CH17 – The Genetic Code

Definition: the correspondence between nucleotide triplets (codons) in mRNA and the 20 standard amino acids in proteins.
Early reasoning on codon length:
- 1-nt code ⇒ $4^1 = 4$ possibilities → insufficient.
- 2-nt code ⇒ $4^2 = 16$ possibilities → still insufficient.
- 3-nt code ⇒ $4^3 = 64$ possibilities → enough to encode 20 amino acids + start/stop signals.
Total codons: 64
- 61 sense (coding) codons.
- 3 nonsense (stop) codons: UAA, UAG, UGA.
Genetic code properties: universal (with rare exceptions), non-overlapping, continuous (comma-less), degenerate (multiple codons per amino acid).

Hypothesized by Francis Crick; experimentally confirmed when small RNAs were found covalently linked to single amino acids.
tRNA length: 73–93 nucleotides.
Function: mediates base-pairing between mRNA codon and its own anticodon while delivering the correct amino acid to the ribosome.

Secondary structure (cloverleaf):
- 4 major arms + optional extra arm in larger tRNAs.
- Anticodon arm: contains anticodon (3 nt) that pairs antiparallel to mRNA (mRNA 5'→3'; tRNA anticodon 5'→3').
- D arm: features dihydrouridine.
- TΨC arm: contains ribothymidine (T) and pseudouridine (Ψ).
- Acceptor (amino-acid) arm: amino acid attached to 3' end (CCA).
Tertiary structure: compact twisted “L” shape formed by coaxial stacking of the D arm & acceptor arm (one leg) and the anticodon arm & TΨC arm (other leg).

Example: Serine encoded by six codons (UCU, UCC, UCA, UCG, AGU, AGC).
Family boxes: codons sharing first two bases often encode the same amino acid; third base can vary (wobble).

Organisms do NOT maintain 61 distinct tRNAs; minimum theoretical set ≈32.
First (5') anticodon nucleotide is the wobble position:
- G at wobble can pair with C or U.
- U at wobble can pair with A or G.
- Inosine (I) derived from adenosine deamination can pair with C, U, or A.
Wobble pairs are non-canonical (weaker) yet tolerated, allowing one tRNA to decode multiple codons.

Open Reading Frame (ORF): sequence from start codon to stop codon.
- Average ORF in E. coli: ≈1000 nt → ≈333 aa.
Start codons:
- Canonical AUG (codes Met).
- Less frequent GUG or UUG also initiate; still insert methionine in that context.
Stop codons: UAA, UAG, UGA.

Silent mutations: nucleotide change that does not alter amino acid (often at 3rd codon position).
- 3rd-base transitions change amino acid only ≈25 % of the time.
Transition in 1st position tends to substitute an amino acid of similar chemical nature, moderating functional impact.
Frameshift mutations (indels of 1 or 2 nt) alter downstream reading frame → severe effects, often introduce premature stop.

Mutant tRNAs whose anticodon mutates to recognize a stop codon → inserts an amino acid, allowing translation to continue.
Example: Tyr-tRNA (original anticodon GUA) mutates to CUA → pairs with UAG stop, inserts tyrosine.
Rare because global read-through of stops is deleterious.

Single-nt substitutions alter only one amino acid in protein → proves non-overlapping.
Indel experiments show frameshifts affect all downstream codons → proves continuous (no commas).

Brenner & Crick bacteriophage T4 b-gene studies:
- Chemical mutagen induced single insertions ( +1 ) or deletions ( –1 ) → severe phenotype (restricted host range) due to frameshift.
- Combination of +1 and –1 within same gene restored downstream frame and partial function.
- Three insertions (+++1) or three deletions (–––1) restored near-wild-type activity → codon length = 3.

Nirenberg & Matthaei (1961):
- Used polynucleotide phosphorylase to synthesize homopolymers.
- Poly-U template + radiolabeled aa mixtures → only phenylalanine incorporated ⇒ UUU codes Phe.
Filter-binding assay (Nirenberg & Leder):
- Short synthetic trinucleotides (e.g., UUU, AAA, CCC).
- Ribosome + tRNA complex retained on nitrocellulose filter if matching aa-tRNA bound.
- Allowed assignment of ≈50 codons.
Har Gobind Khorana:
- Chemically synthesized repeating RNAs (e.g., ACACAC…, UCUCUC…, etc.).
- Patterns of alternating amino acids in resulting polypeptides resolved remaining codons.
Combined, these approaches fully elucidated the 64-codon table.
- Curiosity: High $Mg^{2+}$ in vitro relaxed need for AUG start, permitting translation on synthetic RNAs lacking proper initiation sites.

Mitochondrial genomes & some microbes exhibit variant codon meanings.
- Example: Vertebrate mitochondrial UGA ⇒ Trp (not stop).
- AGA / AGG ⇒ Stop in vertebrate mitochondria; Ser in Drosophila mitochondria.
Alternative start usage varies by species; nevertheless, resulting N-terminal amino acid is methionine.

21st amino acid: Selenocysteine (Sec, U)
- Encoded by UGA in presence of downstream SECIS (Selenocysteine Insertion Sequence) hairpin.
- Special tRNA-Sec (tRNA^Sec):
- Charged initially with serine by Ser-tRNA synthetase.
- Ser converted to Sec by selenocysteine synthase.
- Low abundance prevents global UGA read-through.
- Requires Sec-specific elongation factor (SelB) recognizing SECIS.
22nd amino acid: Pyrrolysine (Pyl, O)
- Observed mainly in certain archaea.
- Encoded by UAG with a downstream structural element akin to SECIS.
- Special tRNA^Pyl charged directly with pyrrolysine by its own synthetase.