3. Protein Synthesis (Translation)

What step follows transcription in the central dogma?

Translation (RNA → protein)

What is translation?

Process of converting mRNA sequence into a protein (amino acid chain)

What is a codon?

3-base sequence of mRNA that codes for an amino acid

Key properties of the genetic code?

• Universal

• Degenerate (multiple codons → same amino acid)

• Unambiguous (one codon → one amino acid)

What does “degenerate” mean?

Multiple codons code for the same amino acid

Are there exceptions to the genetic code?

Yes — mitochondrial DNA, some protozoa, mycoplasma

Types of mutations affecting protein sequence?

• Silent – DNA change but same amino acid → no effect on protein

  Missense – DNA change → different amino acid → may affect protein function

  Nonsense – DNA change → premature STOP codon → truncated (shortened) protein

  Frameshift – insertion/deletion shifts reading frame → completely altered downstream protein (usually severe)

What causes Huntington’s disease?

autosomal dominant Trinucleotide repeat expansion → abnormal protein

Function of tRNA?

Brings amino acids to ribosome

What part of tRNA recognizes mRNA?

Anticodon

What is aminoacyl-tRNA formation and which enzyme is involved?

Aminoacyl-tRNA synthetase attaches the correct amino acid to its corresponding tRNA → forms a “charged” tRNA (aminoacyl-tRNA)

How does aminoacyl-tRNA synthetase ensure accuracy?

• Recognizes specific tRNA + amino acid

• Matches based on anticodon

• Proofreads → removes incorrect amino acids

What is special about tRNAᵐᵉᵗ?

tRNAᵐᵉᵗ carries methionine → forms methionyl-tRNAᵐᵉᵗ (initiator tRNA in translation)

Key features of 5’ UTR? Leader sequence

• Regulates translation initiation in both proks and euks

• Contains ribosome binding sequences

Shine-Dalgarno sequence (prokaryotes)? 5’ UTR

Ribosome binding site upstream of start codon

Kozak sequence (eukaryotes)? 5’ UTR

Sequence surrounding AUG start codon

Function of 3’ UTR? Trailer sequence

• Controls mRNA stability and translational efficiency in proks and euks

• Contains poly(A) signal in euks

• termination signals in proks

Poly-A signal/function?

AAUAAA → poly-A tail → stability/translation efficiency.

What causes Fragile X syndrome (triplet expansion)?

CGG repeat expansion in the 5’ UTR

• Normal: 6–50 repeats

• Gray zone: 50–54 repeats

What causes Myotonic Dystrophy (triplet expansion)?

CUG repeat expansion in the 3’ UTR of the DMPK gene

• Normal: 5–35 repeats

• Affects skeletal muscle (DMPK protein function)

Steps of translation?

Initiation → elongation → termination.

Start & stop codons?

Start: AUG; Stop: UAA, UAG, UGA.

Directionality of translation?

mRNA read 5′→3′; protein made N→C.

Ribosome structure differences?

Prok: 70S (30S+50S); Euk: 80S (40S+60S).

Ribosome sites?

A (arrival), P (peptide), E (exit).

What happens in each ribosome site?

A = tRNA entry; P = peptide bond; E = exit.

What happens during prokaryotic translation initiation?

• 30S binds mRNA via Shine-Dalgarno

• Initiator tRNA placed in P site

• 50S joins to form complete ribosome

Role of IF-1, IF-2, IF-3 in prok initiation?

• IF-1 blocks A site

• IF-2 brings initiator tRNA (GTP)

• IF-3 prevents premature subunit binding

What happens during prokaryotic elongation?

• charged tRNA enters A site via EF-Tu

• Peptide bond formed by peptidyl transferase

• Ribosome translocates via EF-G (specific to prok) and GTP

What is translocation?

• Ribosome moves one codon forward

• tRNA shifts A → P → E

• Requires GTP

Role of EF-G?

Drives translocation using GTP.

What happens during prokaryotic translation termination?

• Ribosome reaches stop codon

• Release factors bind

• Peptidyl transferase releases polypeptide

What happens after polypeptide release in prokaryotes?

• Ribosome recycling factor + EF-G remove 50S subunit

• 30S subunit, mRNA, and tRNA dissociate

What happens during eukaryotic initiation?

• 40S binds Met-tRNA + eIFs

• mRNA cap recognized and scanned for AUG

• 60S joins after GTP hydrolysis

How does eukaryotic initiation begin?

• 40S subunit + eIF3 binds; Met-tRNA + eIF2 joins.

How does mRNA bind in eukaryotes?

5’ cap binds eIF4F → complex scans for AUG.

What completes eukaryotic initiation?

GTP hydrolysis → factors released → 60S joins.

What happens in eukaryotic elongation?

eEF1A delivers tRNA → peptide bond → ribosome translocates.

What catalyzes peptide bond formation?

Peptidyl transferase activity of rRNA (28S in eukaryotes).

What happens during eukaryotic termination?

Stop codon → release factors → bond hydrolyzed → protein released. and ribosome dissociate

Why can’t tRNA bind stop codons?

No anticodon exists for stop codons.

Key difference in translation location?

Prokaryotes: cytoplasm; Eukaryotes: cytoplasm (after nuclear transcription).

What are protein synthesis inhibitors (general)?

Compounds that block translation by targeting ribosomal function.

How do some inhibitors work?

Mimic 3’ end of aa tRNA or bind 50s subunit ribosome → block translocation.

What is diphtheria toxin’s mechanism?

ADP-ribosylates EF-2 → inhibits elongation in eukaryotes.

What are polysomes?

Multiple ribosomes translating one mRNA simultaneously.

Why are polysomes important?

Increase efficiency and rate of protein synthesis.

What inhibitor mimics the 3′ end of aminoacyl-tRNA?

Puromycin and it enters the A site. It causes premature termination of translation by releasing the growing polypeptide.

What does clindamycin do in translation?

binds the 50S ribosomal subunit and inhibits translocation

What does chloramphenicol do?

Inhibits peptidyl transferase (50S). Blocks peptide bond formation.

What does erythromycin do?

Blocks translocation (50S). Stops ribosome movement.

What do tetracyclines do?

Block tRNA entry to A site (30S). Stops elongation.

What does streptomycin do?

Causes mRNA misreading (30S). Produces faulty proteins.