MB 4. Genetic code and translation
MB(ASCP) study notes · Lesson 4
Translation converts the nucleotide sequence of an mRNA into the amino-acid sequence of a polypeptide. For the exam, keep four directions straight: the ribosome reads mRNA 5′→3′, codon and anticodon pair antiparallel, the protein grows N-terminus→C-terminus, and new aminoacyl-tRNAs normally enter the A site. The initiator tRNA is the exception: it starts in the P site.
Original study notes for MB(ASCP) and MB(ASCPi) preparation, reconstructed from the supplied Lesson 4 guide. The diagrams and questions are original. This is not an official ASCP publication and does not reproduce examination questions.
The genetic code
A codon is a group of three nucleotides in mRNA. Four bases can make (4^3 = 64) codons: 61 sense codons specify the 20 standard amino acids, and three are stop signals.
| Property | What it means | Exam consequence |
|---|---|---|
| Triplet | Three bases form one codon | Read the mRNA in groups of three |
| Degenerate | Several codons can specify one amino acid | UUU and UUC both mean phenylalanine |
| Nearly universal | Most organisms use the same assignments | Mitochondrial and specialized exceptions exist |
| Non-overlapping | A base belongs to one codon in a reading frame | A frameshift changes every downstream triplet |
| Commaless | Codons are read continuously | The start point determines the frame |
AUG is the usual start codon. It specifies methionine; bacterial initiation uses N-formylmethionine (fMet). The stop codons are UAA, UAG, and UGA. A stop codon does not add an amino acid. A release factor recognizes it.
Example:
5′-AUG UUU GGC UAA-3′ → Met–Phe–Gly–STOP
The product contains three amino acids. UGA can be recoded as selenocysteine in the correct sequence context with specialized machinery, so “universal” and “always stop” are useful rules, not exception-free laws.
Original schematic. A four-codon message produces a three-amino-acid peptide because stop is a signal, not a residue.
Codon, anticodon, and wobble
tRNA is the adaptor between nucleic acid and protein. Its anticodon recognizes the mRNA codon. Its conserved 3′-CCA end carries the amino acid on the terminal adenosine.
Codon and anticodon pair antiparallel:
- mRNA codon:
5′-AUG-3′ - Paired anticodon:
3′-UAC-5′ - The same anticodon written conventionally 5′→3′:
5′-CAU-3′
Always inspect the printed direction before choosing an anticodon. Complementing AUG to UAC without checking direction is the common trap.
The first two codon positions usually demand standard Watson–Crick geometry. The third codon base is more flexible. This is wobble, corresponding to the first base at the 5′ end of the anticodon. Wobble lets one tRNA recognize several synonymous codons and reduces the number of distinct tRNAs a cell needs.
| 5′ base of anticodon | Possible 3′ base(s) of codon |
|---|---|
| C | G |
| A | U |
| U | A or G |
| G | C or U |
| Inosine (I) | U, C, or A |
An anticodon 5′-IGC-3′ can recognize 5′-GCU-3′, 5′-GCC-3′, and 5′-GCA-3′. The code has not changed; the pairing mechanism is flexible.
Degeneracy and wobble are not synonyms. Degeneracy is the fact that several codons encode the same amino acid. Wobble is how one anticodon can physically recognize several of those codons.
Charging the tRNA
Before a tRNA enters translation, an aminoacyl-tRNA synthetase (aaRS) attaches the correct amino acid. Charging is a separate accuracy checkpoint from ribosomal decoding.
- Amino acid + ATP → aminoacyl-AMP + PPi
- Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
ATP is converted to AMP, and PPi is hydrolyzed. That costs the equivalent of two high-energy phosphate bonds. The amino acid’s carboxyl group forms an ester bond to a hydroxyl on the terminal A of the 3′-CCA end. Its amino group remains free for peptide-bond formation.
Many synthetases have an editing site that removes an incorrectly activated amino acid or hydrolyzes a mischarged tRNA. This matters because the ribosome mainly checks codon–anticodon pairing. If the correct anticodon carries the wrong amino acid, the ribosome may accept it and put the wrong residue into the protein.
| Checkpoint | What it verifies | Error if it fails |
|---|---|---|
| Aminoacyl-tRNA synthetase | Correct amino acid on its cognate tRNA | Correct anticodon delivers the wrong amino acid |
| Ribosomal decoding center | Correct codon–anticodon geometry | Near-cognate tRNA enters the chain |
Charging prepares the adaptor. Decoding later checks the anticodon. Those are different fidelity checkpoints.
Ribosomes and the A, P, and E sites
The small subunit decodes mRNA. The large subunit contains the peptidyl-transferase center.
| Bacterial | Eukaryotic cytoplasmic | |
|---|---|---|
| Complete ribosome | 70S | 80S |
| Small subunit | 30S: 16S rRNA | 40S: 18S rRNA |
| Large subunit | 50S: 23S + 5S rRNA | 60S: 28S + 5.8S + 5S rRNA |
S means Svedberg sedimentation coefficient. It depends on size, shape, and density and is not additive: 30S + 50S makes 70S, not 80S.
The three sites are easiest as one sentence: A accepts, P carries the peptide, E exits.
- A site: incoming aminoacyl-tRNA
- P site: tRNA carrying the growing polypeptide
- E site: deacylated tRNA before departure
In bacteria, 16S rRNA belongs to the 30S decoding subunit. 23S rRNA belongs to the 50S subunit and performs the central peptidyl-transferase chemistry. The ribosome is therefore a ribozyme.
Initiation fixes the reading frame
Initiation puts the start codon in the correct position and places the special initiator tRNA directly in the P site.
Bacteria
- The 30S subunit binds initiation factors.
- The mRNA Shine–Dalgarno sequence base-pairs with 16S rRNA and positions AUG.
- IF-2–GTP delivers fMet-tRNA to the P site.
- IF-1 organizes the complex; IF-3 prevents premature 50S joining.
- The 50S subunit joins, GTP is hydrolyzed, and a 70S initiation complex remains.
Eukaryotic cytoplasm
The 40S complex is recruited to the 5′ cap through initiation factors including eIF4E in eIF4F. eIF2–GTP helps load non-formylated initiator Met-tRNA. The complex scans 5′→3′ for an AUG in a favorable Kozak context, then the 60S subunit joins to form an 80S ribosome.
| Feature | Bacteria | Eukaryotes |
|---|---|---|
| Start positioning | Shine–Dalgarno pairs with 16S rRNA | 5′ cap recruitment, scanning, Kozak sequence |
| Initiator amino acid | fMet | Met, not formylated |
| Initiator delivery | IF-2–GTP | eIF2–GTP |
| Completed ribosome | 70S | 80S |
“All tRNAs enter the A site” is false. Elongator tRNAs do. The initiator tRNA starts in P.
Elongation: delivery, bond, movement
The cycle must stay in this order:
- Delivery and decoding. Bacterial EF-Tu–GTP carries an aminoacyl-tRNA to A. Correct pairing triggers GTP hydrolysis and accommodation. EF-Ts regenerates EF-Tu–GTP.
- Peptide-bond formation. The amino group on the A-site amino acid attacks the ester-linked peptide on the P-site tRNA. The peptide transfers to the A-site tRNA. Bacterial 23S rRNA catalyzes the reaction.
- Translocation. EF-G–GTP moves the ribosome one codon. Peptidyl-tRNA moves A→P; empty tRNA moves P→E and exits. A is free again.
| Bacterial factor | Role | Eukaryotic counterpart |
|---|---|---|
| EF-Tu | Delivers aminoacyl-tRNA to A | eEF1A |
| EF-Ts | Exchanges GDP for GTP on EF-Tu | eEF1B complex |
| EF-G | Translocation | eEF2 |
Each elongation cycle uses two GTP in this simplified accounting: one for EF-Tu selection and one for EF-G translocation. This is in addition to the ATP→AMP cost of charging.
After bond formation the peptide is briefly on the A-site tRNA. EF-G then moves that tRNA into P.
Termination and recycling
Termination begins when UAA, UAG, or UGA reaches A. There is no ordinary charged tRNA for stop. A release factor enters, and water hydrolyzes the ester bond between the completed chain and the P-site tRNA.
| Factor | Recognition |
|---|---|
| Bacterial RF1 | UAA and UAG |
| Bacterial RF2 | UAA and UGA |
| Bacterial RF3 | GTPase assisting release-factor turnover |
| Eukaryotic eRF1 | All three stop codons |
| Eukaryotic eRF3 | GTPase assisting termination |
After release, bacterial RRF, EF-G, and IF-3 recycle the complex. Eukaryotes use ABCE1 and additional factors.
A premature termination codon can trigger nonsense-mediated mRNA decay (NMD). NMD lowers the amount of an mRNA that could produce a truncated protein. It is context-dependent and is an mRNA surveillance pathway, not ordinary termination.
Translation errors, variants, and laboratory evidence
A translation error affects a particular protein molecule without necessarily changing DNA or mRNA. A genetic variant changes the encoded sequence and can recur in every transcript or protein produced from that allele.
| Event | Sequence effect | Likely protein consequence |
|---|---|---|
| Missense variant | One sense codon becomes another | One amino-acid substitution |
| Nonsense variant | Sense codon becomes stop | Truncation and possibly NMD |
| Frameshift | Insertion/deletion not divisible by three | New downstream frame, often early stop |
| In-frame indel | Insertion/deletion divisible by three | Residues added or removed; frame preserved |
| Translation error | DNA may be unchanged | Wrong residue in an individual protein molecule |
One method rarely answers every clinical question:
| Method | Best question | Limitation |
|---|---|---|
| Sanger sequencing | Is a targeted DNA variant present? | Does not prove protein dysfunction |
| NGS | Which variants are present across many regions? | Coverage and interpretation limits remain |
| RT-PCR/RNA analysis | Is transcript structure or splicing altered? | Requires suitable RNA and assay design |
| qPCR | Is nucleic-acid abundance changed? | Abundance does not prove protein function |
| Western blot | Is protein abundance or apparent size altered? | Does not establish activity |
| Enzyme assay | Is function impaired? | May not identify the causal DNA change |
For a premature stop, separate three questions: confirm the DNA variant; test whether the RNA is spliced differently or degraded by NMD; then determine whether protein is absent, shortened, unstable, or inactive.
Say these without looking
- The ribosome reads mRNA 5′→3′; protein grows N→C.
- AUG starts. UAA, UAG, and UGA stop. Bacterial initiator amino acid is fMet.
- Wobble is the third codon base and first 5′ anticodon base. Degeneracy is not wobble.
- Aminoacyl-tRNA synthetase charges 3′-CCA. ATP→AMP costs two high-energy bond equivalents.
- 70S = 30S + 50S; 80S = 40S + 60S. S values are not additive.
- 16S decodes; 23S catalyzes peptide-bond formation.
- A accepts, P holds peptide, E exits. The initiator tRNA starts in P.
- Shine–Dalgarno is bacterial. Cap scanning and Kozak context are eukaryotic.
- EF-Tu delivers, EF-Ts reloads EF-Tu, EF-G translocates.
- Peptide bond first, translocation second.
- RF1 recognizes UAA/UAG; RF2 recognizes UAA/UGA; eRF1 recognizes all three.
- Missense substitutes, nonsense stops, frameshift changes the downstream frame.
Previous: Transcription and RNA processing. All lessons: MB(ASCP) study notes.
Check yourself
Ten original questions. Answer all of them before checking; the explanation appears with the key.
1. Which statement correctly describes translation directionality?
The ribosome advances along mRNA 5′→3′. Each new residue is added to the C end, so the polypeptide grows from N terminus to C terminus.
2. The mRNA codon is 5′-AUG-3′. Which anticodon is correctly written 5′→3′?
The paired anticodon is 3′-UAC-5′. Reverse it when writing 5′→3′: 5′-CAU-3′.
3. Wobble occurs between which positions?
Codon and anticodon are antiparallel. Flexibility at codon position 3 corresponds to the 5′-most anticodon base.
4. Why can a mischarged tRNA cause an incorrect amino acid to be incorporated despite correct codon pairing?
Aminoacyl-tRNA synthetase establishes amino-acid identity. The ribosome can accept a correctly pairing anticodon even when the attached amino acid is wrong.
5. Which bacterial rRNA contains the principal peptidyl-transferase catalytic activity?
23S rRNA is in the 50S large subunit and catalyzes peptide transfer. The 16S rRNA belongs to the 30S decoding subunit.
6. During normal initiation, the initiator tRNA first occupies which site?
The special initiator tRNA starts in P. Later aminoacyl-tRNAs enter through A.
7. Which bacterial factor delivers an aminoacyl-tRNA to the A site?
EF-Tu–GTP delivers and helps select the charged tRNA. EF-G translocates after peptide-bond formation.
8. Immediately after peptide-bond formation but before translocation, the growing peptide is attached to the tRNA in which site?
Peptide transfer moves the chain from P-site tRNA to A-site tRNA. EF-G then translocates that peptidyl-tRNA A→P.
9. Which bacterial release factor recognizes UAA and UGA?
RF2 recognizes UAA and UGA. RF1 recognizes UAA and UAG. Eukaryotic eRF1 recognizes all three stops.
10. A one-nucleotide deletion in a coding region most directly causes which variant class?
An indel not divisible by three shifts the downstream reading frame. A three-base deletion would be in-frame.
Sources and figure note
All three figures are original SVG study schematics created for this page. The primary lesson source is the supplied MB(ASCP) Lesson 4 — Genetic Code and Translation PDF, which identifies Lela Buckingham’s Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications, Chapter 1 (especially printed pp. 45–51 and Figures 1.38–1.41), as its main textbook basis. Eukaryotic initiation, factor equivalents, NMD, and diagnostic-method comparisons are supplementary exam context. Use the current ASCP Board of Certification content guideline to confirm the scope for your examination date.