Molecular Biology

Protein Synthesis

Transcription and translation — DNA's instructions become functional proteins

Protein synthesis converts DNA's genetic information into functional proteins through two stages — transcription (DNA to mRNA in nucleus) and translation (mRNA to polypeptide on ribosomes). Transcription uses RNA polymerase II, recognizes promoters, processes pre-mRNA via 5' capping, splicing of introns, and 3' polyadenylation. Translation occurs at ribosomes (60S + 40S in eukaryotes), with charged tRNAs reading 3-base codons. The genetic code has 64 codons coding for 20 amino acids plus stop signals; degenerate but unambiguous. Drugs target every step — actinomycin D (transcription), aminoglycosides and macrolides (bacterial translation), chloramphenicol, tetracyclines, linezolid. Mutations cause disease — sickle cell (single base change), cystic fibrosis (Phe508 deletion), Duchenne (frame shift).

  • Codons64 total; 61 code amino acids; 3 stop
  • Eukaryotic ribosome80S = 60S large + 40S small subunit
  • Bacterial ribosome70S = 50S + 30S
  • Initiation codonAUG (methionine; fMet in bacteria)
  • SplicingSpliceosome removes introns from pre-mRNA
  • Antibiotic targetsAminoglycosides 30S; macrolides 50S

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Why protein synthesis matters

  • Genetic disease. Mutations in coding sequence underlie thousands of inherited disorders.
  • Antibiotics. Targeting bacterial ribosomes spares human cells.
  • Cancer therapy. Targeted inhibitors exploit translation dependencies in tumor cells.
  • Hemoglobinopathies. Sickle cell, thalassemias result from abnormal globin synthesis.
  • Vaccines. mRNA vaccines hijack ribosomes to produce antigen in vivo.
  • Drug development. Recombinant insulin, growth hormone, monoclonal antibodies.
  • Toxicology. Ricin and diphtheria toxin kill cells by halting translation.

Common misconceptions

  • One gene equals one protein. Alternative splicing creates multiple proteins from one gene; humans have ~20,000 genes but ~100,000 proteins.
  • tRNAs translate alone. Aminoacyl-tRNA synthetases charge tRNAs — that's where amino acid specificity is enforced.
  • Translation always starts at first AUG. Kozak sequence and other context determine which AUG is used.
  • Stop codons code amino acids. They terminate translation; some organisms recode UGA for selenocysteine.
  • Genetic code is identical everywhere. Mitochondria use slightly different code; some ciliates reassign codons.
  • mRNA is short-lived everywhere. Half-lives range from minutes (regulatory) to days (highly expressed); cells regulate mRNA stability.

Frequently asked questions

How does transcription work?

RNA polymerase II binds the promoter (TATA box ~25 bp upstream), aided by transcription factors (TFIID with TBP, TFIIA-H). Helicase activity in TFIIH unwinds DNA. Polymerase synthesizes mRNA 5' to 3' using template strand. The CTD (C-terminal domain) of Pol II recruits processing factors. 5' capping adds 7-methylguanosine for ribosome binding and stability. Splicing removes introns at GU-AG boundaries via spliceosome. 3' end gets cleaved at AAUAAA signal, then polyadenylated (~250 A residues). Mature mRNA exits nucleus through pores.

How does the ribosome translate mRNA?

Initiation: 40S subunit with initiator Met-tRNA scans mRNA from 5' cap to find AUG; 60S joins forming 80S. Elongation: aminoacyl-tRNA enters A site, peptide bond forms (peptidyl transferase activity in 23S/28S rRNA — a ribozyme), translocation moves tRNA to P site, deacylated tRNA exits E site. Each cycle uses 2 GTP. Termination: release factor recognizes stop codon (UAA, UAG, UGA), peptide released. Multiple ribosomes can translate one mRNA simultaneously (polysome).

What is the genetic code?

3-base codons specify 20 amino acids. 64 codons total: 61 code amino acids, 3 are stop (UAA, UAG, UGA). Degenerate (multiple codons per amino acid, especially in 3rd position — wobble pairing) but unambiguous (each codon specifies one amino acid). Universal across nearly all life with rare exceptions (mitochondria, some ciliates). Reading frame matters — frameshift mutations are devastating. Start codon AUG also codes methionine internally; bacteria use formylmethionine for initiation.

How do antibiotics target translation?

Bacterial 70S ribosome differs from eukaryotic 80S — selectively targetable. Aminoglycosides (gentamicin, streptomycin) bind 30S, cause misreading and inhibit initiation. Tetracyclines block A-site tRNA binding. Chloramphenicol inhibits peptidyl transferase on 50S. Macrolides (erythromycin, azithromycin) and clindamycin block 50S exit tunnel. Linezolid blocks initiation. Resistance: ribosomal mutations, methylation (erm genes), efflux pumps, drug-modifying enzymes. Mitochondrial ribosomes resemble bacterial — explains some drug toxicities.

How do mutations cause disease?

Point mutations: silent (no change), missense (different amino acid, e.g. sickle cell HbS Glu→Val), nonsense (premature stop), splice site (altered splicing). Frameshift (insertions or deletions not divisible by 3) destroys downstream protein. Cystic fibrosis ΔF508 deletes one amino acid causing protein misfolding. Duchenne muscular dystrophy: frameshift in dystrophin (severe); Becker MD: in-frame deletion (milder). Trinucleotide repeat expansions (Huntington CAG, fragile X CGG) cause progressive disease.

What are the post-translational modifications?

Many proteins require processing after translation. Cleavage of signal peptides (secreted proteins), proteolytic activation (insulin from proinsulin, zymogens to enzymes), glycosylation (N-linked in ER, O-linked in Golgi), phosphorylation (kinases, regulates activity), ubiquitination (targets for proteasomal degradation), acetylation, methylation, hydroxylation (collagen requires vitamin C for proline/lysine hydroxylation), and disulfide bond formation. Misfolded proteins go to proteasome via ubiquitin tags. Protein quality control diseases: prion, Alzheimer, cystic fibrosis.

How does the cell regulate protein synthesis?

Multiple levels — transcription factors control which genes are read (steroid receptors, NF-κB, p53). Epigenetic: DNA methylation, histone modifications. mRNA stability and miRNA-mediated degradation. Translation: eIF2α phosphorylation by PKR (in viral infection) and PERK (ER stress) globally suppresses translation. mTOR drives growth and translation when nutrients are available. Selective translation through internal ribosome entry sites (IRES). Misregulation contributes to cancer, neurodegenerative disease, and developmental disorders.