insulin present):
Fasted/exercise (AMPK active):
📘 AMPK and ACC regulation – ScienceDirect
The Fatty Acid Synthase (FAS) complex catalyzes the de novo synthesis of palmitate (C16:0) from acetyl-CoA and malonyl-CoA, using NADPH as a reducing agent. This process occurs in the cytosol and consists of a cyclic reaction sequence repeated 7 times to build the 16-carbon saturated fatty acid.
Loading phase
Condensation
Reduction
Dehydration
Reduction
Chain transfer
Repeat cycle
Termination
| Step | Enzyme Domain | Substrate / Action | Product / Outcome |
|---|---|---|---|
| 1. Loading | - Acetyl transferase (AT) loads
acetyl-CoA onto the KS domain
(β-ketoacyl synthase) - Malonyl transferase (MT) loads malonyl-CoA onto ACP (acyl carrier protein) |
Acetyl-KS and Malonyl-ACP formed | |
| 2. Condensation | β-Ketoacyl Synthase (KS) | Acetyl group + Malonyl group → 4-carbon β-ketoacyl-ACP + CO₂ (decarboxylation drives the reaction) | β-ketobutyryl-ACP |
| 3. Reduction | β-Ketoacyl reductase (KR) | Reduces β-keto group to hydroxyl using NADPH | β-Hydroxyacyl-ACP |
| 4. Dehydration | Dehydratase (DH) | Removes H₂O → forms a double bond (trans-Δ²-enoyl-ACP) | Enoyl-ACP |
| 5. Reduction | Enoyl reductase (ER) | Reduces double bond using NADPH → saturated acyl-ACP | Butyryl-ACP (4-carbon chain) |
| 6. Chain Transfer | Transfer the growing acyl chain from ACP to KS, freeing ACP for the next malonyl-CoA | Butyryl-KS formed | |
| 7. Repeat | Another malonyl-CoA is loaded to ACP, and steps 2–6 repeat 6 more times | Adds 2C per cycle until 16C palmitoyl-ACP | |
| 8. Termination | Thioesterase (TE) cleaves palmitate from ACP | Final product: Palmitate (C16:0) |
Inputs per cycle: 1 acetyl-CoA (start), 7 malonyl-CoA, 14 NADPH
Final product: Palmitate (C16:0)
Byproducts: CO₂, H₂O, NADP⁺
Fatty Acid Synthase builds a 16-carbon saturated fatty acid through 7 cycles of condensation, reduction, dehydration, and reduction, using malonyl-CoA as the 2-carbon donor and NADPH as the reducing power, terminating in palmitate release.
Explanation:
3 fatty acyl-CoA + glycerol-3-phosphate → triacylglycerol (TAG)
Requires FA-CoA, not free FA
G3P from:
Liver: via glycerol kinase
Adipose: from glycolysis (DHAP → G3P)
Esterification enzymes and FAS are upregulated by insulin
📘 TAG synthesis pathway – LibreTexts
The esterification process you’re referring to is the biosynthesis of triacylglycerol (TAG), where three fatty acyl-CoA molecules are esterified to a glycerol backbone (specifically glycerol-3-phosphate) to form a TAG (also called a triglyceride).
\[ \text{Glycerol-3-phosphate} + 3\ \text{Fatty acyl-CoA} \longrightarrow \text{Triacylglycerol (TAG)} + 3\ \text{CoA-SH} + \text{Pi} \]
Fatty acids are first “activated” by linking to Coenzyme A (CoA):
\[ \text{Fatty acid} + \text{CoA} + \text{ATP} \rightarrow \text{Fatty acyl-CoA} + AMP + PPi \]
This forms a high-energy thioester bond, making the fatty acid reactive for esterification.
The first fatty acyl-CoA is esterified to the sn-1 hydroxyl of glycerol-3-phosphate:
\[ \text{Glycerol-3-P} + \text{Fatty acyl-CoA} \rightarrow \text{Lysophosphatidic acid} + \text{CoA-SH} \]
A second fatty acyl-CoA is esterified to the sn-2 hydroxyl:
\[ \text{Lysophosphatidic acid} + \text{Fatty acyl-CoA} \rightarrow \text{Phosphatidic acid} + \text{CoA-SH} \]
The phosphate group is removed from phosphatidic acid:
\[ \text{Phosphatidic acid} \rightarrow \text{Diacylglycerol (DAG)} + \text{Pi} \]
A third fatty acyl-CoA is added to the sn-3 hydroxyl:
\[ \text{DAG} + \text{Fatty acyl-CoA} \rightarrow \text{Triacylglycerol (TAG)} + \text{CoA-SH} \]
| Step | Enzyme |
|---|---|
| 2 | Glycerol-3-phosphate acyltransferase (GPAT) |
| 3 | 1-Acylglycerol-3-phosphate acyltransferase (AGPAT) |
| 4 | Phosphatidic acid phosphatase (lipin) |
| 5 | Diacylglycerol acyltransferase (DGAT) |
TAG synthesis involves the sequential esterification of three fatty acyl-CoAs to a glycerol-3-phosphate backbone, forming a highly energy-dense molecule used for long-term energy storage in adipose tissue. This process occurs primarily in the liver and adipose tissue.
Pathways involved:
Glycolysis: glucose → pyruvate + G3P
PDH: pyruvate → acetyl-CoA
Krebs cycle: citrate formed → exported
ATP-citrate lyase: citrate → acetyl-CoA + OAA
ACC: acetyl-CoA → malonyl-CoA
PPP: provides NADPH
Gluconeogenesis (glyceroneogenesis): G3P production
📘 Integrated map – Stryer Biochemistry
Explanation:
Insulin activates PDH phosphatase → dephosphorylates PDH → active
PDH converts pyruvate to acetyl-CoA, promoting substrate availability for lipogenesis
📘 PDH regulation – KEGG Pathway
Since PDH activated, we will have lots of Ac-CoA forming
Two fates:
we require it to enter Krebs because we need it to fully oxidise and make lots of NADH to generate more ATP
we also need ATP for Malynol-CoA
firstly, need Ac-CoA to make its way into cytoplasm –> so we need to allow it to enter Krebs to join onto OAA to form citrate
this allows us to get the CoA back so that PDH can continue to convert pyruvate to Ac-CoA and it needs a CoA to do that
we also allow the formed citrate to flow back out into cytoplasm for lipogenesis
Decided by:
Energy status (ATP/AMP): High ATP → citrate exported
Hormonal state (insulin vs glucagon)
Availability of OAA for citrate formation
📘 Metabolic fate of acetyl-CoA – PMC
Source L4.1 pg 18 - diagram here
Cytosolic citrate:
Activates ACC (↑ fat synthesis)
Inhibits PFK-1 (↓ glycolysis)
Converted to acetyl-CoA + OAA (via ACL)
Converted to malonyl-CoA → inhibits CPT-1 → ↓ β-oxidation
From Lecture:
we want some ac-CoA out in the cytoplasm so we can use it as a 2-carbon building block
the CoA can’t cross membranes so we’ll allow it to enter Krebs cycle
it gets converted to citrate
citrate is transported out to cytoplasm
break the ac-CoA back off it again so now it becomes a 4-carbon OAA again
OAA gets reduced by NADH to give malate
malate gets converted to pyruvate to re-enter the mitochondria and in this process it gives us some NADPH
NADPH is the reductant needed to reduce FA in our cytoplasm
see diagram in lecture
Explanation:
OAA can’t cross the inner mitochondrial membrane
Converted to:
Malate, then back to OAA in mitochondria
Or pyruvate via malic enzyme (generates NADPH)
Pyruvate re-enters mitochondria → carboxylated to OAA by pyruvate carboxylase
📘 OAA shuttle diagram – Nature
Purpose:
Generates NADPH for reductive biosynthesis (e.g., lipogenesis)
Provides ribose-5-phosphate for nucleotide synthesis
Fit with lipogenesis:
Demand for NADPH ↑ → G6PDH (key PPP enzyme) activated
PPP enzymes return 3C and 6C sugars back to glycolysis
Uncategorised: Malonyl-CoA inhibits beta oxidation:
Malynol CoA inhibits CAT-1.
We’re inhibiting transport of FA into the mitochondria where they would undergo beta oxidation
we don’t want to be building FA while we’re oxidising FA inside the mitochondria.
so when ac-CoA activated by turning it into malynol-coA (activated by insulin), the malynol-CoA can inhibit CAT1
this stops us from transporting fatty acyl coA back into mitochondria as acyl carnitines
Key substrates and checkpoints:
| Substrate | Enzyme/Control | Role |
|---|---|---|
| Glucose | GLUT4/HK | Entry/trapping |
| Pyruvate | PDH | Mitochondrial conversion |
| Citrate | ACL | Exported carbon source |
| Acetyl-CoA | ACC | Malonyl-CoA production (RLS) |
| NADPH | G6PDH/PPP | Reductant for FAS |
| G3P | Glyceroneogenesis | Backbone for esterification |
| FA-CoA + G3P | Esterification enzyme | Forms triacylglycerol |
📘 [Integrated regulatory map – Figure in lecture slide 28]
ANNOTATE DIAGRAM ON PAGE 25 L4.1:
Um, this, that citrate, what remains the oxalo acetate needs to get back into the mitochondria to, to stay in the, the reb cycle. Going back in, that will give us some more NADPH. You could annotate even more things along the way, that acetylchoA gets converted to Malanol CA. Um, the fatty acids are going to get userified together to form a triase or glycerol. So you’ve got lots and lots of things feeding, all just so that this fatty acid synthesis can occur.
Explanation:
Dietary fats are hydrophobic, forming large lipid droplets.
Digestion problem: enzymes (like lipase) are water-soluble and can’t access fat interior.
Bile salts (amphipathic molecules made from cholesterol in the liver and stored in gall bladder):
Provide surface area for enzyme action
Release: When a meal containing fat is consumed, the gall bladder contracts and releases bile salts into small intestine.
Emulsify fats into micelles: bile salts mix with dietary fats in small intestine
cholesterol excretion via loss of bile salts in faeces is the only way body eliminates cholesterol
Recycled via enterohepatic circulation: bile salts are efficiently reabsorbed (98-99%) in the small intestine and recycled back into the body
cholesterol is either lost as bile salts or retained in the body, cannot be oxidised.
Explanation:
Pancreatic lipases are enzymes secreted into small intestine that act on fat emulsion created by bile salts. –> enzymes break down triglycerides into smaller absorbable components (monoacylglycerol, diacylglycerol, fatty acids, and glycerol).
Pancreatic lipase is secreted into the duodenum.
Acts on triglycerides inside micelles → releases:
3 Free fatty acids
Monoacylglycerols
Glycerol
Products diffuse into enterocytes → re-esterified into TAG → packaged into chylomicrons
Examples:
Gallstones block bile flow → impaired micelle formation (prevents release of bile salts into small intestine) → poor fat digestion → malabsorption of dietary fats
Orlistat: lipase inhibitor (inhibits digestion and absorption of dietary fats from small intestine) → undigested fat → steatorrhea (oily stools)
Olestra: artificial fat (FA + sucrose) not digestible (allows consumption of fried foods without adding calories)→ passes through gut; can deplete fat-soluble vitamins
📘 Clinical case: Orlistat side effects
source: L4.2 pg 14 - diagram here
Explanation:
Lipoproteins = spherical particles that carry fats in blood
Core: TAG and cholesterol esters
Surface: phospholipids + apoproteins
Differ by:
Density (chylomicrons → HDL)
Size
Apoprotein content (e.g., ApoB-48, ApoC-II, ApoE)
| Lipoprotein | Function | Origin | Fate |
|---|---|---|---|
| Chylomicrons | Transport dietary TAG & cholesterol | Intestine | Lipolysis by LPL → remnants to liver |
| VLDL | Distributes liver-made TAG | Liver | Becomes IDL → LDL |
| LDL | Delivers cholesterol to cells | VLDL remnant | Endocytosed via LDL receptor |
| HDL | Removes cholesterol from tissues | Liver | Returns to liver (reverse transport) |
Clinical Significance: Measuring blood cholesterol involves assessing the levels and ratios of different lipoproteins (VLDL, LDL, HDL) rather than just free-floating cholesterol
Characteristic: the largest and least dense lipoprotein
Formation: formed in the intestinal muscosa to transport dietary fats and cholesterol from the small intestine to the rest of the body
Composition: packed with dietary fats (triglycerides) and cholesterol.
To effectively transport cholesterol within lipoproteins, it must be rendered completely hydrophobic.
This involves modifying cholesterol by attaching a fatty acid to it, forming a cholesterol ester (this makes it fully hydrophobic)
Acyl-CoA Cholesterol Acyltransferase (ACAT): The enzyme that catalyzes the esterification of cholesterol. Transfers a fatty acyl group from acyl-CoA to cholesterol
Entry into circulation: enter the lymph system instead of directly to the bloodstream
Destination: Transport fats (dietary triglycerides and cholesterol) to various tissues throughout body
Plasma Appearance: Blood plasma appears turbid or milky after a fatty meal due to chylomicrons
Metabolism through Lipoprotein lipase (LPL):
LPL is an enzyme located on the surface of blood vessels cappilary walls that hydrolyses triglycerides in lipoproteins –> releasing fatty acids and glycerol for uptake by cells
re-esterification requires a Glycerol 3-phosphate (to store fat away). liver converts glycerol to G3P using glycerol kinase (enzyme only present in liver). any other cells that want to store that feet needs to steal from glycolysis.
Chylomicron remnants: after LPL renoves trigylcerides from chylomicrons, remaining particle is called chylomicron remnant –> gets returned to liver
-gets taken up by liver via endocytosis –> liver repackages with newly synthesised fats and glycerol
Characteristic: pretty much does same thing. Goes around the body and any cells that want the fat of the VLDL are going to express LPL at capillary wall
cells digest some of the fat out of it and send it back to liver again.
if enough fat is taken out of the VLDL it becomes LDL, if a little bit taken it becomes IDL
Characteristic: rich in cholesterol and delivers it to cells and tissues throughout body
Delivery: Cells take up LDLs via LDL receptors on cell surface. Cells internalise LDL via endocytosis
Destination: cells break down LDLs to use the cholesterol and fat inside
Characteristic:
Formation:
Composition:
Entry into circulation:
Destination:
| Lipoprotein | Size | Density | Primary Cargo | Origin | Destination |
|---|---|---|---|---|---|
| Chylomicrons | Largest | Lowest | Dietary Triglycerides | Intestinal Mucosa | Lymph, then tissues |
| VLDL | Large | Low | Endogenous Triglycerides | Liver | Tissues |
| IDL | Medium | Intermediate | Triglycerides, Cholesterol | VLDL Metabolism | LDL or Liver |
| LDL | Small | High | Cholesterol | VLDL/IDL Metabolism | Tissues, can lead to plaque formation |
| HDL | Smallest | Highest | Cholesterol | Liver, Intestine | Liver (Reverse Cholesterol Transport) |
Source: L4.2 pg 22 - diagram here
Dietary fat intake: Fat coming in the diet
small intestine: fat is emulsified with bile salts and broken down by pancreatic lipase
absorption: fatty acids and glycerol are absorbed across intestinal walls
chylomicron formation: intestinal cells repackage fats into chylomicron lipoproteins
lymphatic system: chylomicrons enter the lymph and then the blood
LPL action: any cells that want fats would use LPL in the lining of capillaries to digests tryglycerides from chylomicrons
chylomicron remnant uptake: chylomicron remnants return to the liver
liver processing: the liver uses cholesterol to make bile salts or packages fats into VLDLs
VLDL metabolism: VLDLs deliver fat to cells via LPL, eventually becoming LDL
LDL delivery: LDLs deliver cholesterol to cells via LDL receptors. any remaining will return to the liver again for repackaging
Most cells can synthesize their own cholesterol, often in amounts exceeding dietary intake. The body tightly regulates cholesterol synthesis, and statins, one of the most prescribed drugs, target the initial enzyme in this pathway.
Source: L4.2 pg 23
Pathway:
Acetyl-CoA → HMG-CoA → Mevalonate → Cholesterol
Rate-limiting enzyme: HMG-CoA Reductase
Regulated by:
Insulin (↑)
Cholesterol (↓ via feedback)
Statins (competitive inhibitors)
Circadian rhythm
By inhibiting cholesterol synthesis, statins force cells to uptake LDLs from the blood, lowering LDL concentration
LDL:
Oxidised LDL (oxLDL) taken up by macrophages → foam cells → contributing to plaque formation in arteries by trigerring immune response (atherosclerosis)
Pro-inflammatory, plaque forming
HDL:
Removes excess cholesterol from tissues → delivered to liver for processing
Anti-inflammatory, protective against CVD
HDL Synthesis: HDLs are produced in the liver.
Reverse Cholesterol Transport: HDLs scavenge excess cholesterol from tissues and transport it back to the liver.
Liver Processing: The liver can use the cholesterol from HDLs to synthesize bile salts or repackage it into VLDLs.
Desirable Levels: High HDL levels are generally considered beneficial.
| Strategy | Mechanism |
|---|---|
| Statins | Inhibit HMG-CoA reductase → ↑ LDL receptor expression |
| Phytosterols | Compete with cholesterol for gut absorption |
| Resins | Bind bile salts → ↑ conversion of cholesterol to bile acids |
| Diet | Reduce saturated fat → improves HDL/LDL ratio |
| CETP inhibitors (experimental) | Block cholesterol transfer from HDL → VLDL |
📘 Cholesterol treatment guidelines – AHA
Explanation:
Cholesterol modulates membrane fluidity:
Prevents crystallisation of saturated FAs
Adds stability in fluid unsaturated membranes
Embedded between phospholipids
Essential for lipid raft formation, receptor function, membrane proteins
Cholesterol is a vital structural component of animal cell membranes. It helps maintain the balance between fluidity and rigidity, ensuring that membranes stay functional and stable under different conditions.
✅ Acts like a buffer — stabilizing membrane behavior across temperature changes.
Cholesterol is essential for maintaining the structure, fluidity, and function of cell membranes. It acts as a fluidity buffer, barrier, and organizer, allowing the membrane to be both flexible and strong — critical for cell survival and communication.
Types:
Monounsaturated (MUFA): olive oil (oleic acid)
Polyunsaturated (PUFA):
Omega-6 (linoleic acid): plant oils
Omega-3 (alpha-linolenic acid): flaxseed, fish oil
Function:
Increase membrane fluidity
Cardioprotective effects
Some essential (must be obtained from diet)
Unsaturated fatty acids are fats that contain one or more double bonds in their carbon chains. These are generally considered heart-healthy and are liquid at room temperature.
Example:
Examples:
ALA (alpha-linolenic acid) – plant-based
EPA and DHA – marine-based
First double bond at the 6th carbon from the methyl end
Can be pro-inflammatory in excess but are essential
Examples:
Linoleic acid
Arachidonic acid
| Type | Fatty Acid | Sources |
|---|---|---|
| MUFAs | Oleic acid | Olive oil, avocados, almonds, peanuts, canola oil |
| PUFAs (Omega-3) | ALA | Flaxseeds, chia seeds, walnuts, soybean oil |
| EPA, DHA | Fatty fish (salmon, mackerel, sardines), fish oil | |
| PUFAs (Omega-6) | Linoleic acid | Sunflower oil, corn oil, soybean oil, walnuts |
| Arachidonic acid | Meats, eggs, poultry (small amounts) |
Unsaturated fatty acids include monounsaturated (MUFAs) and polyunsaturated (PUFAs) fats. They are found in plant oils, fish, nuts, and seeds, and are crucial for cell membranes, inflammation control, and cardiovascular health.
Explanation:
β-oxidation can proceed normally after cis→trans conversion
Requires enoyl-CoA isomerase
Oxidation restarts from the trans-intermediate
Trans fats (industrial) are not handled efficiently → raise LDL:HDL ratio
📘 [FA oxidation pathway – Stryer Biochem Fig. 17-9]
Unsaturated fatty acids undergo β-oxidation, just like saturated fatty acids, but with extra steps to handle their double bonds, which can interfere with standard oxidation enzymes.
Used for: Fatty acids with odd-numbered double bonds (e.g. oleic acid)
Used for: Fatty acids with even-numbered double bonds (e.g. linoleic acid)
| Issue Caused by Double Bond | Enzyme Needed | Action |
|---|---|---|
| Cis-Δ³ double bond | Enoyl-CoA isomerase | Converts it to trans-Δ² |
| Conjugated Δ²,Δ⁴ system | 2,4-Dienoyl-CoA reductase + Enoyl-CoA isomerase | Reduces and rearranges to trans-Δ² |
Same as saturated FA oxidation: Acetyl-CoA, FADH₂, NADH
But less ATP is produced than with saturated fatty acids of the same length because:
Oxidation of unsaturated fatty acids requires extra enzymes to rearrange or reduce double bonds so they can enter the normal β-oxidation cycle. These steps ensure that even with double bonds, the fatty acid can still be broken down for energy.
Total body protein pool: ~6 kg in a 70 kg adult
Daily turnover: ~200 g/day (~4%)
This is the recycling of existing proteins into new ones (not full degradation).
Varies by tissue:
| Route | Amount (approx.) |
|---|---|
| Urea (urine) | ~14 g nitrogen/day |
| Faeces | ~2 g nitrogen/day |
| Skin | ~0.5 g nitrogen/day |
➡ Total nitrogen loss ≈ 16.5 g/day, matching intake → neutral nitrogen balance
Daily nitrogen balance reflects 100 g/day protein intake (~16.5 g nitrogen) matched by equivalent nitrogen loss, mostly as urea. Around 200 g of body protein is recycled daily, and although muscle holds most protein, it’s slow to turn over compared to rapidly renewing tissues like the intestine. Amino acids can’t be stored, so any excess must be degraded and detoxified.
Starvation or fasting
High-protein diet
High-turnover tissues (e.g. intestinal epithelium, liver)
Well-fed state with balanced diet
Resting muscle
Processing is driven by need: We process amino acids when:
The liver handles most of the metabolic sorting:
Amino acid processing spikes during starvation, excess protein intake, and in fast-renewing tissues like the intestine. In these states, amino acids are broken down for energy, glucose, or fat, and nitrogen is converted to urea. Processing is lowest in well-fed, non-stressed states, especially in muscle, which has low protein turnover despite being the largest protein reserve.
Explanation: Amino acid processing involves handling the nitrogen (amine group) and the carbon skeleton of amino acids once they are released from proteins (via turnover, dietary intake, or catabolism).
Core Principles:
Amine group (–NH₂) → toxic as free ammonia (NH₃) → must be detoxified via the urea cycle.
Carbon skeleton → used for energy, glucose synthesis, or fatty acid synthesis
Unlike fat or glycogen, amino acids must be used, recycled, or degraded
Excess amino acids are broken down; their nitrogen is excreted and carbon skeletons repurposed
First organ to receive dietary amino acids via the hepatic portal vein
Performs:
Transamination to collect nitrogen into glutamate/alanine
Urea synthesis (from ammonia and aspartate)
Carbon skeleton processing (oxidation, gluconeogenesis, lipogenesis)
Normal turnover: proteins degraded and rebuilt; unused amino acids are oxidised or excreted
Starvation: proteolysis increases, amino acids used for gluconeogenesis
High-protein intake: excess amino acids → detoxified and used for energy or fat synthesis
Leucine, isoleucine, valine
Not metabolised significantly by the liver
Released into circulation → taken up and oxidised by muscle
TLDR Summary: Amino acid processing revolves around detoxifying nitrogen (via the urea cycle) and reusing carbon skeletons (via gluconeogenesis, lipogenesis, or oxidation). The liver is the central organ, coordinating nitrogen disposal and carbon fate. Amino acids can’t be stored, so any excess must be metabolised. BCAAs are the exception, bypassing liver metabolism and serving as an energy source in muscle.
Amino acid processing refers to how the body handles, breaks down, and uses amino acids — especially when they’re not needed for protein synthesis. This involves separating their nitrogen (amino group) from their carbon skeleton, and using those parts for different purposes.
The amino group is transferred from the amino acid to α-ketoglutarate, forming glutamate.
Catalyzed by aminotransferase enzymes (requires pyridoxal phosphate, PLP).
Result:
\[ \text{Amino acid} + \alpha\text{-ketoglutarate} \rightarrow \text{α-keto acid} + \text{glutamate} \]
The leftover carbon skeleton (α-keto acid) is used for:
| Type | Converted Into |
|---|---|
| Glucogenic amino acids | Pyruvate or TCA intermediates → glucose |
| Ketogenic amino acids | Acetyl-CoA or acetoacetate → ketones or fat |
Amino acid processing involves removing nitrogen (via transamination and urea cycle) and reusing the carbon skeleton for energy, glucose, or fat. This process is essential during fasting, low-carb intake, or protein-rich diets.
Not rate-limiting: These enzymes don’t control the speed of degradation; they act when [amino acid] is high.
This makes amino acid degradation:
Ensures that any amino acids not immediately needed are efficiently degraded and detoxified
If excess amino acids are present, they are rapidly degraded, regardless of metabolic state — because the enzymes are already there and waiting to act.
The enzymes that break down amino acids have high Km, meaning they only work when amino acid concentrations are high. This ensures the body preserves amino acids during deficiency but efficiently degrades excess when present. These enzymes are not tightly regulated, making amino acid degradation dependent on availability, not on hormonal control.
Understood. Here’s the comprehensive explanation for learning objectives 5, 6, and 7, using both the transcript and lecture slides, without emojis or informal elements:
Transaminases (aminotransferases) are enzymes that transfer amino groups from one amino acid to an α-keto acid. This process is central to:
The amino group from an amino acid is transferred to an α-keto acid.
The major acceptors of amino groups are:
These reactions allow the nitrogen to be safely collected and shuttled toward the liver for disposal via the urea cycle.
Transaminases enable the safe, reversible transfer of nitrogen, allowing amino acids to be broken down or re-synthesised. They also help channel nitrogen into glutamate, alanine, or aspartate for entry into the urea cycle.
The urea cycle disposes of excess nitrogen (from amino acids) by converting toxic ammonia into urea, which is excreted via urine.
Occurs primarily in the liver
Takes place in both the mitochondrial matrix and the cytosol
Requires input of two nitrogen atoms:
Involves the carrier molecule ornithine, analogous to oxaloacetate in the TCA cycle
Produces urea and fumarate (which feeds back into the TCA cycle)
The urea cycle detoxifies ammonia by forming urea, using inputs from glutamate and aspartate. It requires energy, is compartmentalised in mitochondria and cytosol, and links to central carbon metabolism via fumarate.
1. With the TCA (Krebs) Cycle:
2. With Amino Acid Metabolism:
3. With Gluconeogenesis:
4. With Energy Metabolism:
The urea cycle is deeply interconnected with the TCA cycle, amino acid metabolism, gluconeogenesis, and energy metabolism. It shares intermediates (e.g., fumarate, aspartate) and relies on nitrogen-carrying amino acids to dispose of excess nitrogen, integrating nitrogen disposal with carbon and energy homeostasis.
Amino Acid Catabolism
↓ ↑
(Transamination) (Amino acid synthesis)
↓
Glutamate
↓
Glutamate Dehydrogenase
↓
NH₃ →→→→→→→→→→→→→→
↓ ↓
Urea Cycle Aspartate (from OAA)
↓ ↑
Urea (excreted) ←←←← Fumarate
↓
TCA Cycle → OAA
↓
GluconeogenesisThe urea cycle is a nitrogen-disposal system that is deeply interwoven with carbon metabolism, especially the TCA cycle and gluconeogenesis. It ensures that while amino acids are used for energy or glucose, their nitrogen is safely excreted.
Amino acids are classified based on whether their carbon skeletons can be converted into glucose (gluconeogenic) or ketone bodies/fatty acids (ketogenic).
Glucogenic: Carbon skeletons can be converted into intermediates of the Krebs cycle and used for gluconeogenesis
Carbon skeletons → pyruvate, α-KG, OAA, fumarate, succinyl-CoA
Can feed gluconeogenesis
Ketogenic: Carbon skeletons are converted into acetyl-CoA, which can be used for fatty acid synthesis or ketone body formation
→ acetyl-CoA or acetoacetate
Used in ketone synthesis or energy only
Both:
📘 AA fate chart – Lumen Learning
The classification of amino acids as ketogenic or glucogenic depends on the metabolic fate of their carbon skeletons after the amino (–NH₂) group is removed during amino acid catabolism.
| Type | What they can be converted into | Final metabolic fate |
|---|---|---|
| Glucogenic | → Pyruvate or TCA cycle intermediates | Used to make glucose via gluconeogenesis |
| Ketogenic | → Acetyl-CoA or acetoacetate | Used to make ketone bodies or fat |
| Both | → Some carbon goes to glucose, some to ketones | Have dual metabolic pathways |
The human body needs to:
So depending on where an amino acid enters the metabolic map, it will be classified based on its carbon fate.
| Entry Point | Type | Why? |
|---|---|---|
| Pyruvate | Glucogenic | Pyruvate can → glucose |
| Oxaloacetate, α-ketoglutarate, fumarate, succinyl-CoA | Glucogenic | All are TCA cycle intermediates that can → glucose |
| Acetyl-CoA, acetoacetate | Ketogenic | Cannot → glucose (carbons lost as CO₂ in TCA cycle) |
| Amino Acid | Type | Reason |
|---|---|---|
| Leucine, Lysine | Ketogenic only | Carbon skeleton → acetyl-CoA/acetoacetate |
| Alanine, Glutamate, Aspartate | Glucogenic only | Carbon skeleton → pyruvate or TCA intermediates |
| Isoleucine, Phenylalanine, Tyrosine, Tryptophan | Both | Yields both glucogenic and ketogenic products |
Ketogenic amino acids cannot be used to make glucose because acetyl-CoA cannot be converted back to pyruvate — its carbons are lost as CO₂ in the TCA cycle.
Explanation:
Humans lack enzymes to synthesise certain AA side chains
Essential AAs often require:
Multiple steps
Cofactors (e.g., folate, B₆)
9 essential AAs (e.g., lysine, threonine, leucine)
Conditional essentiality: e.g., arginine in children
Protein Synthesis Requirement: All 20 amino acids are required to synthesize any protein. (00:44:06)
Protein Composition: Most proteins contain hundreds of amino acids, with each of the 20 amino acids represented. (00:44:17)
Ribosome Function: Ribosomes translate mRNA into proteins by adding amino acids to the growing polypeptide chain.
The process of protein synthesis is highly dependent on the availability of all 20 amino acids. If even one amino acid is missing, the ribosome stalls, and the incomplete polypeptide chain is released and degraded. This is unlike language learning, where understanding can still be gleaned even if a word is unknown. In protein synthesis, the absence of a required amino acid halts the entire process.
Key Points:
Ribosomal Stalling: If an aminoacyl tRNA is not available to deliver the required amino acid, the ribosome stalls.
Incomplete Protein Degradation: The ribosome releases the partially constructed polypeptide, which is then broken down.
All 20 Amino Acids Required: The absence of even one amino acid prevents the cell from synthesizing any proteins.
📘 Essential AAs – NIH Fact Sheet
Explanation:
e.g., 6-mercaptopurine inhibits PRPP amidotransferase
Affects de novo purine synthesis
High-impact in rapidly dividing cells (cancer, bone marrow)
Used in leukemia therapy
📘 Purine inhibitors – Cancer.gov
Synthesizing nucleotides de novo is energetically expensive. Therefore, cells prioritize salvaging and recycling existing purines rather than synthesizing them from scratch every time they are needed. This is particularly important for rapidly dividing cells, such as cancer cells, which require vast amounts of nucleotides to replicate their DNA. Salvage pathways are thus a key target for certain medicines, especially in cancer treatment. (00:46:56-00:48:31)
High Energy Cost of Synthesis: De novo nucleotide synthesis is energetically demanding.
Purine Salvage: Cells recycle purines from dead cells to conserve energy and resources.
Target for Medicines: Salvage pathways are targeted by drugs, especially in cancer therapy.
Purine synthesis inhibitors block the production of adenine (A) and guanine (G) nucleotides, which are essential building blocks of DNA, RNA, and ATP/GTP. Because of this, these inhibitors have profound effects on rapidly dividing and metabolically active cells.
📌 Effect: Strongly affects rapidly dividing cells (e.g. cancer, immune, bone marrow, gut lining).
📌 Effect: Cellular fatigue, slowed metabolism, cell death (apoptosis) in severe cases
📌 Effect: Widespread dysregulation of intracellular signaling
High turnover tissues are most vulnerable:
| Tissue | Effect |
|---|---|
| Bone marrow | ↓ white/red blood cells → immunosuppression, anemia |
| GI tract | ↓ epithelial renewal → nausea, diarrhea, ulcers |
| Hair follicles | ↓ cell division → hair loss |
| Immune system | ↓ lymphocyte proliferation → immunosuppression (used therapeutically) |
Methotrexate, azathioprine, mycophenolate mofetil = used as:
Purine synthesis inhibitors impair cell survival and division by blocking the production of purine nucleotides needed for DNA, RNA, energy, and signaling. Their impact is most severe in rapidly dividing or highly active cells, leading to immunosuppression, cytotoxicity, and organ-specific side effects.
Explanation:
AMP → IMP → uric acid via degradation
During energy stress, AMP ↑ due to adenylate kinase
\[ 2ADP ⇌ ATP + AMP \]
Chronic stress → excess urate → crystallises as gout
Exacerbated by low hydration, kidney dysfunction, alcohol
📘 Gout mechanism – Mayo Clinic
A chronic energy charge crisis means the cell has persistently low ATP and high AMP/ADP — a sign that it’s under metabolic stress, like in ischemia, hypoxia, or starvation. In these conditions, uric acid builds up, especially in tissues like muscles, kidneys, and joints. Here’s why:
In energy stress, ATP is rapidly broken down:
\[ \text{ATP} → \text{ADP} → \text{AMP} \]
AMP levels rise, and the cell needs to manage this buildup.
When AMP accumulates, it’s degraded through the purine nucleotide degradation pathway:
\[ \text{AMP} → \text{Inosine} → \text{Hypoxanthine} → \text{Xanthine} → \text{Uric Acid} \]
These reactions are catalyzed by:
Under stress, xanthine oxidase becomes more active.
It catalyzes:
\[ \text{Hypoxanthine} → \text{Xanthine} → \text{Uric Acid} \]
Both steps generate reactive oxygen species (ROS), adding to oxidative damage.
| Cause | Effect |
|---|---|
| Low ATP | Increases AMP breakdown |
| AMP degradation | Produces hypoxanthine, xanthine → uric acid |
| Xanthine oxidase | Converts purine bases → uric acid + ROS |
| Lack of uricase | Uric acid can’t be broken down further |
| Chronic stress (e.g. ischemia) | Sustained AMP degradation → sustained uric acid buildup |
In a chronic energy crisis, the cell breaks down excess AMP through the purine degradation pathway, leading to uric acid accumulation — especially because humans cannot degrade uric acid further.
Autoimmune destruction of pancreatic β-cells
Onset typically in childhood or adolescence
Symptoms:
Rapid progression without treatment:
Diagnosis:
Measured by C-peptide levels (marker of endogenous insulin production)
Type I diabetes is caused by autoimmune destruction of pancreatic β-cells, leading to complete insulin deficiency. This results in severe metabolic imbalance: hyperglycemia, ketone production, and rapid weight loss. Without insulin therapy, it is fatal. Key diagnostic features include low or absent C-peptide and autoantibodies against β-cells.
First phase (Rapid)
Second phase (Sustained)
Insulin secretion occurs in two phases: a rapid first phase (pre-stored insulin) and a slower second phase (new insulin synthesis). Measuring C-peptide reflects β-cell function and distinguishes endogenous from injected insulin.
Begins with genetic predisposition (e.g. HLA associations, family history)
Progresses through a latent autoimmune phase:
First defect: loss of first-phase insulin secretion
Pre-diabetes stage: mild hyperglycemia; insulin secretion no longer sufficient to control glucose
Clinical onset:
Type I diabetes develops over time, starting with autoantibodies, progressing to partial β-cell loss, and culminating in insulin dependence. Early intervention in pre-diabetes (e.g. immunosuppression) may delay onset. Diagnosis occurs when endogenous insulin secretion becomes inadequate.
Insulin promotes glucose uptake into certain tissues by triggering the translocation of GLUT-4 transporters to the cell membrane.
This is essential in:
Insulin enables glucose uptake in muscle and fat by promoting GLUT-4 translocation to the membrane. The liver and brain remain insulin-independent. In Type I diabetes, GLUT-4 remains inactive, reducing tissue glucose uptake and causing hyperglycemia.
Insulin doesn’t just help glucose enter the cell — it also activates metabolic pathways to trap and use glucose:
Insulin is essential not only for glucose uptake into cells but also for promoting glucose usage via glycolysis, glycogen synthesis, and fat synthesis. Without insulin, glucose disposal halts, leading to G6P accumulation, feedback inhibition of hexokinase, and continued hyperglycemia.
Insulin inhibits lipolysis in adipose tissue by:
Uncontrolled lipolysis:
Contributes to ketogenesis in liver:
Leads to metabolic acidosis (ketotic acidosis)
Lipid breakdown drives:
Among insulin’s metabolic roles, anti-lipolysis is critically important in preventing:
Insulin strongly inhibits fat breakdown. Without it, lipolysis becomes uncontrolled, releasing glycerol and fatty acids that drive gluconeogenesis, suppress glucose oxidation, and fuel ketone production. This contributes to both hyperglycemia and ketoacidosis, making insulin’s anti-lipolytic role central to metabolic stability.
Insulin inhibits gluconeogenic enzymes in the liver.
Suppresses:
Insulin also reduces substrate availability for gluconeogenesis.
Loss of inhibition leads to uncontrolled gluconeogenesis.
Additionally:
Thus, liver continues to make glucose despite hyperglycemia.
Hepatic glucose production increases via:
Contributes directly to sustained hyperglycemia
Without insulin, the liver fails to suppress gluconeogenesis. Elevated glycerol, amino acids, and lactate supply abundant substrates. Hepatic glucose output rises even when blood glucose is already high, worsening hyperglycemia in Type I diabetes.
In hypoinsulinemia:
Ketone bodies (acetoacetate, β-hydroxybutyrate) produced in liver and exported.
Brain and peripheral tissues:
PDH is inhibited by high acetyl-CoA and low insulin, so:
Ketosis causes tissues (including the brain) to switch from glucose to ketone oxidation. This suppresses glucose usage, reinforces hyperglycemia, and drives the shift toward fat metabolism. Ketones become the dominant energy source in uncontrolled diabetes.
| System Affected | Metabolic Effect |
|---|---|
| Carbohydrate metabolism | ↓ Glucose uptake (GLUT-4 inactive) ↓ Glycolysis ↓ Glycogenesis ↑ Hepatic glucose production |
| Lipid metabolism | ↑ Lipolysis ↑ Fatty acid oxidation ↑ Ketone body production |
| Protein metabolism | ↑ Proteolysis ↑ Amino acid release ↑ Substrate supply for gluconeogenesis |
| Energy metabolism | Tissues rely on fat and ketones, not glucose |
Hypoinsulinemia unleashes unregulated catabolism: glucose uptake drops, fat and protein breakdown accelerate, and hepatic glucose production surges. The result is profound hyperglycemia, ketone body overproduction, metabolic acidosis, and rapid tissue wasting characteristic of uncontrolled Type I diabetes.
In hypoinsulinemia, excess fatty acids are released and oxidised to acetyl-CoA.
With TCA intermediates depleted (e.g. oxaloacetate used for gluconeogenesis), acetyl-CoA is diverted to ketogenesis.
Results in overproduction of ketone bodies:
Ketotic acidosis in Type I diabetes arises from uncontrolled ketone production due to high fatty acid oxidation and low TCA cycle capacity. Alongside lactate and fatty acids, ketone accumulation causes systemic acidosis that can be fatal without intervention.
| Symptom | Underlying Cause |
|---|---|
| Polyuria (frequent urination) | Glucose exceeds renal threshold → osmotic diuresis |
| Polydipsia (excessive thirst) | Water loss from tissues → dehydration triggers thirst |
| Weight loss | Uncontrolled lipolysis and proteolysis |
| Fatigue, weakness | Muscle wasting, loss of energy substrates |
| Ketotic breath | Acetone from spontaneous decarboxylation of acetoacetate |
| Hyperglycemia | ↓ Glucose uptake (GLUT-4), ↑ Hepatic gluconeogenesis |
| Glycosuria | Glucose appears in urine once plasma glucose > 10 mM |
This clinical picture is described as “starvation in the face of plenty”—cells cannot use glucose despite high blood levels.
Hypoinsulinemia causes osmotic diuresis, tissue dehydration, muscle and fat breakdown, hyperglycemia, and ketone accumulation. The result is rapid weight loss, thirst, ketotic breath, and fatigue—all hallmark symptoms of untreated Type I diabetes.
Avoid acute complications:
Prevent chronic complications caused by sustained hyperglycemia:
Diabetes control aims to prevent acute crises (like DKA) and long-term complications (e.g. vascular and nerve damage). The goal is stable glycemia with minimal excursions, using HbA1c and symptom tracking as indicators.
Finger-prick glucose testing
Continuous glucose monitoring (CGM)
HbA1c
Subcutaneous injections:
Insulin pumps:
Newer formulations:
Diabetes management includes glucose monitoring (finger-prick, CGM, HbA1c) and insulin delivery (injections, pumps, analogs). Monitoring guides treatment decisions. Long-term success depends on maintaining stable glucose while avoiding hypoglycemia.
Explanation:
📘 Spectrophotometry & Standard Curves – Khan Academy
Explanation:
The working range is the absorbance range where the standard curve remains linear
Readings outside this range:
Ensuring your unknowns fall within this range prevents invalid interpolation
Dilutions may be necessary to bring unknowns into the valid range
📘 Understanding the Beer-Lambert Law – LibreTexts
Explanation:
In this assay, ethanol is oxidised by ethanol oxidase to acetaldehyde, producing H₂O₂
H₂O₂ is used by peroxidase to convert 4-AAP and phenol into a red chromogen
Reaction:
\[ \text{Ethanol} + O₂ → \text{Acetaldehyde} + H₂O₂ \\ \text{4-AAP} + \text{Phenol} + 2H₂O₂ → \text{Red complex} + H₂O \]
Absorbance of red product measured at 500 nm (in glucose ELMA it was 540 nm)
The assay is adapted to test blood alcohol concentration (BAC) using red samples—so blanks are critical to correct for colour interference
📘 Spectrophotometry animation – YouTube
Assay volume limit: 200 µL (microplate format)
Dilutions needed to bring test samples into linear absorbance range
Must determine reaction completion time (~40 min for glucose ELMA)
Blank solution: all reagents except analyte (ethanol) → used to subtract background absorbance
Manually blank your data:
\[ A_{\text{corrected}} = A_{\text{sample}} - A_{\text{blank}} \]
📘 Using spectrophotometric blanks – ThermoFisher Guide
The central dogma outlines the general information flow:
Reverse transcription (RNA → DNA) also occurs in some cells (e.g. retroviruses).
DNA is the long-term store, RNA is a short-term intermediate, and proteins (and some RNAs) are functional molecules.
Each cell has the full genome but expresses only what’s necessary based on context (e.g. skin cells don’t express insulin).
Genetic information flows from DNA to RNA to protein within cells and is faithfully copied during cell division between generations. Most cells carry the complete genome, but only express genes as needed, ensuring efficiency and specificity. Experimental evidence firmly established DNA (not protein) as the hereditary material.
Defined as the entire DNA content of a cell.
Nearly all cells (except RBCs) have a full genome.
Genome includes:
In eukaryotes, only a small portion codes for proteins—the rest may have regulatory or unknown functions.
The complete set of RNA molecules transcribed from the genome at a given time.
Includes:
Transcriptome is dynamic: varies by cell type, developmental stage, and environmental conditions.
The entire set of proteins expressed in a cell or organism at a given time.
Proteins include:
Reflects not only gene expression but also post-transcriptional and post-translational modifications.
More complex than the genome due to alternative splicing and protein folding.
While every cell contains the same genome, the transcriptome and proteome differ based on cell function and environmental cues. mRNA makes up only a tiny fraction of RNA; most is rRNA and tRNA. Proteins diversify cell functions and can be far more varied due to regulation, splicing, and folding. These molecular layers highlight the complexity beyond the DNA sequence itself.
| Layer | Contents | Constant? | Function |
|---|---|---|---|
| Genome | All DNA | Yes (in all nucleated cells) | Information storage |
| Transcriptome | All RNA expressed | No (varies by cell type/time) | Regulates protein synthesis |
| Proteome | All proteins present | No (cell-specific, dynamic) | Executes cellular functions |
| Component | Analogy | Function |
|---|---|---|
| DNA | Hard drive (permanent storage) | Long-term, stable |
| mRNA | RAM (temporary access) | Transient access |
| Protein | Application or device | Functional unit |
DNA and RNA components:
Sugar: DNA = deoxyribose (no 2′-OH), RNA = ribose (2′-OH present)
Bases:
Phosphate backbone: negatively charged, links 5′-phosphate of one nucleotide to 3′-OH of the next
Chargaff’s Rules (for double-stranded DNA):
📘 Detailed: https://doi.org/10.1016/S0021-9258(19)50884-5
Phosphodiester bond:
N-glycosidic bond:
These are covalent bonds, making the backbone stable.
📘 Video on forces: https://www.youtube.com/watch?v=UY8jXo1KwUY
DNA’s double-stranded, complementary structure allows accurate replication and repair. The use of deoxyribose and thymine enhances its chemical stability and fidelity, making it an ideal long-term information storage molecule. Its ability to be compacted further supports its role as the cell’s genetic archive.
The DNA double helix has two grooves:
Arise due to the asymmetrical positioning of the sugar-phosphate backbone around the helical axis.
Major groove:
Exposes base-pair edges in a readable format
Allows sequence-specific binding by proteins such as:
Proteins can “read” the DNA sequence via hydrogen bond donors/acceptors and shape
Enables precise gene regulation
Minor groove:
The major and minor grooves in DNA expose specific chemical patterns that proteins use to recognize and bind to target sequences. The major groove, in particular, allows sequence-specific regulation, making it critical for gene expression control.
| Feature | DNA | RNA |
|---|---|---|
| Sugar | Deoxyribose (no 2′-OH) | Ribose (has 2′-OH) |
| Bases | A, T, G, C | A, U, G, C |
| Strandedness | Double-stranded (dsDNA) | Typically single-stranded |
| Stability | More stable | Less stable (prone to hydrolysis) |
| Function | Long-term genetic storage | Temporary message, catalysis, regulation |
| Helical Form | B-form (right-handed helix) | Many forms, often complex secondary structure (e.g. loops, stems) |
DNA is a stable, double-stranded molecule with deoxyribose and thymine, optimised for information storage. RNA is typically single-stranded, contains ribose and uracil, and functions dynamically in gene expression and regulation. Their differences reflect their distinct biological roles.
| Category | Description |
|---|---|
| Substrates | Deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis (e.g., dATP, dGTP, dCTP, dTTP); NTPs for RNA synthesis |
| Products | Newly synthesized nucleic acid strands: DNA strands with complementary base pairing to the template strand |
| Strand | Direction |
|---|---|
| Template strand | Read 3’ to 5’ |
| New strand | Synthesized 5’ to 3’ |
DNA and RNA synthesis is thermodynamically unfavourable, but energy is supplied by hydrolysis of dNTPs/NTPs:
| Enzyme/Protein | Function |
|---|---|
| Helicase (DnaB) | Unwinds DNA at the replication fork |
| Single-Stranded Binding Proteins (SSBs) | Stabilize single strands after separation |
| Primase (DnaG) | Synthesizes short RNA primers to provide 3’ OH group |
| DNA Polymerase III | Main replicative polymerase; adds nucleotides and proofreads (3’→5’ exonuclease) |
| Sliding Clamp (β-clamp) | Holds DNA Pol III in place on DNA |
| Clamp Loader | Loads sliding clamp onto DNA |
| DNA Polymerase I | Replaces RNA primers with DNA; has both 5’→3’ and 3’→5’ exonuclease activities |
| DNA Ligase | Seals nicks between adjacent nucleotides after RNA replacement |
| Topoisomerase II (DNA Gyrase) | Relieves supercoiling ahead of replication fork |
| Topoisomerase IV | Separates circular chromosomes after replication |
| DnaA | Recognizes oriC (origin of replication) and initiates DNA unwinding |
1. Initiation
└─ DnaA binds oriC and unwinds DNA
└─ Helicase (DnaB) unwinds DNA → replication bubble
└─ SSBs stabilize single strands
2. Primer Synthesis
└─ Primase (DnaG) synthesizes RNA primers on both strands
3. Elongation
└─ DNA Pol III adds nucleotides 5'→3' from RNA primers
└─ Leading strand: continuous
└─ Lagging strand: discontinuous (Okazaki fragments)
└─ Sliding clamp ensures processivity
└─ Clamp loader assists clamp attachment
4. Primer Removal & Replacement
└─ DNA Pol I removes RNA (5’→3’ exonuclease) and fills gap with DNA
5. Ligation
└─ DNA ligase forms final phosphodiester bond
6. Termination
└─ Tus-Ter complex halts replication forks
└─ Topoisomerase IV separates circular chromosomesReplication in prokaryotes presents several biochemical and structural challenges. Here’s how the cell solves each one:
Issue: DNA is a stable double helix that must be separated into single strands for copying.
Solution:
Issue: As helicase unwinds DNA, positive supercoils form ahead of the replication fork, halting progression.
Solution:
Issue: DNA polymerase cannot initiate synthesis de novo; it requires a 3′-OH group.
Solution:
Issue: DNA is antiparallel, but DNA polymerase only works 5′→3′. The lagging strand runs the “wrong way.”
Solution:
Issue: DNA polymerase could fall off the template during replication.
Solution:
Issue: Prokaryotic chromosomes are circular and become interlinked after replication.
Solution:
Replication requires overcoming structural barriers like supercoiling, antiparallel strand orientation, and polymerase limitations. Helicase, topoisomerases, primase, DNA polymerases, clamps, and ligase coordinate to unwind, copy, and rejoin the genome with high efficiency and structural integrity.
Replication must be extremely accurate, far more so than transcription, for the following reasons:
| Reason | Explanation |
|---|---|
| Mutations are permanent | Errors in DNA replication become fixed in the genome and passed on to daughter cells. |
| Protein errors are transient | Mistakes in transcription or translation affect only that molecule, not future generations. |
| Genome is only copied once per cycle | There’s little opportunity to “average out” errors like there is in mRNA/protein production. |
| Property | Function |
|---|---|
| Base selection | DNA Pol III selects the correct dNTP based on base-pairing geometry. |
| Proofreading activity | 3′→5′ exonuclease activity removes mismatched bases as they are added. |
| Post-replication repair | Additional mechanisms fix occasional mismatches (e.g., methyl-directed mismatch repair). |
| Feature | DNA Polymerase III | RNA Polymerase |
|---|---|---|
| Proofreading | Yes (3′→5′ exonuclease) | No proofreading |
| Error rate | 10⁻⁷ (or better) | 10⁻⁴ |
| Consequence of error | Inherited mutation | Misfolded or nonfunctional protein (temporary) |
DNA polymerases are highly accurate due to nucleotide selectivity and proofreading. This high fidelity is essential because replication errors are heritable, unlike transcription errors, which are transient. Additional post-replication repair mechanisms further reduce mutation rates, preserving genomic stability.
| Phase | Description |
|---|---|
| G₁ (Gap 1) | Decision to divide or enter quiescence (G₀) |
| G₀ | Non-dividing state; can be reversible (quiescent) or irreversible (senescent) |
| S (Synthesis) | DNA replication occurs |
| G₂ (Gap 2) | Final preparations for mitosis |
| M (Mitosis) | Cell division into two daughter cells |
Cyclins + CDKs: Cyclin-dependent kinases regulate progression by phosphorylating target proteins. Cyclin levels fluctuate; CDK levels stay relatively constant.
Checkpoints:
| Class | Function | Cancer Relevance |
|---|---|---|
| Proto-oncogenes | Stimulate cell growth | Mutated → Oncogenes → gain-of-function → unregulated growth |
| Tumour Suppressors | Inhibit cell division or promote apoptosis | Loss-of-function in both alleles required for dysfunction |
Example: Retinoblastoma Protein (pRb)
The cell cycle is tightly regulated by cyclins, CDKs, and checkpoints to ensure cells only divide when safe. Mutations in growth-promoting or inhibitory genes (proto-oncogenes or tumour suppressors) can disrupt this balance, leading to unregulated division and cancer.
| Feature | Prokaryotic (e.g., E. coli) | Eukaryotic (e.g., Human) |
|---|---|---|
| Chromosome Structure | Single, circular | Multiple, linear |
| Genome Size | ~4.6 million bp | ~6 billion bp (diploid) |
| Origins of Replication | 1 per genome | 30,000–50,000 per genome |
| Replication Rate | Fast (~1000 nt/s) | Slower (~50 nt/s) |
| Polymerases | DNA Pol III (main) | DNA Pol δ (main) |
| Proofreading | Yes (3’→5’ exonuclease) | Yes (DNA Pol δ) |
| Packaging | Supercoiling | Histone wrapping, chromatin structure |
| Re-initiation Control | oriC control via DnaA | CDKs prevent reactivation of origins |
While the core mechanism of DNA replication is conserved across life—bidirectional synthesis, priming, and proofreading—eukaryotes face added complexity due to larger, linear genomes and the need for coordinated cell cycle regulation. Multiple origins and slower polymerases compensate for size, and histone packaging adds a layer of regulation.
Multiple origins of replication must be selected and activated precisely.
Pre-replicative complex (pre-RC) assembled in G₁:
Activation in S phase:
Regulation:
DNA must be re-wrapped around histones after replication.
Histone genes:
Telomerase:
Ribonucleoprotein with:
Extends 3′ end → allows primer addition and lagging strand fill-in
Eukaryotic replication begins from multiple regulated origins, requiring orchestration via cyclin-dependent kinases. Replication also demands rapid histone production for DNA packaging. Linear chromosomes pose a unique challenge: end regions cannot be replicated conventionally, which is solved by telomerase extending telomeres—a mechanism vital for cell immortality and highly active in cancer cells.
| Cell Type | Telomerase Activity |
|---|---|
| Germ cells | High |
| Stem cells | Moderate |
| Somatic cells | Very low or absent |
| Cancer cells | Reactivated and high |
Telomerase extends telomeres, allowing cells to bypass the end replication problem. In most somatic cells, its inactivity leads to gradual telomere shortening and cellular ageing. In contrast, germline and cancer cells maintain telomerase activity, granting them the ability to divide indefinitely—contributing to tissue renewal or tumour immortality, respectively.
| Strategy | Mechanism |
|---|---|
| Direct enzyme inhibitors | Block telomerase’s reverse transcriptase activity |
| Antisense oligonucleotides | Target the RNA template to prevent extension |
| Immunotherapy | Target telomerase-expressing cells (e.g., vaccines, T-cells) |
| Small molecules | Stabilise telomeric structures (e.g., G-quadruplexes) to inhibit access |
Telomerase is a prime target in cancer because it’s active in most tumours but largely absent in healthy somatic cells. Inhibiting telomerase could limit cancer cell division. However, challenges include delayed therapeutic effects, potential off-target impacts, and alternative telomere maintenance pathways in some cancers.
Complementary base pairing (A–T, G–C) via hydrogen bonds allows:
Example: In PCR, primers bind to their complementary DNA sequence through hydrogen bonding. Correct annealing is required for successful amplification.
π–π interactions between adjacent planar bases stabilise the double helix.
Important for:
Example: ssDNA absorbs more UV light than dsDNA because base stacking is disrupted when strands are separated.
| Technique | Role of Base Pairing/Stacking |
|---|---|
| PCR | Primer annealing depends on base pairing; melting point influenced by GC content and stacking |
| Gel electrophoresis | DNA denaturation is affected by GC content (more GC = higher Tm) |
| Spectrophotometry | Based on absorbance change when dsDNA melts into ssDNA |
Base pairing allows sequence-specific primer binding in molecular biology methods, while base stacking contributes to DNA stability and UV absorbance properties. Together, these weak interactions underpin key lab techniques like PCR, electrophoresis, and quantification by spectrophotometry.
| Technique | Relevance |
|---|---|
| Gel electrophoresis | DNA migrates toward the positive electrode because of its negatively charged phosphate backbone |
| Primer specificity | Repulsion between negatively charged strands promotes specificity; magnesium ions (Mg²⁺) shield these repulsions and affect binding |
| DNA stability | High salt concentrations (e.g., Mg²⁺) shield repulsive forces, increase Tm, but can reduce specificity in PCR if excessive |
DNA Strand (-) Mg²⁺ DNA Strand (-)
↓ ⇄ ↓
Repulsion Shielding → Improved annealing
↓
May reduce specificityThe phosphate backbone’s negative charge drives DNA migration in electrophoresis and influences hybridisation. Controlling ionic conditions (especially Mg²⁺) is essential for tuning strand annealing and primer specificity in techniques like PCR.
A technique to amplify specific DNA regions using thermal cycling.
| Component | Role |
|---|---|
| Template DNA | Contains the target region to be amplified |
| DNA polymerase | Adds dNTPs to extend from primers (e.g., Taq polymerase) |
| Primers (forward & reverse) | Short DNA sequences that define the region of interest |
| dNTPs | Building blocks for new DNA (dATP, dTTP, dCTP, dGTP) |
| Buffer + Mg²⁺ | Provides optimal pH and ionic conditions |
Step 1: Denaturation (~95°C)
- Heat separates dsDNA → ssDNA
Step 2: Annealing (~55–65°C)
- Primers bind to complementary sequences
Step 3: Extension (~72°C)
- Polymerase adds nucleotides to 3′ end of primer
→ Repeat 25–35 times (exponential amplification)| Feature | PCR | Cellular DNA Replication |
|---|---|---|
| Strand separation | Heat | Helicase |
| Primer | Synthetic DNA | RNA primer by primase |
| Enzyme | Taq polymerase | DNA Pol III / δ |
| Proofreading | No (Taq) | Yes (high-fidelity polymerases) |
| Scope | Targeted sequence | Whole genome |
| Re-initiation | Repeats per cycle | Only once per S phase |
PCR mimics natural DNA replication in vitro but uses heat to separate DNA strands and synthetic primers for targeted amplification. Taq polymerase extends DNA from primers without proofreading. Exponential DNA synthesis through repeated cycles makes PCR a rapid and powerful molecular biology tool.
DNA polymerases differ in fidelity, processivity, thermostability, and speed. The choice of polymerase directly impacts the success and specificity of PCR.
| Property | Relevance to PCR |
|---|---|
| Thermostability | Essential to withstand repeated heating cycles during denaturation (95 °C). Taq polymerase remains active after >30 cycles. |
| 5′→3′ polymerase activity | Extends DNA strand from the primer. Core enzymatic function. |
| Lack of 3′→5′ exonuclease (in Taq) | Increases speed but reduces fidelity (no proofreading). |
| Processivity | Ability to synthesise long stretches of DNA without falling off the template. |
| Optimal activity at 72 °C | Matches PCR extension temperature; maximises polymerase function. |
| Enzyme | Notes |
|---|---|
| Taq polymerase | Most common; high thermostability, low fidelity (error-prone) |
| Pfu polymerase | From Pyrococcus furiosus; proofreading ability, higher fidelity |
| High-fidelity blends | Engineered polymerases combining Taq with proofreading enzymes for both speed and accuracy |
DNA polymerases used in PCR must be thermostable and active at high temperatures. Taq polymerase, the most common, is fast and heat-resistant but lacks proofreading, making it less accurate. For high-fidelity applications, proofreading polymerases (e.g. Pfu) are used. The choice of polymerase influences the accuracy, speed, and success of PCR.
| Source | Notes |
|---|---|
| Retroviruses (e.g., MMLV, AMV) | Naturally encode reverse transcriptase |
| Engineered variants | Optimised for thermal stability and reduced RNase activity |
| Component | Function |
|---|---|
| RNA template | Usually mRNA extracted from cells |
| Primer | Provides a 3′-OH; may be: – Oligo(dT) (binds poly-A tail) – Random hexamers – Gene-specific primers |
| dNTPs | Building blocks for DNA |
| Reverse transcriptase | Synthesises DNA from RNA template |
| RNase inhibitor | Prevents degradation of RNA |
1. RNA + primer anneal
2. Reverse transcriptase synthesises cDNA (5′→3′)
3. RNA is degraded (optionally by RNase H)
4. Result: single-stranded cDNAThe cDNA can then be amplified by PCR using gene-specific primers (→ RT-PCR).
mRNA (poly-A tail)
↓ primer (oligo-dT or random hexamer)
Reverse Transcriptase
↓
cDNA synthesis → cDNA + PCR = gene expression profilingReverse transcription synthesises complementary DNA from RNA templates using reverse transcriptase enzymes. This enables analysis of gene expression and RNA viruses via standard DNA-based tools like PCR. The primer choice determines whether all mRNA or specific genes are converted into cDNA.
Developed by Frederick Sanger in 1977, this method remains foundational in molecular biology for sequencing short to medium-length DNA fragments.
Sanger sequencing uses selective incorporation of dideoxynucleotides (ddNTPs), which terminate DNA synthesis when incorporated.
| Molecule | Structure | Result |
|---|---|---|
| dNTP | Has 3′-OH | Allows elongation |
| ddNTP | No 3′-OH | Terminates DNA strand extension |
| Component | Function |
|---|---|
| Template DNA | Sequence to be determined |
| Primer | Anneals to template; provides 3′-OH |
| DNA polymerase | Extends primer using dNTPs |
| dNTPs | Normal nucleotides for elongation |
| Fluorescently-labelled ddNTPs | Chain terminators (one for each base) |
| Buffer + Mg²⁺ | Required for polymerase activity |
1. DNA template + primer + polymerase
2. Add dNTPs + fluorescent ddNTPs
3. Chain termination at random positions
4. Size separation via capillary electrophoresis
5. Laser reads terminal base → sequence reconstructedSanger sequencing uses labelled ddNTPs to terminate DNA synthesis at specific bases, creating fragments of varying lengths. These are separated and detected via fluorescence, revealing the DNA sequence. The method is accurate, robust, and still widely used for short sequences.
| Property | Why It Matters in Sequencing |
|---|---|
| 5′→3′ polymerase activity | Adds nucleotides from primer for strand synthesis |
| Efficient ddNTP incorporation | Must be able to use both dNTPs and ddNTPs as substrates |
| No 3′→5′ proofreading | Proofreading would remove ddNTPs, ruining termination accuracy |
| High processivity | Ensures full extension until termination point |
Sanger sequencing polymerases are often engineered to lack exonuclease (proofreading) activity to preserve ddNTP incorporation events.
DNA polymerases used in Sanger sequencing must efficiently incorporate both dNTPs and ddNTPs without proofreading activity, to allow accurate chain termination. Enzymes like Sequenase or modified Taq polymerase are preferred for their high processivity and compatibility with fluorescent ddNTPs.
| Type | Description |
|---|---|
| Introns | Non-coding regions within genes; spliced out during mRNA processing. Can comprise >90% of a gene’s length. |
| Repetitive DNA | Includes telomeres, centromeres, and Variable Number Tandem Repeats (VNTRs); important for structural and forensic purposes. |
| Transposons | Mobile DNA sequences; can copy and insert themselves into new genomic locations (e.g. LINEs, SINEs). |
| Pseudogenes | Non-functional copies or remnants of once-active genes, often arising from duplication or retrotransposition. |
| Regulatory elements | Sequences controlling gene expression, including enhancers, silencers, and insulators. |
The human genome contains vast amounts of non-coding DNA, including introns, repetitive sequences, and pseudogenes. While only ~1–2% of the genome encodes proteins, the rest plays structural, regulatory, or unknown roles—illustrating the complexity beyond simple coding functions.
| Feature | Description |
|---|---|
| Chromatin | Complex of DNA + proteins (primarily histones). |
| Nucleosome | The fundamental unit of chromatin: ~145 bp of DNA wrapped around a histone octamer. |
| Histone Octamer | 2 each of H2A, H2B, H3, and H4. |
| Linker DNA | ~50 bp between nucleosomes; bound by H1 histone. |
DNA is tightly wrapped around histone octamers to form nucleosomes, the basic units of chromatin. Electrostatic interactions between positively charged histone tails and negatively charged DNA enable compact storage of ~2 meters of DNA in a ~10 μm nucleus, while still allowing access for gene expression.
| Modification | Enzymes | Effect on DNA Packing | Impact on Gene Expression |
|---|---|---|---|
| Acetylation | HATs (Histone Acetyltransferases) | Loosens DNA-histone interaction (neutralises lysine’s positive charge) | Activates transcription by increasing accessibility |
| Deacetylation | HDACs (Histone Deacetylases) | Restores positive charge on histones → tighter DNA wrapping | Represses transcription |
| Methylation | HMTs (Histone Methyltransferases) | Depends on which residues are modified | Can activate or repress, depending on context and reader proteins |
Histone modifications such as acetylation and methylation modulate how tightly DNA is packed, thereby controlling access to genetic information. Acetylation generally enhances transcription, while methylation’s effect depends on context. These modifications are reversible and dynamic, enabling fine-tuned regulation of gene expression.
| Enzyme | Function | Mechanism |
|---|---|---|
| HATs (Histone Acetyltransferases) | Add acetyl groups to lysine residues on histone tails | Neutralise positive charge → weaken interaction with DNA |
| HDACs (Histone Deacetylases) | Remove acetyl groups | Restore positive charge on lysine → strengthen interaction with negatively charged DNA |
HATs and HDACs modify histone charge states to regulate DNA-histone interactions. Acetylation loosens chromatin for gene expression; deacetylation restores tight packing and silences genes. This electrostatic regulation is central to dynamic transcriptional control.
| Chromatin State | Features | Gene Activity |
|---|---|---|
| Euchromatin | Loosely packed, accessible | Active transcription |
| Heterochromatin | Tightly packed, inaccessible | Repressed transcription |
Specific combinations of histone modifications (the “histone code”) recruit remodelling complexes.
These proteins interpret the code and restructure chromatin by:
| Modification | Example Effect |
|---|---|
| H3K9 acetylation | Promotes open chromatin, active transcription |
| H3K9 methylation | Recruits HP1, promotes heterochromatin formation |
| H3K27 methylation | Repressive mark, key in development (e.g., Polycomb silencing) |
ATP-dependent chromatin remodellers respond to histone marks and reposition nucleosomes to:
Histone modifications act as molecular signals that recruit chromatin remodelling proteins. These complexes reshape nucleosome positioning to either loosen or condense chromatin structure, modulating accessibility and regulating gene expression accordingly.
| Cell Type | Chromatin State | Gene Access |
|---|---|---|
| Pluripotent Stem Cells | Broadly open chromatin | Can access and potentially express most genes |
| Terminally Differentiated Cells | Highly restricted chromatin | Many genes are silenced and inaccessible |
Pluripotent stem cells have open chromatin and can access most of the genome, enabling them to differentiate into any cell type. Terminally differentiated cells have restricted chromatin, limiting gene access to only lineage-appropriate genes. Histone modifications enforce this transcriptional memory and cell identity.
Transcription is the DNA-to-RNA step of the central dogma, carried out in prokaryotes by a single RNA polymerase enzyme.
Signals to stop transcription arise from the RNA, not DNA.
Two major mechanisms:
Transcription in prokaryotes involves three stages: initiation (promoter recognition by RNA polymerase + σ), elongation (RNA synthesis 5′→3′, no primer), and termination (via intrinsic or Rho-dependent signals in the RNA). It’s a precise but faster and slightly more error-prone process than DNA replication.
Positioning: –35 and –10 sequences are ~17 bp apart (optimal spacing for RNA polymerase binding).
5′ - TTGACA ------17bp------ TATAAT - +1 - 3′
↑ ↑
–35 region –10 regionProkaryotic promoters contain conserved –10 and –35 regions upstream of the transcription start site. These are recognised by the RNA polymerase σ subunit and are essential for correct and efficient transcription initiation.
Determined by:
Strong promoters:
Weak promoters:
Analogy used in lecture: A familiar-looking bus stop (strong promoter) vs. an ambiguous one (weak promoter) affects how likely RNA pol is to stop.
Promoters determine where and how frequently a gene is transcribed. Strong promoters closely match the consensus and promote frequent transcription; weak ones lead to low expression levels. The promoter’s sequence and spacing are critical for efficient RNA polymerase binding.
| Factor | Effect |
|---|---|
| Match to consensus sequences | Closer match = stronger binding = more frequent transcription |
| Spacing between –10 and –35 regions | Optimal spacing (~17 bp) allows best positioning of RNA pol |
| DNA supercoiling and local sequence context | May influence accessibility or σ subunit recognition |
Important note: Promoter strength is constitutive, meaning it’s intrinsic to the DNA sequence. Regulation (e.g. via repressors) is layered on top of this.
Promoter strength determines how often transcription is initiated, based on the match to consensus and spacing of the –10/–35 boxes. Strong promoters drive frequent transcription; weak ones yield rare transcription.
| Feature | Lac Operon | trp Operon |
|---|---|---|
| Inducible or repressible? | Inducible (turned on when needed) | Repressible (turned off when product abundant) |
| Default state | Off (blocked by repressor) | On (until trp builds up) |
| Inducer/Corepressor | Allolactose (removes repressor) | Tryptophan (activates repressor) |
| Repressor binding site | Operator (overlaps with promoter) | Operator |
No lactose → repressor binds → transcription OFF
Lactose → allolactose binds repressor → repressor releases → transcription ONLow Trp → repressor inactive → transcription ON
High Trp → Trp binds repressor → repressor binds operator → transcription OFFThe lac operon is inducible—turned ON in the presence of lactose, while the trp operon is repressible—turned OFF in the presence of tryptophan. These classic operons demonstrate how bacteria regulate gene expression based on metabolic needs through repressor-operator interactions.
| Type | Example | Binding Trigger | Result |
|---|---|---|---|
| Inducible | Lac repressor | Inactivated by allolactose | Transcription ON |
| Repressible | trp repressor | Activated by tryptophan | Transcription OFF |
Repressors are proteins that bind to operators and prevent RNA polymerase from initiating transcription. Their activity is modulated by small molecules (e.g. allolactose or tryptophan) to regulate operons like lac and trp in response to environmental conditions.
| Feature | Prokaryotic Transcription | Eukaryotic Transcription |
|---|---|---|
| Location | Cytoplasm | Nucleus (then mRNA exported to cytoplasm) |
| RNA Polymerase | One type | Three distinct polymerases (I, II, III) |
| Initiation | Requires σ factor | Requires many general transcription factors (TFII family) |
| Promoters | Simple: –10 (TATAAT), –35 (TTGACA) | Complex: TATA box, Inr, CAAT, GC, DPE |
| Transcription and Translation | Coupled (simultaneous) | Separated in space and time |
| RNA Processing | None (mRNA often used immediately) | Extensive: 5′ capping, splicing, 3′ poly-A tail |
| Termination | Rho-dependent or intrinsic | Cleavage + polyadenylation; distinct mechanisms |
Eukaryotic transcription is more complex than prokaryotic transcription due to compartmentalisation, extensive RNA processing, multiple RNA polymerases, and reliance on large protein complexes. Prokaryotes streamline transcription with a single RNA polymerase and direct mRNA translation.
| RNA Polymerase | Location | Function | Sensitivity to α-amanitin |
|---|---|---|---|
| Pol I | Nucleolus | Synthesises 45S rRNA precursor → 18S, 5.8S, 28S rRNAs | Insensitive |
| Pol II | Nucleoplasm | Synthesises mRNA, some snRNA, miRNA | Highly sensitive |
| Pol III | Nucleoplasm | Synthesises tRNA, 5S rRNA, some snRNA | Moderately sensitive |
Eukaryotic cells use three RNA polymerases: Pol I (rRNA), Pol II (mRNA and snRNA), and Pol III (tRNA, 5S rRNA). Pol II is the most important for gene expression and is highly sensitive to transcriptional inhibitors like α-amanitin.
| Promoter Element | Location | Function |
|---|---|---|
| TATA box | ~–25 | Highly conserved; facilitates transcription initiation via TBP (TATA-binding protein) |
| CAAT box | –40 to –150 | Enhancer-like; binds regulatory proteins |
| GC box | –40 to –150 | Often found in housekeeping genes; binds Sp1 TF |
| Initiator (Inr) | ~+1 | Defines the transcription start site; can function without TATA box |
| Downstream Promoter Element (DPE) | +30 | Found in TATA-less promoters; functions with Inr |
Promoter combinations vary, adding flexibility and complexity to gene regulation.
5’ ——– GC/CAAT —— TATA —— +1 (Inr) —— DPE —— 3’Eukaryotic promoters are more diverse and modular than prokaryotic ones. They often include a TATA box, CAAT or GC boxes, and additional downstream elements. Their arrangement affects transcription strength, tissue specificity, and RNA Pol II recruitment.
Cis-regulatory elements that increase the rate of transcription initiation.
Can act:
Bind activator proteins (enhancer-binding transcription factors).
Through DNA looping, they are brought into proximity with the core promoter, facilitating:
| Feature | Enhancer | Promoter |
|---|---|---|
| Location | Can be distant | Close to TSS |
| Orientation | Works bidirectionally | Orientation-specific |
| Role | Regulatory – boosts transcription | Core – initiates transcription |
Enhancers are distant regulatory DNA elements that increase transcription by looping into contact with the promoter. They work by binding activators that help stabilise the transcriptional machinery, making them crucial for gene-specific regulation.
| Type | Function |
|---|---|
| General Transcription Factors (GTFs) | Required for all Pol II transcription (e.g., TFIID, TFIIB, TFIIE, TFIIF, TFIIH) |
| Regulatory (Specific) Transcription Factors | Bind enhancers, silencers, or response elements to regulate gene-specific transcription |
| Factor | Role |
|---|---|
| TFIID | Includes TBP (TATA-binding protein); binds the TATA box |
| TFIIH | Helicase activity to unwind DNA; kinase activity to phosphorylate RNA Pol II |
| TFIIB, TFIIE, TFIIF | Recruit and stabilise RNA Pol II at the promoter |
| Domain | Function |
|---|---|
| DNA-binding domain (DBD) | Recognises specific DNA sequences (e.g., zinc fingers, helix-turn-helix) |
| Activation/repression domain | Interacts with coactivators or corepressors to modulate transcription |
| Dimerisation domain | Allows homo-/heterodimer formation for increased binding specificity |
Transcription factors are proteins that control gene expression by guiding RNA polymerase to specific promoters (GTFs) or regulating its activity via enhancers (regulatory TFs). Their modular structure enables DNA recognition and interaction with other regulatory proteins.
1. Hormone diffuses into cell
2. Binds nuclear receptor → conformational change
3. Receptor-hormone complex binds **hormone response element (HRE)** in DNA
4. Recruits coactivators or corepressors → modifies gene expression| Hormone | Receptor | Response Element | Target Effect |
|---|---|---|---|
| Cortisol | Glucocorticoid receptor | GRE | Anti-inflammatory genes |
| Estrogen | Estrogen receptor | ERE | Reproductive and growth genes |
Steroid hormones regulate gene expression by binding to intracellular receptors that act as transcription factors. These hormone–receptor complexes target specific DNA response elements to turn genes on or off based on physiological signals.
| Drug | Target | Type | Effect |
|---|---|---|---|
| Tamoxifen | Estrogen receptor (ER) | SERM (Selective Estrogen Receptor Modulator) | Antagonist in breast → used in ER+ breast cancer |
| Dexamethasone | Glucocorticoid receptor | Agonist | Suppresses inflammation by activating anti-inflammatory genes |
| RU486 (Mifepristone) | Progesterone receptor | Antagonist | Blocks progesterone → used in medical abortion |
Hormone-based drugs modulate gene expression by targeting nuclear receptors. Some (like dexamethasone) mimic hormones to stimulate gene expression, while others (like tamoxifen or RU486) block hormone receptors to suppress specific gene programs, including those involved in cancer and inflammation.
| Step | Description |
|---|---|
| 1. Nucleotide Modification | Methylation and pseudouridine formation on specific bases or sugars; enhances ribosome stability and function |
| 2. Assembly with Ribosomal Proteins | Forms large ribonucleoprotein (RNP) complex |
| 3. Cleavage | 45S pre-rRNA is cleaved into 18S (small subunit), 5.8S and 28S (large subunit components) |
5S rRNA is transcribed separately by RNA polymerase III, outside the nucleolus.
The 18S, 5.8S, and 28S rRNAs are transcribed as a 45S precursor by RNA Pol I, modified, assembled with proteins, and cleaved into mature forms. The 5S rRNA is transcribed separately by RNA Pol III. These steps ensure proper ribosome assembly and function.
| Step | Description |
|---|---|
| 1. 5′ and 3′ End Trimming | RNases remove extra sequences from both ends |
| 2. Addition of CCA at 3′ End | Universal 3′-CCA tail is added enzymatically (not encoded) → site of amino acid attachment |
| 3. Base Modifications | Includes methylation and pseudouridylation (modifications enhance stability and function) |
| 4. Intron Removal and Ligation | If present, intron is spliced and exons ligated to form a functional anticodon loop |
Pre-tRNAs undergo end trimming, 3′ CCA addition, base modifications, and splicing of introns. These steps are essential to produce a mature tRNA capable of delivering amino acids during translation.
| Feature | Description |
|---|---|
| When | After ~25 nt are transcribed |
| How | 3 enzyme steps → adds a 7-methylguanosine cap via 5′–5′ linkage |
| Function | Protects mRNA from 5′ exonucleases; enhances translation efficiency |
| Feature | Description |
|---|---|
| Signal | AAUAAA motif triggers cleavage |
| Process | Cleaved by nucleases, then ~250 adenosines added by poly(A) polymerase (not transcribed from DNA) |
| Function | Enhances mRNA stability and translation; does not affect nuclear export |
| Exception | Histone mRNAs: no poly(A) tail → form stem-loop structures instead |
| Feature | Description |
|---|---|
| Purpose | Remove non-coding introns and join exons |
| Signals | 5′ splice site (GU), branch point (A), 3′ splice site (AG) |
| Catalysed by | Spliceosome (snRNAs + proteins) |
| Coupled with transcription | Via phosphorylation of RNA Pol II CTD tail |
Eukaryotic mRNAs are capped at the 5′ end, polyadenylated at the 3′ end, and spliced to remove introns. These modifications protect mRNA, promote translation, and are essential for proper gene expression. All processing steps are tightly coupled with transcription.
| Feature | Description |
|---|---|
| Structure | 7-methylguanosine (m⁷G) cap linked via 5′–5′ triphosphate bridge |
| Added by | Capping enzyme complex soon after transcription begins (~25 nt) |
| Functions | |
| • Protects mRNA from degradation | |
| • Aids nuclear export | |
| • Required for ribosome recognition and translation initiation (via cap-binding proteins) |
| Feature | Description |
|---|---|
| Triggered by | AAUAAA signal sequence upstream of cleavage site |
| Process |
The 5′ cap protects mRNA and is essential for translation initiation, while the 3′ poly(A) tail stabilises mRNA and boosts translation efficiency. Both are added co-transcriptionally and are crucial for proper gene expression in eukaryotic cells.
| Feature | Description |
|---|---|
| Purpose | Removes introns (non-coding) and joins exons (coding) |
| Signals | |
| • 5′ splice site: GU | |
| • Branch point: A | |
| • 3′ splice site: AG | |
| Catalysed by | Spliceosome (complex of snRNAs: U1, U2, U4, U5, U6 + proteins) |
| Coupled with transcription | Via phosphorylation of RNA Pol II C-terminal domain (CTD) |
| Energetics | Requires ATP, though cleavage and ligation are transesterification reactions |
1. U1 binds 5′ splice site
2. U2 binds branch point A
3. U4/U5/U6 recruited
4. Intron looped and cut → lariat structure
5. Exons joined, lariat degradedSplicing removes introns and joins exons via the spliceosome, using conserved GU–AG boundaries and a branch point A. This editing step is critical for generating functional proteins and is tightly linked to transcription.
| Type | Example |
|---|---|
| Exon skipping/inclusion | Exon 3 included in one isoform but skipped in another |
| Mutually exclusive exons | Only one of two exons used |
| Alternative 5′/3′ splice sites | Different start/end sites for the same exon |
| Intron retention | Introns may be retained and translated in some contexts |
Alternative splicing allows a single gene to produce multiple protein isoforms. It is regulated by RNA-binding proteins and is essential for tissue specificity, adaptability, and biological complexity in eukaryotes.
| Step | Description |
|---|---|
| Export form | Mature mRNA is exported as messenger ribonucleoprotein (mRNP) complex |
| Gate | Transported through nuclear pore complexes (NPCs) |
| Signals | Cap-binding proteins (CBPs), exon junction complexes (EJCs), and poly(A)-binding proteins help mark mRNA as export-ready |
| Directionality | Export is unidirectional (nucleus → cytoplasm) |
| Post-export changes | CBPs are replaced by eIF4E, allowing translation initiation in cytoplasm |
Once capped, polyadenylated, and spliced, mRNA is exported from the nucleus through nuclear pores as a mature mRNP. Export is highly regulated to ensure only fully processed transcripts reach the cytoplasm for translation.
| Property | Nucleic Acids | Amino Acids |
|---|---|---|
| Function | Store and transmit genetic information | Build functional proteins, enzymes, signalling molecules |
| Structure | Similar backbones with variable bases (A, U/T, C, G) | Diverse side chains (charged, polar, nonpolar, aromatic, etc.) |
| Chemical Makeup | Sugar-phosphate backbone + nitrogenous bases | Central carbon with amino, carboxyl, hydrogen, and R-group |
| Charge | Negatively charged due to phosphate groups | Varies (acidic, basic, neutral) depending on R-group |
| Role in Biology | Information storage & transfer (DNA/RNA) | Functional diversity in metabolism and cellular structure |
TLDR: Nucleic acids are chemically uniform and function as information carriers, while amino acids are structurally diverse to support a wide range of biochemical roles.
| Feature | Description |
|---|---|
| Triplet Code | 3 nucleotides = 1 codon = 1 amino acid |
| Universal | Used by nearly all organisms with few exceptions (e.g. protozoa) |
| Non-overlapping | Codons are read in strict triplets, without overlaps |
| No punctuation | No nucleotide set aside as a “stop” base; stop codons are specific triplets |
| Degeneracy | Most amino acids are encoded by >1 codon (e.g. serine: 6 codons) |
| Directionality | Always read 5′ to 3′ |
TLDR: The genetic code is a robust, redundant system that translates RNA into protein with high reliability. Degeneracy buffers against harmful mutations.
| Feature | Detail |
|---|---|
| Length | ~73–93 ribonucleotides |
| Structure | Cloverleaf 2D shape, L-shaped 3D conformation |
| Key Sites | Anticodon loop (binds mRNA), 3′ CCA tail (binds amino acid) |
| Post-transcriptional Modifications | Methylation, base alterations help recognition |
TLDR: tRNAs act as molecular adaptors, matching mRNA codons to their respective amino acids. Their structure ensures compatibility with ribosomes, mRNA, and synthetases.
| First Anticodon Base (5′ end of tRNA) | Recognised 3rd Codon Bases (3′ end of mRNA) |
|---|---|
| C | G |
| A | U |
| U | A or G |
| G | U or C |
| I (Inosine) | U, C, or A |
TLDR: Wobble base pairing explains how cells use fewer tRNAs than codons. It provides flexibility in codon recognition without compromising translational accuracy.
Amino acid + ATP → aminoacyl-AMP + PPi
Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP| Feature | Description |
|---|---|
| Substrate Specificity | Recognises both amino acid and tRNA anticodon loop |
| Proofreading | Uses double-sieve mechanism to prevent incorrect charging |
| Energy Use | Requires ATP (2 high-energy bonds used per aminoacylation) |
| Impact on Translation | Errors at this step cause misincorporation of amino acids, not corrected during translation |
TLDR: Aminoacyl-tRNA synthetases ensure the genetic code is correctly interpreted by precisely pairing each tRNA with its corresponding amino acid, forming aminoacyl-tRNA in an ATP-dependent, highly specific process.
| Subunit | Composition | Function |
|---|---|---|
| 30S | 16S rRNA + proteins | Binds mRNA, decodes codons |
| 50S | 23S + 5S rRNA + proteins | Catalyses peptide bond formation |
| 70S | 30S + 50S (assembled unit) | Coordinates initiation, elongation, termination |
| Site | Function |
|---|---|
| A (Aminoacyl) | Incoming aminoacyl-tRNA binds here |
| P (Peptidyl) | Holds the growing peptide chain |
| E (Exit) | Where deacylated tRNA exits the ribosome |
TLDR: Ribosomes orchestrate translation by positioning mRNA and tRNAs, forming peptide bonds, and ensuring directionality and fidelity of protein synthesis through their structured A, P, and E sites.
1. Initiation
- 30S binds Shine-Dalgarno (mRNA)
- Initiator tRNA (fMet) binds P site
- 50S joins → 70S complex forms
2. Elongation (repeats per codon)
- EF-Tu brings aminoacyl-tRNA to A site
- Peptide bond formed (23S rRNA)
- EF-G drives ribosome translocation
3. Termination
- Stop codon enters A site
- Release factors hydrolyse peptide-tRNA bond
- Ribosome dissociates| Factor | Role |
|---|---|
| IF1, IF2, IF3 | Initiation factors (assembly & accuracy) |
| EF-Tu, EF-G | Elongation (delivery and movement) |
| RF1, RF2, RF3 | Termination (release of peptide) |
TLDR: Translation consists of initiation (ribosome assembly), elongation (codon-by-codon peptide synthesis), and termination (release of finished protein). Each stage uses GTP and accessory factors to maintain speed and accuracy.
| Feature | Prokaryotes | Eukaryotes |
|---|---|---|
| mRNA recognition | Shine-Dalgarno sequence pairs with 16S rRNA | Ribosome scans from 5′ cap to start codon (AUG in Kozak sequence) |
| Initiator tRNA | fMet-tRNA (formylated) | Met-tRNA (non-formylated) |
| Coupling | Transcription and translation coupled | Occur in different compartments (nucleus vs cytoplasm) |
| mRNA structure | Often polycistronic | Always monocistronic |
| Ribosome size | 70S (30S + 50S) | 80S (40S + 60S) |
TLDR: Prokaryotic translation is faster and coupled with transcription, using fMet and Shine-Dalgarno sequences. Eukaryotic translation is separated in space and time, involves scanning from a cap, and uses more initiation factors.
| Drug / Toxin | Target | Mechanism | Application / Effect |
|---|---|---|---|
| Streptomycin | 30S subunit | Inhibits initiation, causes misreading | Antibiotic (bactericidal) |
| Tetracycline | 30S subunit | Blocks A-site tRNA binding | Antibiotic |
| Chloramphenicol | 50S subunit | Inhibits peptidyl transferase | Broad-spectrum antibiotic |
| Erythromycin | 50S subunit | Blocks peptide exit tunnel | Macrolide antibiotic |
| Cycloheximide | 80S (eukaryotic) | Inhibits elongation | Lab use; toxic to humans |
| Ricin | 28S rRNA (eukaryotic) | Depurinates rRNA, inactivates ribosome | Lethal toxin |
| Diphtheria toxin | eEF-2 (eukaryotic) | Blocks elongation by ADP-ribosylation | Causes cell death |
TLDR: Many antibiotics exploit differences in ribosomes to block bacterial translation. Others, like ricin and diphtheria toxin, target eukaryotic translation and can be lethal. These mechanisms underpin drug action and biotoxin lethality.
Most eukaryotic mRNAs require a 5′ cap for ribosome binding and translation initiation.
Some mRNAs (especially viral or mitochondrial) lack a 5′ cap but still undergo translation using:
| Feature | Description |
|---|---|
| Structure | Complex RNA loop in 5′ UTR before start codon |
| Function | Recruits initiation factors or 40S subunit directly, bypassing the 5′ cap |
| Discovered in | Polio virus (cap-independent translation) |
| Benefit to virus | Can still be translated even if host cap-dependent translation is shut down |
Co-expression of multiple proteins from a single mRNA (poly-cistronic in eukaryotes):
Used in tracking gene expression in real time (e.g. with GFP or luciferase reporters).
Alternative translation start sites via IRES enable cap-independent translation. Found in viruses and essential host genes, IRES sequences are also exploited experimentally for bicistronic expression in eukaryotes.
Splicing deposits Exon Junction Complexes (EJCs) downstream of exon-exon boundaries.
During the first round of translation:
Remaining EJCs signal decay:
NMD removes faulty mRNAs containing early stop codons. Exon junction complexes downstream of the premature stop mark them for degradation, preventing translation of truncated, potentially harmful proteins.
Non-stop decay removes transcripts that lack a stop codon by recognizing stalled ribosomes at the 3′ end. Both the faulty mRNA and its abnormal protein product are rapidly degraded.
| Cause | Example |
|---|---|
| Rare codons | No matching tRNAs in host cell |
| Secondary structure | Stable stem-loops block ribosome movement |
| Chemical damage | Modified bases interfering with translation |
No-go decay eliminates mRNAs that cause ribosome stalling (e.g., due to rare codons or structured RNA). It rescues stalled ribosomes and degrades the faulty mRNA to maintain translation fidelity.
Cells must tightly regulate iron levels to avoid:
| Protein | Function |
|---|---|
| Ferritin | Iron storage protein (synthesised when Fe²⁺ is high) |
| Transferrin receptor (TfR) | Iron uptake receptor (synthesised when Fe²⁺ is low) |
| Iron Level | IRP Activity | Effect on mRNAs |
|---|---|---|
| Low Iron | IRPs bind IREs | Block ferritin mRNA translation (don’t store iron); stabilize TfR mRNA (more iron uptake) |
| High Iron | IRPs do not bind | Ferritin translated (store excess iron); TfR mRNA degraded (reduce uptake) |
Cellular iron homeostasis is regulated post-transcriptionally. IRPs bind IREs on ferritin and transferrin receptor mRNAs depending on iron levels, controlling translation or stability to balance iron storage and uptake.
| Feature | Detail |
|---|---|
| IRE location | 5′ UTR |
| Low iron | IRP binds IRE → blocks ribosome scanning → no ferritin made |
| High iron | IRP detaches → ribosome initiates translation → ferritin made to store excess iron |
| Feature | Detail |
|---|---|
| IRE location | 3′ UTR (multiple IREs) |
| Low iron | IRP binds → protects mRNA from endonuclease cleavage → stable mRNA → more receptor |
| High iron | IRP dissociates → mRNA degraded → less receptor → reduced iron import |
[Low Iron]
- IRP binds IREs on ferritin mRNA → translation OFF
- IRP binds IREs on TfR mRNA → stabilisation → translation ON
[High Iron]
- IRP released from IREs → ferritin mRNA translated
- TfR mRNA unprotected → degradedThe position of IREs dictates their effect: 5′ UTR binding blocks translation (ferritin), while 3′ UTR binding stabilizes mRNA (TfR). This elegant switch allows rapid post-transcriptional regulation of iron metabolism.
| Class | Size | Function |
|---|---|---|
| miRNA (microRNA) | ~21–25 nt | Endogenous regulators of gene expression |
| siRNA (short interfering RNA) | ~21 nt | Often exogenous; used in RNA interference |
| piRNA (piwi-interacting RNA) | ~24–30 nt | Suppress transposons in germ cells |
miRNA is transcribed, processed by Drosha and Dicer.
Incorporated into RISC (RNA-Induced Silencing Complex).
Guides RISC to target mRNAs by partial base pairing (usually 3′ UTR).
If match is:
Small RNAs (like miRNAs) regulate gene expression post-transcriptionally by binding mRNAs and either blocking translation or promoting degradation. They enable fine-tuned, rapid control of protein synthesis in response to cellular signals.
Recombinant DNA (rDNA) technology involves manipulating and recombining DNA from different organisms to produce new genetic combinations.
| Application Area | Examples |
|---|---|
| Protein production | Insulin, growth hormone, clotting factors, hepatitis B vaccine |
| Research tools | Restriction enzymes, polymerases, glucose oxidase, fluorescent proteins (GFP), antibodies |
| Visualisation techniques | Immunofluorescence, Western blotting, optogenetics, in vivo GFP tagging (neuronal networks, transgenic animals, tissue-specific expression) |
| Commercial products | GloFish, fluorescent proteins in fish and mice |
| Agriculture | Bt cotton (Cry proteins from Bacillus thuringiensis), golden rice (β-carotene genes), sterile mosquito vectors |
Recombinant DNA technology is used across medicine, research, and agriculture. It enables protein production, creation of visualisation tools, and genetic enhancement of crops and animals. Examples include insulin, fluorescent tagging, Bt cotton, and sterility-based mosquito control.
Because the genetic code is universal, genes from any organism can be expressed in any other, enabling recombinant techniques. This foundational principle underpins all genetic engineering across species.
| Feature | Genomic DNA | Complementary DNA (cDNA) |
|---|---|---|
| Source | Entire genome (nucleus) | Synthesised from processed mRNA (cytoplasm) |
| Contains introns | Yes | No (introns removed via mRNA splicing) |
| Represents | Full genetic potential (all genes) | Only expressed genes at the time of RNA extraction |
| Use cases | GWAS, mutation analysis | Gene expression studies, cloning for bacteria |
| Usability in bacteria | Not suitable directly (contains introns) | Ideal for prokaryotic expression (no introns) |
Genomic DNA includes all genetic material, both coding and non-coding. cDNA is made from mRNA and contains only spliced, expressed genes, making it ideal for recombinant expression in systems like bacteria.
Add random primers (for whole transcriptome) or gene-specific primers (for targeted amplification).
Perform PCR to amplify cDNA, creating either:
| Enzyme | Function |
|---|---|
| Reverse Transcriptase | Synthesizes DNA from RNA (RdDp) |
| Taq Polymerase | Amplifies DNA (DdDp) |
cDNA is created by extracting mRNA, isolating it using its poly(A) tail, and reverse-transcribing it into DNA using reverse transcriptase. This cDNA can then be amplified for expression studies or cloning, especially useful in systems that cannot process introns (e.g., bacteria).
Used to carry and propagate foreign DNA in host cells (e.g., E. coli).
| Component | Function |
|---|---|
| OriV (Origin of Replication) | Allows plasmid to replicate independently within host cell |
| Antibiotic Resistance Gene | Provides selectable marker; only transformed cells survive in antibiotic |
| Multiple Cloning Site (MCS) | Cluster of unique restriction sites to insert gene of interest |
| Promoter | Drives transcription of inserted gene (can be constitutive or inducible) |
[ OriV ]───[ Antibiotic Resistance ]───[ Promoter ]───[ MCS ]───(Insert Gene Here)Cloning vectors like plasmids are engineered with OriV for replication, an antibiotic resistance gene for selection, a promoter to drive gene expression, and a multiple cloning site for gene insertion.
Restriction enzymes cleave DNA at specific sequences, usually palindromic.
Cuts can result in:
Competency induced with CaCl₂ and heat shock:
Very low efficiency (<10%) but sufficient for downstream work.
Recombinant DNA is generated by isolating plasmids, cutting both vector and insert with restriction enzymes, ligating them, and transforming them into bacteria. Selection uses antibiotics; screening distinguishes recombinants from empty vectors.
| Property | Description |
|---|---|
| Source | Naturally found in bacteria as a defense against phages |
| Recognition Sites | Usually palindromic DNA sequences |
| Cutting Mechanism | Hydrolyse phosphodiester bonds in DNA backbone |
| DNA Methylation Sensitivity | Bacteria methylate their own DNA to prevent self-digestion |
| End Type | Create sticky ends (5′/3′ overhangs) or blunt ends |
| Feature | Purpose in Cloning |
|---|---|
| Sticky ends | Promote directional and specific ligation |
| Blunt ends | Less efficient, more versatile but may accept any insert |
| Multiple Cloning Site (MCS) | Contains many unique RE sites to give flexibility in cloning |
| Tail Addition via PCR | Artificial RE sites added to primers for compatibility |
[Plasmid DNA] + [Restriction Enzyme] → Cut at MCS → Insert Gene with Matching Ends → Ligate → TransformRestriction enzymes are sequence-specific DNA cutters originally from bacteria. They are essential in cloning for preparing plasmids and inserts. Sticky ends allow specificity; blunt ends allow flexibility. The MCS ensures safe, targeted insertion.
Recombinant screening ensures that plasmids carry the intended DNA insert, not just the vector backbone. Since not all transformed bacteria are recombinant, several validation techniques are used.
| Colony Colour | Interpretation |
|---|---|
| White | Recombinant plasmid (insert disrupted LacZ) |
| Blue | Non-recombinant (LacZ intact) |
| Result | Interpretation |
|---|---|
| Larger band | Recombinant (insert included) |
| Smaller/no band | Non-recombinant or no insert |
| Outcome | Meaning |
|---|---|
| Two fragments (larger insert) | Recombinant |
| Two fragments (smaller, no insert) | Non-recombinant |
| Feature | Benefit |
|---|---|
| Fluorescent nucleotides | Direct sequence readout |
| Template specific primers | Targeted verification |
| Method | Confirms Insert? | Speed | Cost | Drawback |
|---|---|---|---|---|
| Blue-white screen | Indirect | Fast | Low | False positives, qualitative only |
| Colony PCR | Yes | Very fast | Low | Can fail if primers poorly designed |
| Restriction digest | Yes (size only) | Medium | Low | Cannot confirm sequence or orientation |
| Sequencing | Yes (definitive) | Slow | High | Costly for large-scale screening |
Screening recombinant DNA involves a tiered validation strategy:
Explanation: An absorption spectrum plots absorbance against wavelength for a substance. By running a scan (typically from 650 nm to 350 nm), you identify the λmax, the wavelength at which the compound absorbs the most light. This value is crucial because measuring absorbance at λmax provides the most sensitive and reliable data.
Procedure Highlights:
Example Diagram:
Video: Absorption Spectra Explained – YouTube (Kurzgesagt style)
Explanation: To accurately quantify a compound, measure at or near its λmax, where sensitivity is highest and interference is minimized. In mixtures, choose wavelengths where the target compound’s absorbance is significant and others’ is minimal.
Example: When mixing red and blue dyes, both may have overlapping spectra. Use unique λmax values to deconvolute contributions.
Key Point: Choose λmax based on the compound of interest, while minimizing overlapping absorbance from other substances.
Equation: \(A = \varepsilon \cdot c \cdot l\) Where:
Example Calculation: If \(A = 0.5\), \(\varepsilon = 5\, \text{mM}^{-1}\text{cm}^{-1}\), and \(l = 1\, \text{cm}\), then:
\[ c = \frac{A}{\varepsilon \cdot l} = \frac{0.5}{5 \cdot 1} = 0.1\, \text{mM} \]
Useful Tool: You can calculate these in Excel or lab software.
Steps:
Key Point: The linearity (R² close to 1.00) confirms reliable measurement. Use this curve to interpolate unknown concentrations.
Diagram Example:
Excel Guide Video: Create a Standard Curve in Excel
Explanation: Using the equation from your standard curve (e.g., \(A = 0.01c\)), you can solve for unknown concentration:
\[ c = \frac{A}{\text{slope of the curve}} \]
Example: If the standard curve equation is \(y = 0.0104x\) and an unknown has an absorbance of 0.52:
\[ x = \frac{0.52}{0.0104} \approx 50\, \text{nmol/mL} \]
Explanation:
Working range: The span of absorbance values where the spectrophotometer provides reliable, linear data (typically between 0.1 and 1.0).
Outside this range:
Example: If your unknown’s absorbance is 1.7, you should dilute it to bring it into the optimal range (~0.5).
Application in ELMA (Glucose Assay):
Measures glucose via a coupled enzyme reaction:
In practice:
| Objective | Key Technique | Example |
|---|---|---|
| Generate absorption spectrum | λ scan 650–350 nm | Blue dye: λmax = 628 nm |
| Select λ | Choose λmax | Yellow dye = 426 nm |
| Beer-Lambert Law | \(A = \varepsilon c l\) | Use ε = 10 to find c |
| Standard curve | Serial dilutions, Excel | y = 0.0104x |
| Interpolate unknowns | Use standard curve eq. | A = 0.52 → x = 50 |
| Working range | 0.1 ≤ A ≤ 1.0 | Above or below = error |
| Biological context | ELMA glucose test | Measures diabetes state |
Goal: Extract human DNA (from cheek cells) in a form suitable for PCR.
Key Steps:
Critical Notes:
🖼️ Diagram: DNA Isolation Workflow – ThermoFisher
| Component | Function |
|---|---|
| Template DNA | Source DNA (e.g. D1S80 locus from cheek cells) |
| Primers | Short DNA oligos that define the amplified region |
| Taq polymerase | Heat-stable enzyme that synthesises new DNA |
| dNTPs | Free nucleotides used to build new DNA strands |
| Buffer (Tris, KCl) | Maintains pH and salt conditions for enzyme activity |
| MgCl₂ | Essential cofactor for DNA polymerase |
| Water | To bring the final volume to 50 µL |
🧪 PCR Master Mix includes everything except primers and template.
| Component | Too Much | Too Little | Omitted |
|---|---|---|---|
| Template DNA | Inhibition due to impurities or competition | Weak product | No amplification |
| Primers | Non-specific binding, primer-dimer formation | Incomplete reactions | No amplification |
| Taq polymerase | Non-specific products, background bands | Inefficient amplification | No amplification |
| Mg²⁺ | Increased error rate, non-specific bands | Reduced yield | No activity |
| dNTPs | Incorporation errors | Incomplete synthesis | No DNA synthesis |
| Buffer | pH imbalance | Poor enzyme function | No amplification |
Principle:
A260/A280 Ratio:
🖼️ Nanodrop Spectrum Example (p. 20–21)
Contaminants and Their UV Absorbance Patterns:
| Contaminant | Absorbance Signature |
|---|---|
| Protein | Elevated 280 nm peak → low A260/A280 |
| Phenol | High 270 nm absorbance, distorts spectrum |
| Chelex | Does not show UV absorbance but can inhibit PCR if carried over |
| Contaminant | Impact on PCR |
|---|---|
| Protein | Binds DNA or enzyme, reducing yield |
| Phenol or SDS | Denatures Taq polymerase |
| Chelex | Chelates Mg²⁺, blocking DNA polymerase |
| Ethanol/salts | Interfere with enzyme activity and denature DNA |
Conclusion: Even small amounts of contaminants can fully inhibit PCR. Always remove Chelex and centrifuge thoroughly to avoid inhibitory carryover.
| Method | What it tells you | Limitations |
|---|---|---|
| Nanodrop (spectrophotometry) | DNA concentration, purity (A260/A280) | Doesn’t show DNA degradation or specific fragment size |
| Agarose gel electrophoresis | Fragment size, integrity, presence of bands | Doesn’t give precise concentration or purity |
🔁 Why use both?
Example:
🖼️ Gel electrophoresis image – example of VNTR PCR
| Objective | Tool/Technique | Outcome |
|---|---|---|
| DNA isolation | Cheek cells, Chelex | PCR-compatible DNA |
| PCR components | Master mix + DNA + primers | Amplification of D1S80 |
| Troubleshooting | Vary each component | Understand function |
| Nanodrop | A260/A280 ratio | Purity + concentration |
| UV spectrum | Identify contaminants | Decide if DNA is usable |
| Gel electrophoresis | Separation by size | Visual confirmation |
| Combined analysis | Spectro + Gel | Reliable, interpretable genotyping |
Method:
Key Concept:
🧰 Excel Tip: Plot log10(bp) (y-axis)
vs migration (mm) (x-axis) → trendline equation gives a tool to
calculate unknown sizes.
📊 Example Tool: Bioline DNA HyperLadder IV: https://www.bioline.com/sg/hyperladder-100bp.html
D1S80 locus structure:
Formula:
\[ \text{Number of repeats} = \frac{\text{Amplicon size} - 324}{16} \]
Example: If amplicon size is 624 bp →
\[ \frac{624 - 324}{16} = 18 \text{ repeats} \]
Applications:
🎥 Video: What is DNA profiling?
| Control | What it contains | Expected result | Purpose |
|---|---|---|---|
| Positive control | Working PCR mix + known DNA | Visible band | Confirms PCR reagents and thermocycler work |
| No-template control (NTC) | PCR mix with water instead of DNA | No bands | Confirms no contamination or primer-dimers |
Common errors if controls fail:
Principle:
Humans inherit one D1S80 allele from each parent.
Gel with:
Key Concept:
Example:
🧬 Video: DNA inheritance explained
Plasmid Map Basics:
🧭 Use SnapGene or NEB Cutter to:
📍 Example:
🧬 Video: Interpreting plasmid maps
Steps:
Double digest → more precise location of cut sites
Interpretation:
Process:
🧩 Matching tip: Use your pre-drawn restriction maps from prework!
Example:
Considerations:
Key Predictions:
Band Pattern Example:
🎥 Video: Gel electrophoresis interpretation
\[ \text{Moles (mol)} = \frac{\text{Mass (g)}}{\text{Molecular Weight (g/mol)}} \]
\[ \text{Concentration (mol/L)} = \frac{\text{Moles (mol)}}{\text{Volume (L)}} \]
How many moles are in 180 mg of glucose (MW = 180.16 g/mol)?
Convert mg to g:
\[ 180\, \text{mg} = 0.180\, \text{g} \]
\[ \text{Moles} = \frac{0.180}{180.16} \approx 0.001\, \text{mol} = 1\, \text{mmol} \]
What is the concentration if you dissolve 0.5 g of NaCl (MW = 58.44 g/mol) in 250 mL of water?
\[ \text{Moles} = \frac{0.5}{58.44} \approx 0.00855\, \text{mol} \]
\[ \text{Concentration} = \frac{0.00855}{0.250} = 0.0342\, \text{mol/L} = 34.2\, \text{mM} \]
\[ \text{Mass (g)} = \text{Moles} \times \text{Molecular Weight (g/mol)} \]
How much Tris base (MW = 121.14 g/mol) do you need to make 100 mL of a 0.5 M solution?
\[ \text{Moles} = 0.5\, \text{mol/L} \times 0.1\, \text{L} = 0.05\, \text{mol} \]
\[ \text{Mass} = 0.05 \times 121.14 = 6.06\, \text{g} \]
How many grams of EDTA (MW = 292.24 g/mol) are required for 500 mL of 20 mM solution?
\[ \text{Moles} = 0.020\, \text{mol/L} \times 0.5\, \text{L} = 0.01\, \text{mol} \]
\[ \text{Mass} = 0.01 \times 292.24 = 2.9224\, \text{g} \]
\[ C_1 V_1 = C_2 V_2 \]
Where:
You have 10X TBE buffer and need 500 mL of 1X buffer.
\[ 10 \times V_1 = 1 \times 500 \Rightarrow V_1 = \frac{500}{10} = 50\, \text{mL} \]
Mix 50 mL of 10X TBE with 450 mL water to make 500 mL of 1X.
You have a 1 M NaOH stock. How much is needed to make 200 mL of 0.1 M NaOH?
\[ 1 \times V_1 = 0.1 \times 200 \Rightarrow V_1 = \frac{20}{1} = 20\, \text{mL} \]
Use 20 mL of 1 M NaOH, top up with 180 mL water to get 200 mL of 0.1 M.
| Unit | Equivalent |
|---|---|
| 1 L | 1000 mL |
| 1 mM | 0.001 M |
| 1 µL | 0.001 mL |
| 1 g | 1000 mg |
| Concept | Formula | Key Units |
|---|---|---|
| Mass ↔︎ Moles | \(\text{mass} = \text{mol} \times \text{MW}\) | g, mol, g/mol |
| Moles ↔︎ Concentration | \(\text{C} = \frac{\text{mol}}{\text{L}}\) | mol/L |
| Dilution | \(C_1 V_1 = C_2 V_2\) | M, mL or L |
