8.1 The code #
The study of The code provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Genetic Code Bias 2
8.1.1 Triplets, wobble, start, and stop #
The code is read in non-overlapping triplets from a fixed start. Crick and Brenner showed the frame with phage mutants: one or two inserted bases destroy it, and three of the same sign restore it.
Wobble is why fewer than 61 tRNA species suffice, on the order of forty. The third codon base pairs loosely with the first anticodon base. U in that position pairs with A or G, G pairs with U or C, and inosine pairs with U, C, or A. C pairs only with G.
| Signal | Codon | What is delivered |
|---|---|---|
| Bacterial start | AUG, rarely GUG or UUG | N-formylmethionine on initiator tRNA |
| Eukaryotic start | AUG in a Kozak context | methionine on initiator tRNA, not formylated |
| Stop | UAA, UAG, UGA | no tRNA; a release factor |
| A degenerate pair | UUU and UUC | both phenylalanine |
The initiator tRNA is not the elongator that reads internal AUG. In bacteria its methionine is formylated, so the P site accepts it as if a peptide had already started. The formyl group, and often the methionine itself, is removed from many finished proteins. A suppressor mutation can change a tRNA so that it reads a stop. That is a mutant tRNA, not a fourth standard stop.
8.2 Charging #
8.2.1 Synthetases proofread; the ribosome does not #
An aminoacyl-tRNA synthetase attaches one amino acid to the 3′ CCA of its tRNAs, using ATP and an aminoacyl-adenylate. Class I synthetases generally first join the amino acid to the 2′-OH of the terminal A, class II synthetases to the 3′-OH.
Ribosome Mrna Translation
The double sieve is the proofreading. The activation site excludes amino acids that are too large. It does not inspect the side chain on a correctly paired tRNA.
8.3 The ribosome and the cycle #
8.3.1 The A, P, and E sites #
A bacterial ribosome is 70S: 30S (16S rRNA plus proteins) and 50S (23S and 5S rRNA plus proteins). A eukaryotic ribosome is 80S: 40S (18S) and 60S (28S, 5.8S, and 5S). Sedimentation values are not masses, so 30 plus 50 is not 70. About the first 30–40 residues are still inside it.
8.3.2 Shine–Dalgarno initiation versus scanning #
Bacterial initiation is on the 30S subunit. IF3 keeps subunits apart and supports start-codon fidelity. IF1 occupies the A-site region. IF2–GTP brings fMet-tRNA into the P site. The Shine–Dalgarno sequence, a purine-rich stretch such as AGGAGG a few nucleotides upstream of AUG, pairs with the anti-Shine–Dalgarno at the 3′ end of 16S rRNA. The 50S subunit joins, GTP is hydrolysed, factors leave, and the A site is free.
Eukaryotes do not use that pairing. A 43S complex — 40S, eIF1, eIF1A, eIF3, eIF5, and eIF2–GTP–Met-tRNAi — is loaded at the cap. eIF4E binds the cap, eIF4G is the scaffold, and eIF4A, an ATP-dependent helicase, unwinds the 5′ leader. eIF4G also binds poly(A)-binding protein, so the mRNA is circularised. The 40S subunit scans to the first AUG in a good Kozak context. A purine, usually A, at −3 and a G at +4 matter most; the consensus is often written GCCRCCAUGG. A weak context allows leaky scanning to a later AUG. eIF5B helps the 60S subunit join. Phosphorylation of eIF2α traps the exchange factor eIF2B and shuts most initiation down, the integrated stress response, while a few special mRNAs continue. The existence of the bypass is the point.
8.3.3 Elongation factors and translocation #
Aminoacyl-tRNA arrives as a ternary complex with EF-Tu–GTP in bacteria, or eEF1A–GTP in eukaryotes. Cognate pairing speeds GTP hydrolysis. Near-cognate tRNAs are usually rejected before hydrolysis or just after it, which is kinetic proofreading. EF-Ts, or eEF1B, recharges EF-Tu with GTP.
After the peptide bond, EF-G–GTP in bacteria, or eEF2–GTP in eukaryotes, drives translocation. The peptidyl-tRNA moves to the P site, the empty tRNA to the E site, and the mRNA advances one codon. Diphtheria toxin ADP-ribosylates eEF2 and stops this step, which is why one modified factor arrests the whole cycle.
8.3.4 The peptidyl transferase is a ribozyme #
The α-amino group of the A-site amino acid attacks the carbonyl that attaches the chain to the P-site tRNA. The chain is transferred to the A-site tRNA.
The catalyst is RNA. The peptidyl-transferase centre is in domain V of 23S rRNA, or 28S rRNA in eukaryotes. The 2′-OH of the P-site tRNA’s terminal adenosine helps shuttle protons, so the substrate assists. They do not supply the catalytic group. The ribosome is a ribozyme, as is the active spliceosome.
8.3.5 Termination, release factors, and polysomes #
A stop codon in the A site is read by a protein, not a tRNA. Bacterial RF1 reads UAA and UAG, RF2 reads UAA and UGA, and RF3 is the GTPase that helps them cycle. Eukaryotic eRF1 reads all three stops, and eRF3 is the GTPase. Subunits are then split, by ribosome recycling factor and EF-G in bacteria, and with the help of ABCE1 in eukaryotes.
A polysome is several ribosomes on one mRNA, each making a polypeptide. Initiation rate sets the spacing. In eukaryotes polysomes form only after export.
8.4 Antibiotics at the ribosome #
8.4.1 Five sites of action #
| Antibiotic | Site | Step that fails |
|---|---|---|
| Streptomycin | 30S decoding centre | Fidelity: misreading; initiation is also perturbed |
| Tetracycline | 30S A site | Binding of aminoacyl-tRNA |
| Chloramphenicol | 50S peptidyl-transferase centre | Peptide-bond formation |
| Erythromycin | 50S exit tunnel | Elongation, once a short chain meets the block |
| Puromycin | A site of the peptidyl-transferase centre, in both domains of life | The chain is transferred to puromycin and released |
Streptomycin, tetracycline, chloramphenicol, and erythromycin select bacterial ribosomes because the sites differ from the cytosolic 80S. Mitochondrial ribosomes, of bacterial ancestry, are sensitive to chloramphenicol, which is the molecular basis of its host toxicity. Puromycin mimics the aminoacylated end of tRNA and is not selective. It is not a selection procedure, and none of these compounds is given here as a dose.
8.5 Coupling #
8.5.1 Only where the two processes share a compartment #
Bacteria and archaea have no nuclear envelope. A ribosome can bind a nascent mRNA, and translation can begin before RNA polymerase has finished. Trp attenuation (Chapter 10) reads the position of that ribosome. Coupling is a regulatory device, not only a way to save time.
Eukaryotic nuclear genes cannot do this. Processing is nuclear and translation is cytosolic, after export. A nuclear operon would not be translated as several proteins from one mRNA by Shine–Dalgarno initiation.
8.6 Key terms, summary, and self-check #
Key terms. Wobble; fMet; Shine–Dalgarno; Kozak; EF-Tu; eEF2; peptidyl transferase; release factor; polysome; puromycin.
Summary. The code is a degenerate triplet table, relaxed at the third position by wobble, with AUG as start and UAA, UAG, and UGA as stop. Synthetases charge tRNA and proofread the amino acid; the ribosome checks only the anticodon. Bacteria place fMet-tRNA in the P site by a Shine–Dalgarno interaction. Eukaryotic 40S subunits scan from the cap to a Kozak AUG. EF-Tu or eEF1A delivers aminoacyl-tRNA, EF-G or eEF2 translocates, and 23S or 28S rRNA makes the peptide bond. Release factors hydrolyse the chain at a stop, and polysomes carry several ribosomes on one mRNA. The five named antibiotics each stop a stated step. Ribosomes translate unfinished transcripts only where there is no nuclear envelope.
Self-check.
1. Which codon position wobbles, and which base of the anticodon is the wobble base?
2. Why does a tRNA charged with the wrong amino acid still pass the ribosome?
3. What base pairing places the bacterial AUG in the P site, and what does the eukaryotic 40S subunit do instead?
4. Which rRNA makes the peptide bond, and which factor, EF-Tu or EF-G, moves the tRNAs afterwards?
5. Which antibiotic releases unfinished chains from both bacterial and eukaryotic ribosomes, and which blocks the 30S A site?
