Difficulty: Intermediate | Prerequisites: Parts 1 and 2 of these notes (Central Dogma, genetics basics); familiarity with general chemistry concepts (atoms, bonds, energy)
This section covers two distinct but connected areas tested on Exam 1. First, the molecular mechanics of gene expression: how DNA is transcribed into RNA and how RNA is translated into protein, plus clinical examples showing what happens when these processes go wrong. Second, the physical chemistry that underpins all of it: the types of bonds holding biomolecules together, the thermodynamic rules governing whether a reaction proceeds, how enzymes accelerate those reactions, and why ATP is the universal energy currency. If Part 1 told you what the Central Dogma is and Part 2 showed you the inheritance patterns genes follow, this part explains how the machinery actually works and what powers it.
Transcription copies DNA into RNA (5' → 3'), and translation reads mRNA in codons to assemble a polypeptide chain. Point mutations in these sequences cause diseases like sickle cell anaemia and cystic fibrosis. All of these processes depend on covalent and non-covalent bonds, are governed by thermodynamic principles (Gibbs free energy, entropy, enthalpy), require enzymes to lower activation energy, and are fuelled by ATP hydrolysis.
Transcription
The process by which RNA polymerase synthesises an RNA strand complementary to a DNA template strand, reading the template 3' → 5' and building the RNA 5' → 3'.
Translation
The process by which ribosomes decode mRNA into a polypeptide chain, reading the mRNA 5' → 3' and assembling amino acids from the N-terminus to the C-terminus.
Chemical bond
An attractive force holding atoms together in a molecule or compound.
Covalent bond
A strong bond formed by the sharing of one or more pairs of electrons between atoms. Covalent bonds have high bond energies (typically 150 to 950 kJ/mol) and define the primary structure of biomolecules.
Weak bonds (non-covalent interactions)
Bonds that are individually much weaker than covalent bonds but collectively stabilise the three-dimensional structures of macromolecules. Includes hydrogen bonds, Van der Waals forces, charge-charge (ionic/electrostatic) interactions, and hydrophobic interactions.
Valence
The number of bonds an atom can form, determined by the number of electrons it needs to gain, lose, or share to fill its outer shell.
Polar (bond or molecule)
A bond or molecule with an uneven distribution of electron density, creating partial positive and partial negative charges.
Dipole
A molecule or bond with two regions of opposite charge (a positive and a negative pole), arising from differences in electronegativity.
Chiral
A molecule that is not superimposable on its mirror image, typically because it has a carbon atom bonded to four different groups. Chirality matters in biology because enzymes are stereospecific.
Stereoisomers
Molecules with the same molecular formula and connectivity but different spatial arrangements of atoms. Includes enantiomers (mirror images) and diastereomers (non-mirror-image stereoisomers).
Bond angle
The angle formed between two bonds that share a common atom. Determines the three-dimensional shape of a molecule.
Bond rotation
Rotation around a single bond, which allows molecules to adopt different conformations. Double bonds restrict rotation.
Peptide bond
A covalent bond formed between the amino group of one amino acid and the carboxyl group of another, with the loss of a water molecule (condensation reaction). Peptide bonds link amino acids into polypeptide chains.
Equilibrium constant (K_eq)
A ratio of product concentrations to reactant concentrations at chemical equilibrium. K_eq > 1 means products are favoured; K_eq < 1 means reactants are favoured.
Gibbs free energy (ΔG)
The energy available to do work in a system at constant temperature and pressure. In simple terms, ΔG tells you whether a reaction will happen spontaneously. If ΔG is negative, the reaction releases free energy and proceeds spontaneously (exergonic). If ΔG is positive, the reaction requires an input of energy (endergonic).
Standard free energy (ΔG°)
The free energy change under standard conditions (1 M concentrations, 1 atm, 25°C, pH 7 for biochemistry, denoted ΔG°'). It reflects the intrinsic tendency of a reaction to proceed, independent of actual cellular concentrations.
Entropy (S)
A measure of the disorder or randomness of a system. The Second Law of Thermodynamics states that the total entropy of the universe tends to increase.
Enthalpy (H)
The total heat content of a system. In biochemistry, changes in enthalpy (ΔH) reflect the heat absorbed or released during bond breaking and formation.
Hydrogen bond
A weak interaction between a hydrogen atom covalently bonded to an electronegative atom (O, N, F) and a lone pair on another electronegative atom. Individually weak (about 4 to 20 kJ/mol), but collectively they stabilise DNA's double helix, protein secondary structures, and water's properties.
Van der Waals forces
Very weak, short-range attractions between all atoms and molecules, caused by transient fluctuations in electron distribution. Individually the weakest of the non-covalent forces, but they contribute to protein folding and molecular recognition when many atoms are in close contact.
Charge-charge (electrostatic/ionic) interactions
Attractions between fully charged groups (e.g., NH₃⁺ and COO⁻). Stronger than hydrogen bonds in a vacuum but weakened significantly by water.
Hydrophobic interactions
The tendency of nonpolar molecules or groups to cluster together in an aqueous environment, driven by the increase in entropy of surrounding water molecules. This is a major force in protein folding and membrane assembly. In simple terms, oily bits avoid water and bunch up together, which actually lets the water molecules be more disordered overall.
Activation energy (E_a)
The minimum energy input required for a reaction to proceed. Enzymes lower E_a, making reactions fast enough for biological timescales.
ATP (adenosine triphosphate)
The primary energy currency of the cell. Energy is released by the hydrolysis of its terminal phosphoanhydride bond (ATP → ADP + Pᵢ), yielding approximately -30.5 kJ/mol under standard conditions.
Transcription occurs in the nucleus (in eukaryotes) and is carried out by RNA polymerase.
Initiation: RNA polymerase binds to the promoter region upstream of the gene. In bacteria, the sigma factor helps polymerase recognise the promoter. In eukaryotes, general transcription factors and the mediator complex recruit RNA Polymerase II to the promoter.
Elongation: RNA polymerase moves along the template strand 3' → 5', synthesising a complementary RNA strand in the 5' → 3' direction. The new RNA is built by adding ribonucleotides to the 3'-OH end.
Termination: transcription ends when RNA polymerase reaches a termination signal. In bacteria, this can be rho-independent (a GC-rich hairpin followed by a poly-U sequence) or rho-dependent (the rho protein catches up to the polymerase and unwinds the RNA-DNA hybrid). In eukaryotes, termination is coupled to polyadenylation and cleavage of the pre-mRNA.
Direction summary: the template strand is read 3' → 5'; the RNA is synthesised 5' → 3'; the coding (non-template) strand has the same sequence as the RNA (with T instead of U).
Translation occurs on ribosomes, in the cytoplasm.
Initiation: the small ribosomal subunit binds to the mRNA near the 5' cap and scans for the start codon (AUG). The initiator tRNA carrying methionine (fMet in bacteria) base-pairs with AUG in the P site. The large subunit joins.
Elongation: aminoacyl-tRNAs enter the A site, the ribosome catalyses peptide bond formation (transferring the growing chain from the P-site tRNA to the A-site amino acid), and the ribosome translocates one codon along the mRNA. The mRNA is read 5' → 3'; the polypeptide grows from N-terminus to C-terminus.
Termination: when a stop codon (UAA, UAG, UGA) enters the A site, no tRNA binds. Release factors enter, trigger hydrolysis of the polypeptide from the last tRNA, and the ribosome dissociates.
Sickle Cell Anaemia
A single nucleotide change in the beta-globin gene (GAG → GTG in DNA, which produces GAG → GUG in mRNA) causes an amino acid substitution: glutamic acid (position 6) is replaced by valine. The resulting haemoglobin S polymerises under low oxygen conditions, distorting red blood cells into a sickle shape. This is autosomal recessive: heterozygous carriers (sickle cell trait) are generally asymptomatic and have some resistance to malaria.
Huntington Disease
Caused by an expansion of a CAG trinucleotide repeat in the huntingtin gene on chromosome 4. The resulting protein has an abnormally long polyglutamine tract that causes it to misfold and aggregate, leading to progressive neurodegeneration. Huntington disease is autosomal dominant: one mutant allele is sufficient. Age of onset typically correlates inversely with the number of repeats.
Cystic Fibrosis
Most commonly caused by a three-nucleotide deletion (ΔF508) in the CFTR gene, which removes a single phenylalanine at position 508 of the CFTR protein. The misfolded protein is degraded before reaching the cell membrane, so chloride ion transport is impaired. This leads to thick, sticky mucus in the lungs, pancreas, and other organs. Cystic fibrosis is autosomal recessive.
Amino acid substitution is the key mechanism connecting a DNA mutation to a disease phenotype. A change in one nucleotide (a point mutation) can alter the codon so that a different amino acid is incorporated, potentially disrupting the protein's structure and function.
Covalent bonds form the backbone of biomolecules:
C-C, C-H, C-O, C-N bonds create the skeletons of sugars, fatty acids, amino acids, and nucleotides.
Peptide bonds link amino acids.
Phosphodiester bonds link nucleotides in DNA and RNA.
Stronger covalent bonds correspond to more negative free energy changes when broken (i.e., more energy is stored in them).
Weak bonds stabilise higher-order structures:
Hydrogen bonds stabilise the DNA double helix (A=T has 2 H-bonds; G≡C has 3), protein alpha helices and beta sheets, and enzyme-substrate interactions.
Van der Waals forces contribute to the stacking of base pairs in DNA and to protein core packing.
Charge-charge interactions (salt bridges) form between oppositely charged amino acid side chains in proteins.
Hydrophobic interactions drive the burial of nonpolar amino acids in the interior of proteins and the assembly of lipid bilayers.
The key distinction: covalent bonds define what a molecule is; non-covalent bonds define its shape and how it interacts with other molecules.
First Law of Thermodynamics: energy cannot be created or destroyed, only converted between forms. In biological systems, chemical energy in food is converted to ATP, heat, and work.
Second Law of Thermodynamics: in any spontaneous process, the total entropy of the universe increases. Living organisms maintain local order (low entropy) by increasing entropy in their surroundings (releasing heat, excreting waste).
Gibbs free energy equation:
ΔG = ΔH - TΔS
ΔH = change in enthalpy (heat released or absorbed)
T = absolute temperature in Kelvin
ΔS = change in entropy
A reaction is spontaneous (exergonic) when ΔG < 0. A reaction is non-spontaneous (endergonic) when ΔG > 0.
Relationship between ΔG° and K_eq:
ΔG° = -RT ln K_eq
Where R = 8.314 J/(mol·K) and T = temperature in Kelvin. A large K_eq (products favoured) corresponds to a large negative ΔG°.
Coupled reactions: an endergonic reaction can be driven forward by coupling it to an exergonic reaction (such as ATP hydrolysis) so that the overall ΔG for the coupled process is negative.
Enzymes accelerate reactions by lowering the activation energy (E_a). They do not change the ΔG of the reaction and do not shift the equilibrium; they only make equilibrium reached faster.
How enzymes lower E_a:
Stabilising the transition state.
Providing an alternative reaction pathway with a lower energy barrier.
Orienting substrates correctly in the active site.
Providing acid-base or covalent catalysis.
Examples: DNA polymerase (catalyses phosphodiester bond formation during DNA replication), RNA polymerase (transcription), aminoacyl-tRNA synthetases (charge tRNAs with the correct amino acid), hexokinase (phosphorylates glucose in the first step of glycolysis).
ATP stores energy in its phosphoanhydride bonds (the bonds between the phosphate groups). Hydrolysis of the terminal phosphate releases energy:
ATP + H₂O → ADP + Pᵢ, ΔG°' ≈ -30.5 kJ/mol
This is a "high-energy" bond not because the bond itself is unusually strong, but because the products (ADP + Pᵢ) are much more stable than the reactants due to charge repulsion relief, resonance stabilisation of Pᵢ, and increased solvation.
Other high-energy molecules in biology include phosphoenolpyruvate (PEP), 1,3-bisphosphoglycerate, and acetyl-CoA, each with a more negative ΔG° of hydrolysis than ATP.
Protein synthesis: ATP is consumed indirectly. GTP powers ribosomal translocation and factor binding, but ATP drives aminoacyl-tRNA synthetases to charge tRNAs (ATP → AMP + PPᵢ, equivalent to two ATP → ADP conversions).
Amino acid activation: aminoacyl-tRNA synthetase cleaves ATP to AMP + PPᵢ, forming an aminoacyl-AMP intermediate. The subsequent hydrolysis of PPᵢ to 2Pᵢ (by pyrophosphatase) pulls the reaction forward, making it effectively irreversible.
DNA synthesis: deoxyribonucleoside triphosphates (dNTPs) provide both the monomers and the energy for the reaction. Each incoming dNTP releases pyrophosphate (PPᵢ) upon incorporation, and the hydrolysis of PPᵢ drives the reaction forward.
ATP synthesis itself: in oxidative phosphorylation, the proton motive force across the inner mitochondrial membrane drives ATP synthase to condense ADP + Pᵢ → ATP. The energy input comes from the electron transport chain, not from ATP.
Actomyosin cycle (muscle contraction): ATP binding to myosin causes it to release actin. ATP hydrolysis cocks the myosin head into a high-energy conformation. Pᵢ release triggers the power stroke (myosin pulls actin). ADP release completes the cycle. Without new ATP, myosin stays locked to actin (rigor).
Gibbs free energy:
ΔG = ΔH - TΔS
Standard free energy and equilibrium:
ΔG° = -RT ln K_eq
Actual free energy under cellular conditions:
ΔG = ΔG° + RT ln ([products]/[reactants])
ATP hydrolysis:
ATP + H₂O → ADP + Pᵢ; ΔG°' ≈ -30.5 kJ/mol
The thermodynamics covered here is the same framework engineers and chemists use to predict whether any chemical reaction will proceed. In drug design, understanding enzyme active sites and transition state stabilisation is how pharmaceutical companies develop enzyme inhibitors (e.g., protease inhibitors for HIV). Sickle cell anaemia and cystic fibrosis are among the most commonly tested examples in medical school genetics precisely because they illustrate how a single nucleotide change can cascade into organ-level disease.
"Enzymes provide energy for reactions." Enzymes do not supply energy. They lower the activation energy barrier, allowing reactions to proceed faster. The energy comes from the substrates and their bonds.
"ATP has 'high-energy bonds' that are especially strong." The term is misleading. The bonds themselves are not unusually strong; the products of hydrolysis are unusually stable. It is the large negative ΔG of hydrolysis, not the bond strength, that makes ATP useful as an energy carrier.
"A negative ΔG° means a reaction happens quickly." ΔG tells you whether a reaction is thermodynamically favourable, not how fast it occurs. A reaction can be strongly favoured (large negative ΔG) but kinetically slow without an enzyme.
"The Second Law of Thermodynamics means living things shouldn't exist because they are ordered." Living things are open systems that increase the entropy of their surroundings (by releasing heat and waste products) more than they decrease their own internal entropy. The total entropy of the universe still increases.
⚠️ Know the direction of transcription (RNA synthesised 5' → 3', template read 3' → 5') and translation (mRNA read 5' → 3', polypeptide assembled N → C terminus). These directions are exam staples.
⚠️ Be able to explain each clinical correlation at the molecular level: what the mutation is, what it does to the protein, and how that causes the disease.
⚠️ The Gibbs free energy equation (ΔG = ΔH - TΔS) and the relationship ΔG° = -RT ln K_eq are likely to appear in calculation or conceptual questions. Know what a negative ΔG means versus a negative ΔG°.
⚠️ Distinguish covalent bonds from each type of non-covalent interaction and give a biological example of each.
⚠️ Understand coupled reactions: be able to show that coupling an endergonic reaction to ATP hydrolysis makes the overall ΔG negative.
⚠️ Compare the role of ATP in at least three different processes (protein synthesis, DNA synthesis, muscle contraction). The exam may ask you to explain what happens at each step.
True or False: RNA polymerase synthesises RNA in the 3' → 5' direction. Answer: False. RNA polymerase synthesises RNA 5' → 3' (it reads the template 3' → 5').
Fill in the blank: In sickle cell anaemia, the amino acid ______ is replaced by ______ at position 6 of beta-globin. Answer: glutamic acid; valine.
True or False: Enzymes shift the equilibrium of a reaction toward the products. Answer: False. Enzymes lower the activation energy and speed up both the forward and reverse reactions equally. They do not change the equilibrium position.
Fill in the blank: ΔG = ΔH - T______. Answer: ΔS.
True or False: Hydrophobic interactions are driven primarily by the entropy of water molecules surrounding nonpolar groups. Answer: True.
Q: Describe the three phases of transcription and identify the direction in which RNA is synthesised.
A: Initiation: RNA polymerase binds the promoter with the help of sigma factor (prokaryotes) or transcription factors (eukaryotes). Elongation: polymerase moves along the template strand 3' → 5', adding ribonucleotides to the 3'-OH end of the growing RNA. The RNA is built 5' → 3'. Termination: polymerase reaches a termination signal (e.g., a hairpin loop followed by poly-U in prokaryotes) and releases the completed RNA.
Q: Explain at the molecular level why sickle cell anaemia is caused by a single base change in DNA.
A: A point mutation in the beta-globin gene changes the codon GAG (glutamic acid) to GUG (valine) at position 6 of the mRNA. Valine is hydrophobic, while glutamic acid is charged. This substitution causes haemoglobin S molecules to polymerise into rigid fibres under low-oxygen conditions, deforming red blood cells into sickle shapes that block capillaries and are destroyed prematurely.
Q: What is the relationship between ΔG° and the equilibrium constant K_eq?
A: They are related by the equation ΔG° = -RT ln K_eq. When K_eq > 1 (products favoured at equilibrium), ΔG° is negative. When K_eq < 1 (reactants favoured), ΔG° is positive. A more negative ΔG° corresponds to a more product-favoured equilibrium.
Q: Compare covalent bonds and hydrogen bonds in terms of strength, function, and a biological example.
A: Covalent bonds are strong (150 to 950 kJ/mol), define the primary structure of molecules, and are exemplified by the peptide bonds linking amino acids or the phosphodiester bonds in DNA. Hydrogen bonds are weak (4 to 20 kJ/mol each), stabilise three-dimensional structure and molecular interactions, and are exemplified by the base pairing between complementary strands of DNA (A=T has two H-bonds, G≡C has three).
Q: Explain how ATP hydrolysis can drive an endergonic reaction through coupling.
A: If an endergonic reaction has a ΔG of +15 kJ/mol and ATP hydrolysis has a ΔG of -30.5 kJ/mol, coupling the two gives an overall ΔG of about -15.5 kJ/mol, which is negative and therefore spontaneous. In practice, the enzyme catalysing the reaction uses ATP hydrolysis to change the pathway so that the thermodynamically unfavourable step is linked to the favourable one, making the combined process proceed spontaneously.
Q: Describe the role of ATP in the actomyosin cycle of muscle contraction.
A: ATP binds to myosin, causing it to release from actin (detachment). ATP is then hydrolysed to ADP + Pᵢ, which cocks the myosin head into a high-energy position (cocking). Myosin binds weakly to a new actin site. Release of Pᵢ triggers the power stroke, pulling actin toward the centre of the sarcomere. ADP is released, completing the cycle. A new ATP molecule must bind for myosin to detach again, otherwise rigor occurs.
The transcription and translation material here is the molecular payoff of the Central Dogma introduced in Part 1. The clinical correlations (sickle cell, Huntington, cystic fibrosis) are Mendelian genetics from Part 2 made tangible at the protein level. The thermodynamics and enzyme sections underpin every reaction in the course going forward: replication, transcription, translation, and all metabolic pathways require enzymes, rely on favourable free energy changes, and are powered directly or indirectly by ATP.
transcription phases, RNA polymerase, initiation elongation termination, translation ribosome, mRNA 5' to 3', sickle cell anaemia HbS, Huntington disease trinucleotide repeat, cystic fibrosis CFTR ΔF508, amino acid substitution, point mutation, covalent bond, hydrogen bond, Van der Waals, hydrophobic interaction, electrostatic interaction, Gibbs free energy, ΔG, ΔG°, entropy, enthalpy, equilibrium constant K_eq, activation energy, enzyme catalyst, ATP hydrolysis, coupled reactions, high-energy bonds, actomyosin cycle, aminoacyl-tRNA synthetase, DNA synthesis dNTPs, First Law of Thermodynamics, Second Law of Thermodynamics, stereoisomers, chirality, peptide bond, UCF Molecular Biology I Exam 1