state that a gene mutation is a change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide

Protein Synthesis – Cambridge International AS & A Level Biology (9700)

Learning Objective

State that a gene mutation is a change in the sequence of base pairs in a DNA molecule that may result in an altered polypeptide.

1. DNA – Structure & Semi‑conservative Replication

• Nucleotides: phosphate + deoxyribose + nitrogenous base (A, T, C, G).
• Double helix: two antiparallel strands; A pairs with T (2 H‑bonds) and C pairs with G (3 H‑bonds); base‑stacking stabilises the helix.
• Semi‑conservative replication (DNA polymerases synthesize 5′→3′):

  1. Origin of replication – DNA helicase unwinds the helix.
  2. Single‑strand‑binding proteins keep the strands separated.
  3. RNA primase lays down a short RNA primer (5′‑phosphate) on each template.
  4. Leading strand synthesis – continuous synthesis in the 5′→3′ direction.
  5. Lagging strand synthesis – discontinuous synthesis producing Okazaki fragments; each fragment begins with an RNA primer.
  6. DNA polymerase replaces the RNA primers with DNA and DNA ligase joins the fragments, giving two identical daughter molecules.

2. Transcription – From DNA to mRNA

2.1 Promoter architecture & initiation

• Core promoter (e.g., TATA box ~25‑30 bp upstream of the transcription start site) – recognised by the TATA‑binding protein (TBP), a subunit of transcription factor II D (TFII‑D).
• Proximal promoter elements such as the CAAT box and GC‑rich regions bind additional transcription factors that stabilise the pre‑initiation complex.
• RNA polymerase II, together with general transcription factors (TFIIA, TFIIB, TFIIF, TFIIE, TFIIH), forms the transcription initiation complex.
• TFIIH possesses ATP‑dependent helicase activity that unwinds ~15‑20 bp of DNA, creating the transcription bubble.

2.2 Elongation

• RNA polymerase moves along the template strand in the 3′→5′ direction, synthesising mRNA 5′→3′.
• Base‑pairing rules: A↔U, C↔G.
• Proof‑reading is limited; misincorporation can give rise to mutations.

2.3 Termination

• Eukaryotes: a poly‑adenylation signal (AAUAAA) downstream of the coding region causes cleavage of the pre‑mRNA and addition of a poly‑A tail.
• Prokaryotes: termination occurs via rho‑dependent or rho‑independent (hairpin) sequences that cause RNA polymerase to dissociate.

2.4 mRNA processing (eukaryotes)

• 5′‑cap – a 7‑methyl‑guanosine added to the first nucleotide; protects the transcript and is required for ribosome binding.
• Splicing – introns are removed by the spliceosome; exons are ligated to produce a continuous coding sequence.
• Poly‑A tail – ~200 adenine residues added to the 3′ end; enhances stability and export from the nucleus.

3. Translation – From mRNA to Polypeptide

3.1 Ribosomal structure – prokaryote vs. eukaryote

3.2 Initiation

1. Prokaryotes: 30S subunit + initiation factors (IF1, IF2, IF3) bind the Shine‑Dalgarno sequence; fMet‑tRNA_i pairs with AUG in the P‑site; 50S subunit joins.
2. Eukaryotes: 40S subunit + eIFs bind the 5′‑cap; the complex scans downstream until it finds an AUG in a favourable Kozak context; Met‑tRNA_i occupies the P‑site; 60S subunit joins to form the 80S ribosome.

3.3 Elongation

1. Charged amino‑acyl‑tRNA (via amino‑acyl‑tRNA synthetase) enters the A‑site; correct codon‑anticodon pairing is checked.
2. Peptidyl‑transferase (rRNA) forms a peptide bond, transferring the growing chain from the P‑site tRNA to the A‑site tRNA.
3. Translocation moves the ribosome one codon downstream: deacylated tRNA shifts to the E‑site, peptidyl‑tRNA to the P‑site; GTP hydrolysis by EF‑G (prokaryotes) or eEF‑2 (eukaryotes) provides energy.

3.4 Termination & recycling

• When a stop codon (UAA, UAG, UGA) occupies the A‑site, release factors (RF1/2 in prokaryotes; eRF1/eRF3 in eukaryotes) promote hydrolysis of the peptide‑tRNA bond.
• Ribosomal subunits dissociate with the help of recycling factors (RRF, EF‑G in prokaryotes; eIF‑6 in eukaryotes) and can be reused.

4. The Universal Genetic Code

There are 64 possible codons: 61 encode the 20 standard amino acids, and 3 are stop signals.

5. Gene Mutations – Types & Detailed Effects on the Polypeptide

5.1 Types of mutations

• Point mutations – alteration of a single base pair:

  • Missense: codon now specifies a different amino acid.
  • Nonsense: codon becomes a stop codon.
  • Silent: codon still codes for the same amino acid (may affect translation efficiency or splicing).

• Frameshift mutations – insertion or deletion of nucleotides not in multiples of three; the reading frame downstream is shifted.
• Chromosomal mutations – large‑scale changes (duplications, inversions, translocations, deletions) that can delete whole genes, create fusion proteins, or alter gene dosage.

5.2 Consequences for the polypeptide

1. Missense mutation – the substituted amino‑acid may:

   • Alter the active‑site geometry → reduced or abolished enzyme activity.
   • Disrupt hydrophobic core or disulfide bonds → misfolding and loss of stability.
   • Introduce a new functional group → gain of a novel activity (rare).

2. Nonsense mutation – premature stop codon produces a truncated protein that is usually non‑functional and often targeted for degradation by nonsense‑mediated decay.
3. Silent mutation – amino‑acid sequence unchanged, but can:

   • Influence mRNA secondary structure, affecting translation speed.
   • Create or abolish splice sites, potentially leading to aberrant splicing.

4. Frameshift mutation – shifts the reading frame, resulting in:

   • A completely different amino‑acid sequence downstream.
   • A premature stop codon within a few dozen residues → severely truncated, typically non‑functional protein.

5. Chromosomal mutation – effects depend on the nature of the change:

   • Deletion of a gene → no protein product.
   • Duplication of a gene → over‑production of the protein (gene dosage effect).
   • Translocation creating a fusion gene → chimeric protein with novel properties (e.g., BCR‑ABL in chronic myeloid leukaemia).

6. Biotechnological Applications – Links to Protein‑Synthesis Mechanisms

• Recombinant DNA technology

  • Gene of interest is inserted into a plasmid vector → transcription of the foreign gene by the host’s RNA polymerase.
  • Host ribosomes translate the mRNA, producing the desired protein (e.g., human insulin in E. coli).

• Hybridoma technique (monoclonal antibody production)

  • Fusion of a specific B‑cell (which has rearranged immunoglobulin genes) with a myeloma cell.
  • The hybridoma transcribes and translates the antibody heavy‑ and light‑chain genes, secreting identical antibodies.

• Gene therapy

  • Delivery of a functional copy of a defective gene into patient cells.
  • The introduced gene is transcribed and translated, restoring production of the missing or defective protein.

• RNA interference (RNAi)

  • Short interfering RNAs (siRNAs) bind complementary mRNA and recruit the RISC complex.
  • The mRNA is cleaved, preventing translation and thus silencing the target gene.

7. Summary Flowchart (Suggested Diagram)

Design a clear, labelled flowchart that shows:

• DNA (double helix) → Replication (semi‑conservative, leading/lagging strands).
• Transcription:

  • Promoter (TATA box, transcription factors) → RNA polymerase II → pre‑mRNA.
  • Processing (5′‑cap, splicing, poly‑A tail) → mature mRNA.

• Export to cytoplasm → Translation (ribosome assembly, initiation, elongation, termination, recycling).
• Polypeptide → folding (chaperones) → functional protein.
• Inset illustrating a point mutation that converts a sense codon to a stop codon (nonsense mutation) and the resulting truncated protein.