CH18 - Protein Synthesis

Overview of the Translation Cycle

Translation = protein synthesis; takes place on ribosomes and proceeds in three major phases
- Initiation → Elongation → Termination (plus ribosome recycling)
Directionality
- mRNA is decoded $5' \rightarrow 3'$
- Polypeptide grows $N\text{-terminus} \rightarrow C\text{-terminus}$
Multiple ribosomes can translate a single mRNA simultaneously → formation of polysomes / polyribosomes (efficient amplification of protein output)

Ribosome: Structure, Composition & Catalytic Activity

Macromolecular ribonucleoprotein complex; two unequal sub-units that associate only during translation

Kingdom	Complete ribosome	Large subunit	Small subunit
Bacteria	$70\,S$	$50\,S$	$30\,S$
Eukaryotes	$80\,S$	$60\,S$	$40\,S$

(Sedimentation coefficients in Svedberg units, $S$ )

rRNA & protein inventory (representative numbers)
- Bacterial 50 S: 33 proteins (L1–L36), rRNAs $5S$ & $23S$
- Bacterial 30 S: 21 proteins (S1–S21), rRNA $16S$
- Eukaryotic 60 S: ≈47 proteins, rRNAs $28S, 5.8S, 5S$
- Eukaryotic 40 S: ≈32 proteins, rRNA $18S$
Functional sites created when sub-units join
- mRNA channel (narrow cleft → only single-stranded RNA; secondary structures removed during passage)
- tRNA binding pockets: A (aminoacyl), P (peptidyl), E (exit)
- All tRNAs except the initiator first bind A → P → E
- Initiator tRNA starts directly in the P site
Ribozyme nature
- $23S$ rRNA (prokaryotes) / $28S$ rRNA (eukaryotes) provides peptidyl-transferase center
- Harry Noller’s experiments
- Point mutations in rRNA abolished peptide-bond formation (no translation)
- Protease/SDS/phenol removal of ribosomal proteins left rRNA intact → peptidyl transfer still occurred
- Concluded RNA alone catalyzes peptide-bond formation (ribozymic activity)
- Peptidyl transfer does not require additional NTP hydrolysis; energy stored in aminoacyl-tRNA ester bond powers reaction

Activation of Amino Acids (tRNA Charging)

Enzymes: 20 distinct Aminoacyl-tRNA synthetases (aaRS) – one per amino acid
Two-step reaction
1. Adenylylation $\text{AA} + \text{ATP} \rightarrow \text{AA{-}AMP} + PP_i$ (carboxyl of AA linked to $\alpha$ -phosphate)
2. tRNA charging $\text{AA{-}AMP} + tRNA \rightarrow \text{AA{-}tRNA} + AMP$
Classes of aaRS
- Class I → initially attaches AA to 2'-OH of terminal A on CCA tail
- Class II → attaches to 3'-OH
- Spontaneous trans-esterification ensures final $AA$ resides on 3'-OH regardless of class
“Second genetic code”
- Specific recognition elements spread across tRNA: anticodon loop and acceptor stem
- Mutagenesis pinpointed crucial nucleotides for identity
Proof-reading / editing (high fidelity ~ $10^{-4}$ error rate)
- Particularly important for structurally similar AAs (Val vs Ile)
- Example: Ile-RS possesses
- Acylation (synthetic) site – fits Ile or (erroneously) Val
- Editing site – smaller pocket; accommodates Val, hydrolyzes mis-charged Val-tRNA $^{Ile}$ ; Ile too bulky to enter → retained
Energetic cost: $1\,ATP \approx 2\,\text{high-energy bonds}$ expended per AA during charging (+ possible extra ATP for editing)

Initiation of Translation

Goal: place start codon (AUG) in P site, position initiator tRNA, then recruit large sub-unit.

Prokaryotes (Bacteria)

mRNA positioning: Shine–Dalgarno (SD) sequence (AGGAGGU consensus) ~8–14 nt upstream of AUG base-pairs with $16S$ rRNA → aligns AUG in P site
Initiator tRNA: $fMet{-}tRNA^{fMet}$ (Met later formylated by Met-tRNA formyl-transferase)
Initiation factors
- IF1 – blocks A site on 30 S
- IF3 – binds E site; prevents premature 50 S association
- IF2-GTP – escorts $fMet{-}tRNA^{fMet}$ to P site
Sequence of events
1. IF1 + IF3 bind free 30 S
2. mRNA binds via SD/ $16S$ pairing ⇒ AUG positioned
3. IF2-GTP delivers $fMet{-}tRNA^{fMet}$ to P site → correct codon–anticodon triggers
4. IF1 & IF3 depart; 50 S joins
5. GTP on IF2 hydrolyzed → IF2-GDP released ⇒ 70 S initiation complex ready for elongation

Eukaryotes

More factors (≥12) & both ATP + GTP hydrolyses
Key eIFs
- eIF1 – blocks A site (A-site inhibitor)
- eIF1A – binds E site (homologous to bacterial IF1?)
- eIF3 – binds 40 S; blocks 60 S joining
- eIF2-GTP – binds initiator $Met{-}tRNA^{iMet}$ (distinct from elongator Met-tRNA)
- eIF4F complex (eIF4E cap-binding protein + eIF4G scaffold + eIF4A RNA helicase) – recognizes 5' cap
Steps
1. 40 S•eIF1•eIF1A•eIF3 assembles
2. eIF2-GTP•Met-tRNA $^{iMet}$ joins → 43 S pre-initiation complex
3. eIF4F binds 5' cap & poly(A)-binding protein (PABP) → circularizes mRNA; recruits 43 S
4. 40 S scans 5'→3' using ATP helicase activity until AUG in Kozak consensus (e.g. GCCRCCAUGG)
5. Correct base-pairing triggers eIF factor release (including eIF3)
6. 60 S joins; eIF5B-GTP aids joining; GTP hydrolysis finalizes 80 S initiation complex; eIF4F remains at cap (cycling polysomes)
Cap-independent initiation
- IRES (Internal Ribosome Entry Site) – complex RNA element upstream of AUG; recruits 40 S directly (± eIF4F)
- La protein binds pyrimidine-rich leaders and delivers them to 40 S

Elongation

Three repeating sub-steps per codon:

Aminoacyl-tRNA delivery
- Bacteria: EF-Tu–GTP binds AA-tRNA → delivers to A site
  - Correct codon/anticodon ⇒ GTP hydrolysis; EF-Tu-GDP released
- EF-Ts = nucleotide-exchange factor (GDP→GTP regeneration)
- Conformational “accommodation” rotates tRNA, placing AA into peptidyl-transferase center
Peptide-bond formation (rRNA catalysis)
- $\alpha\text{-NH}_3^+$ of A-site AA attacks ester bond linking P-site peptide to tRNA ⇒ peptide transferred to A-site tRNA
- Growing chain now located in A site; empty tRNA in P (soon E) site
Translocation
- EF-G–GTP (prokaryotes) / eEF-2–GTP (eukaryotes) binds inter-subunit space; GTP hydrolysis shifts ribosome one codon 3'ward
- Post-shift: peptidyl-tRNA in P site; de-acylated tRNA in E site (then exits); A site open

Energy budget per residue (minimum)
\begin{cases}
\text{tRNA charging} & 2\,\text{~ATP equivalents}\
EF\text{-Tu/eEF1}\alpha & 1\,GTP\
EF\text{-G/eEF2} & 1\,GTP
\end{cases}\;\Rightarrow\; 4\,\text{NTPs / amino acid}
(+1 extra ATP if editing occurs)

Termination & Ribosome Recycling

Stop codons: $UAA,\; UAG,\; UGA$ – no cognate tRNAs

Bacterial Release Factors

RF1 recognizes $UAA$ & $UAG$
RF2 recognizes $UAA$ & $UGA$
RF3-GDP binds RF1/2; GTP exchange & hydrolysis promote factor release after peptide liberation
Reaction: RF1/2 induces peptidyl-transferase to hydrolyze ester bond → polypeptide released

Ribosome Recycling

RRF (Ribosome Recycling Factor) binds vacant A site
RRF + EF-G–GTP trigger dissociation of 50 S & 30 S + release of de-acylated tRNAs
IF3 re-binds 30 S to prevent premature sub-unit re-association until a new initiation event

Quality-Control Pathways Coupled to Translation

1. tmRNA / Trans-translation (Bacteria & organelles)

tmRNA (SsrA) hybrid molecule
- 5' end folds like tRNA $^{Ala}$ → charged with Ala by Ala-RS
- 3' end encodes short ORF ending with stop codon
Mechanism for mRNAs lacking stop codon
1. EF-Tu–GTP delivers Ala-tmRNA to stalled A site
2. Ala incorporated; ribosome shifts to tmRNA ORF (acts as surrogate mRNA)
3. Adds ~10 AA tag ending with stop codon
4. Release factors terminate; tagged peptide recognized & degraded; tmRNA promotes decay of defective mRNA

2. Non-stop mRNA Decay (Eukaryotes)

Ribosome reaches 3' end without stop codon → stalls on poly(A)
Ski7 binds stalled ribosome; recruits exosome 3'→5' exonuclease → mRNA degradation
Translation of poly(A) yields poly-Lys tail that targets aberrant protein for proteolysis

3. Nonsense-Mediated mRNA Decay (NMD)

Premature stop codon upstream of exon-junction complexes (EJCs)
During pioneer round, residual EJC downstream of stop recruits UPF1/2 → decapping enzyme → exonucleases degrade transcript

Additional Key Concepts & Connections

Peptide synthesis polarity (N→C) mirrors transcription polarity (5'→3') but opposite to mRNA reading frame (codon read 5'→3')
Polysomes exist in both bacteria & eukaryotes; electron micrographs can distinguish kingdom by concurrent transcription–translation coupling (bacteria) vs nuclear separation (eukaryotes)
Energetic cost underlines importance of tight regulation; defective products are swiftly removed (quality-control pathways) to conserve resources and protect cell
Clinical/biotech relevance
- Antibiotics (e.g. tetracycline, chloramphenicol, aminoglycosides) exploit structural differences in prokaryotic ribosomes or elongation factors
- IRES elements crucial for viral translation; potential therapeutic targets
- tmRNA tags (SsrA tag) widely used in synthetic biology as built-in degradation signals