5.2 Translation & Transcription

Central Dogma at PA-CAT Depth

Quick Answer: Transcription copies a gene's DNA sequence into mRNA inside the nucleus; translation decodes that mRNA into a polypeptide at ribosomes in the cytoplasm. The PA-CAT Bulletin of Information, rev. 20240815, lists this as prerequisite molecular biology underlying "The Cell" content group.

Transcription: DNA → RNA

Transcription is catalyzed by RNA polymerase, which synthesizes RNA in the 5′→3′ direction using a DNA template. In eukaryotes, three RNA polymerases divide the work: RNA polymerase I makes most rRNA, RNA polymerase II makes mRNA and some snRNA, and RNA polymerase III makes tRNA and 5S rRNA.

Transcription has three phases:

1. Initiation. RNA polymerase II binds the promoter, a region upstream of the transcription start site. Eukaryotic promoters often contain a TATA box (~25–30 bases upstream) recognized by TATA-binding protein (TBP), part of transcription factor IID (TFIID). General transcription factors assemble into the pre-initiation complex.
2. Elongation. RNA polymerase unwinds DNA locally (transcription bubble) and adds ribonucleotides complementary to the template strand, reading the template 3′→5′. The newly synthesized RNA trails behind as a single strand.
3. Termination. Eukaryotic protein-coding genes are terminated by a polyadenylation signal (AAUAAA in the RNA). Cleavage and polyadenylation occur ~10–30 bases downstream, releasing the transcript.

Post-Transcriptional Processing

Eukaryotic pre-mRNA is modified before leaving the nucleus:

• 5′ cap (7-methylguanosine, added cotranscriptionally) — protects from exonucleases and enables ribosome binding.
• 3′ poly-A tail (~150–200 adenines) — stabilizes mRNA and aids nuclear export.
• Splicing — the spliceosome (snRNPs U1–U6) removes introns and ligates exons. Alternative splicing lets one gene produce multiple isoforms.

Only processed mRNA is exported through the nuclear pore complex to the cytoplasm, where translation occurs.

Translation: mRNA → Protein

Translation occurs at ribosomes. The genetic code maps 64 codons (3-nucleotide sequences) to 20 amino acids plus stop signals. It is degenerate (most amino acids have multiple codons), unambiguous (each codon specifies only one amino acid), and nearly universal (the same code applies across nearly all organisms).

The stages of translation:

1. Initiation. In eukaryotes, the 40S small subunit + initiator met-tRNAiᵐᵉᵗ scans from the 5′ cap to the first AUG start codon. The 60S large subunit then joins, forming an 80S ribosome with the initiator tRNA in the P site.
2. Elongation. Aminoacyl-tRNA enters the A site matching the next codon. Peptidyl transferase (a ribozyme activity of the 28S rRNA) forms a peptide bond, transferring the growing chain to the A-site tRNA. Translocation moves the ribosome one codon downstream; the deacylated tRNA exits via the E site. Elongation factors eEF1A (delivery) and eEF2 (translocation) participate.
3. Termination. A stop codon (UAA, UAG, UGA) enters the A site. No tRNA recognizes it; instead, a release factor (eRF1) binds, hydrolyzing the bond between the polypeptide and the last tRNA. The ribosome dissociates.

tRNA and Aminoacylation

Each tRNA has an anticodon complementary to a codon and a 3′ CCA end where its specific amino acid is attached by an aminoacyl-tRNA synthetase. There is one synthetase per amino acid (20 total); each charges its tRNAs with high fidelity — a mischarged tRNA would insert the wrong amino acid.

Post-Translational Modification

Newly made polypeptides are often nonfunctional until modified. Common modifications:

• Phosphorylation (by kinases) — activates or inactivates enzymes (e.g., glycogen phosphorylase).
• Glycosylation (in RER and Golgi) — folds and targets membrane/secreted proteins.
• Proteolytic cleavage — converts zymogens (e.g., trypsinogen → trypsin) or prohormones (e.g., proinsulin → insulin).
• Ubiquitination — marks proteins for proteasomal degradation.

Clinical and Exam Relevance

Several drugs and toxins act at defined steps:

• Actinomycin D intercalates DNA and blocks RNA polymerase (inhibits transcription).
• Rifampin inhibits bacterial RNA polymerase (used in tuberculosis treatment).
• Chloramphenicol inhibits the 50S peptidyl transferase.
• Tetracycline blocks aminoacyl-tRNA binding to the A site.
• Cycloheximide inhibits eukaryotic 60S translocation.

A PA-CAT-style question might ask which step is blocked by a compound that prevents mRNA export from the nucleus — answer: post-transcriptional processing (splicing/capping/polyadenylation), since unprocessed pre-mRNA cannot exit through nuclear pores.

Understanding transcription and translation also frames later physiology topics: insulin synthesis (preproinsulin → proinsulin → insulin via signal peptide cleavage in RER and proteolytic processing in secretory vesicles), hemoglobin production (globin genes transcribed in erythroid precursors), and hormone receptors (steroid receptors are transcription factors activated by ligand binding).

Prokaryotic vs Eukaryotic Translation and Exam Pitfalls

PA-CAT physiology overlaps with microbiology, so you must keep the two translational machineries straight. Prokaryotic ribosomes are 70S (30S small + 50S large subunits), whereas eukaryotic ribosomes are 80S (40S + 60S). This size difference is the basis of antibiotic selectivity: drugs like tetracycline (blocks 30S A-site binding) and chloramphenicol (inhibits 50S peptidyl transferase) target bacterial ribosomes with limited effect on human 80S ribosomes, whereas cycloheximide specifically inhibits eukaryotic 60S translocation and spares bacteria — a useful discriminating clue on mechanism-of-action questions.

Initiation also differs. Prokaryotic mRNA is polycistronic and uses the Shine-Dalgarno sequence (AGGAGG, upstream of the start codon) which base-pairs with 16S rRNA to position the start codon in the P site; the initiator is N-formylmethionine (fMet), carried by a specialized fMet-tRNA. Eukaryotic mRNA is monocistronic and cap-dependent: the 40S subunit with met-tRNAi loads at the 5′ 7-methylguanosine cap and scans to the first AUG. The eukaryotic initiator methionine is not formylated. A common PA-CAT trap lists a drug that blocks the Shine-Dalgarno interaction and asks for the affected organism — the correct answer is a bacterium, since eukaryotes lack Shine-Dalgarno sequences entirely.

Wobble explains why ~61 sense codons can be decoded by fewer than 61 tRNAs. The first two codon positions pair strictly (Watson-Crick), but the third position tolerates non-standard pairing (G-U, I-U, I-C, I-A) at the 5′ anticodon position. This degeneracy means many silent third-position mutations change the codon but not the amino acid. tRNA charging fidelity rests on the aminoacyl-tRNA synthetases, each of which edits misactivated amino acids in a proofreading step; a mischarged tRNA will insert the wrong residue because the ribosome reads the anticodon, not the amino acid — the basis of misleading incorporation experiments.

Mutations map onto translation output predictably. A silent mutation changes the codon but not the amino acid (often a third-position swap). A missense mutation substitutes one amino acid, as in sickle cell disease where an A-to-T transversion in the β-globin gene converts GAG (Glu) to GTG (Val). A nonsense mutation introduces a premature stop codon, producing a truncated, usually nonfunctional protein (e.g., certain factor XI deficiency alleles). A frameshift mutation (insertion or deletion not divisible by three) shifts the entire downstream reading frame, typically yielding a premature stop soon after. PA-CAT questions often give a partial sequence and ask for the consequence of a single-nucleotide change — always rewrite the codons and check the amino acid table rather than reasoning by intuition.

A worked antibiotic reasoning example: a compound inhibits bacterial RNA polymerase without affecting human RNA polymerase II. Which step is blocked, and why is host transcription spared? The blocked step is transcription initiation/elongation in bacteria, and selectivity comes from structural differences between bacterial core polymerase (with its σ factor) and eukaryotic Pol II (with general transcription factors). The drug described is rifampin, which binds the β-subunit of bacterial RNA polymerase and prevents the transition from initiation to elongation — it does not inhibit translation, splicing, or post-translational modification, so answers invoking those steps are distractors.