Protein Synthesis, Gene Expression, and Mutations

DNA & Chromosomes

• DNA is the hereditary molecule that holds genetic information in eukaryotic nuclei.
• Compacts into tightly packed chromosomes; each chromosome contains many genes.

Genes & Genome

• Gene = sequence of DNA that codes for one protein.
  • Provides the “recipe” (nucleotide code) for the amino-acid sequence of a protein.
• Complete genetic information of an organism = genome.

From Gene to Protein: The Central Dogma

• Three core stages to convert genetic code into functional protein:
  1. Transcription – DNA → RNA (nucleus)
  2. RNA Processing – pre-RNA → mature mRNA; exported to cytoplasm.
  3. Translation – mRNA → polypeptide (cytoplasm/ribosome)
• After translation the polypeptide:
  • Folds into functional 3-D shape.
  • May undergo further chemical modification.
  • Is transported to its site of action (cytosol, membrane, secretion, etc.).

DNA vs. RNA: Key Differences

• Nitrogenous bases:
  • DNA = A, G, C, T
  • RNA = A, G, C, U
• Sugar:
  • DNA = deoxyribose
  • RNA = ribose
• Structure:
  • DNA = double-stranded helix
  • RNA = usually single-stranded (can form hairpins/enzymatic structures)
• Localization & stability:
  • DNA stays in nucleus; long-term storage (stable).
  • RNA can serve as hereditary material in viruses & acts catalytically, but is less stable – not ideal for long-term coding.

Functional Types of RNA

• mRNA (messenger RNA)
  • Complementary copy of gene; carries genetic instructions nucleus → ribosome.
• rRNA (ribosomal RNA)
  • Combines with proteins to form ribosome (enzyme complex) & helps decode mRNA.
• tRNA (transfer RNA)
  • Binds specific amino acid & “translates” codon → amino acid via anticodon-codon pairing.

Functional Summary

• DNA = master code (secure in nucleus).
• mRNA = mobile blueprint.
• rRNA = structural & catalytic core of ribosome.
• tRNA = amino-acid courier/translator.

Transcription (DNA → pre-mRNA)

• Purpose: Produce complementary RNA copy of gene.
• Overall result: single-stranded RNA transcript.
• 3 Phases:
  1. Initiation
  • Gene possesses a promoter region (upstream regulatory sequence).
  • Transcription factors + RNA polymerase II bind promoter.
  • DNA unwinds; template strand (3′→5′) exposed.
  1. Elongation
  • RNA polymerase synthesizes RNA 5′→3′ by complementary base pairing (A↔U, G↔C).
  • Nucleotides added to 3′ OH of growing RNA.
  1. Termination
  • Polymerase encounters termination signal (intrinsic hair-pin loop or Rho-dependent cue).
  • RNA and polymerase detach; DNA re-zips.

RNA Processing (Eukaryotes)

• Converts pre-mRNA to mature mRNA capable of translation.

1. End Modifications
   • 5′ cap = modified guanine; aids ribosome binding & protects from exonucleases.
   • 3′ poly-A tail ≈ 150+ adenines; stabilizes & regulates nuclear export.
2. Splicing
   • Introns (intervening sequences) removed; exons (expressed) ligated.
   • Catalyzed by spliceosome (snRNP complex).
3. Outcome = exon-only mRNA, multiple transcripts can be produced simultaneously when gene is open → high-demand proteins made rapidly.

Translation (mRNA → Polypeptide)

• Location: cytoplasm on free or ER-bound ribosomes.
• “Language switch” from nucleotide code to amino acids.
• Requires: mature mRNA, tRNA, ribosomes (rRNA + proteins), energy (GTP).

Genetic Code Principles

• Codon = 3 consecutive RNA bases.
• Total combinations = 4^3 = 64 codons for 20 amino acids → redundancy/degeneracy (protective against mutations).
• Universal among living cells.
• Special codons:
  • Start = AUG (codes Met; sets reading frame).
  • Stops = UAA, UGA, UAG (no amino acid).

tRNA Structure & Function

• Cloverleaf RNA; two critical sites:
  1. Amino-acid attachment site (3′ CCA tail).
  2. Anticodon – 3 bases complementary to mRNA codon.
• Example: Anticodon CGG pairs with codon GCC → amino acid Alanine.

Ribosome Architecture

• Large + small subunits (assemble on mRNA start site).
• 3 binding sites:
  • A (acceptor) – incoming charged tRNA.
  • P (peptidyl) – tRNA holding growing chain.
  • E (exit) – uncharged tRNA leaves.

Translation Steps

1. Initiation
   • Small subunit binds 5′ cap & scans to AUG.
   • Initiator tRNA (Met, anticodon UAC) occupies P site.
   • Large subunit joins; initiation complex ready.
2. Elongation
   • Correct tRNA enters A site (anticodon-codon pairing verified).
   • Peptidyl transferase (rRNA catalyst) forms peptide bond between chain (P) & new amino acid (A).
   • Ribosome translocates one codon 5′→3′ along mRNA → tRNA shift: A→P, P→E.
   • Process repeats; chain elongates, spontaneously begins folding.
3. Termination
   • Ribosome reaches stop codon.
   • Release factor binds; water hydrolyzes bond, releasing polypeptide (typically 20\text{–}200 aa in examples, but can be thousands).
   • Subunits dissociate; mRNA may be re-used by other ribosomes (polyribosome/polysome).

Post-Translational Processing & Trafficking

• Proteins destined for secretion, membranes, or organelles enter rough ER during synthesis.
• Glycosylation (addition of sugars) → glycoproteins.
• Further modification/sorting in Golgi apparatus → vesicles → final destination.

DNA Mutations

• Mutation = random, permanent DNA sequence change.
• Can be single nucleotide to large chromosomal alterations.
• Often neutral; when in gene → may be deleterious or advantageous.

Frequency & Repair

• DNA polymerase proofreading corrects most errors.
• Uncorrected rate ≈ 1/3\times10^7 bases → \sim100{-}200 new mutations per human generation.

Causes

• Spontaneous replication errors.
• Chemicals: pesticides, drugs (e.g., AZT), food additives, household cleaners, plastics.
• Radiation: X-ray, UV → thymine or cytosine dimers (notably in p53 tumor suppressor gene).

Point Mutations (Single-base substitutions)

• Silent – alters codon to synonym; peptide unchanged.
• Missense – new codon → different amino acid.
  • Conservative: similar properties (minor effect).
  • Non-conservative: different properties (potentially drastic).
  • Example: Sickle-cell anemia = non-conservative missense in hemoglobin β-chain (Glu→Val).
• Nonsense – mutation converts sense codon → premature STOP; truncated non-functional protein.

Practice Examples (from transcript)

• CCT→CCC (Gly→Gly) → Silent.
• CCT→CTT (Gly→Glu) → Missense (non-conservative bonus answer).
• CCT→ACT (Gly→STOP) → Nonsense.

Frameshift Mutations

• Insertion/Deletion (indel) shifts reading frame → downstream codons altered.
• Diseases:
  • Fragile X syndrome: expansion of \text{CGG} repeats on X-chromosome; repeat number ↑ each generation; intellectual disability, distinct phenotype, male-biased severity.
  • Cystic Fibrosis: in CFTR chloride channel gene, exon 7 contains 1-base deletion + 2-base insertion → truncated non-functional protein; autosomal recessive, lethal by 20s–30s (respiratory failure).
• UV-induced thymine dimers can also cause frameshifts.

Mutagen Detection: The Ames Test

• Uses auxotrophic bacteria that require histidine.
• Expose to potential mutagen on medium without histidine.
• Growth (reversion to His+) indicates chemical caused mutation → positive mutagen.

Mutation Effects by Cell Type

• Somatic mutations – in body cells; not inherited; can lead to cancer if tumor-suppressor genes silenced.
• Germ-line mutations – in gametes; heritable.
  • Spectrum: silent → minor trait variation → major phenotypic change.
  • ~3\% of U.S. births have major birth defects; \approx25\% due to genetic mutations.
  • Teratology studies environmental agents (teratogens) causing congenital malformations.

Thought & Conceptual Notes

• Redundant genetic code buffers against point mutations; silent & conservative missense often harmless.
• Polysomes allow high-demand protein production by simultaneous translation of one mRNA.
• Post-translation trafficking ensures proteins reach correct cellular compartments; mis-localization can mimic loss-of-function.
• Evolution leverages mutation: while most are neutral/negative, rare advantageous mutations drive adaptation (e.g., sickle-cell allele confers malaria resistance).