TC - Translation (Elongation) Flashcards
Describe the directionality of mRNA decoding and protein synthesis
- mRNA is decoded from the 5’- to 3’-end
- Proteins are synthesized starting from their amino-terminus to their carboxyl-terminus
What are ribosomes, and where does protein synthesis occur?
Ribosomes are complex ribonucleoprotein particles that bind mRNA and tRNA, containing rRNA and proteins
- Protein synthesis occurs in ribosomes
Key features of the bacterial (E. coli) ribosomal subunits (6)
30S subunit – 16S rRNA + proteins S1- S19
- Contains the “decoding centre” (DC)
- Helix 44 of 16S rRNA forms the A and P tRNA binding sites
- 3’ of 16S rRNA complements the Shine-Dalgarno
50S subunit - 23S and 5S rRNAs + proteins L1– L31
- 23S rRNA forms 6 domains (I-VI)
- Contains the peptidyl transferase centre (PTC)
- Contains the peptide exit tunnel
How does the ribosome catalyze peptide bond formation?
The ribosome catalyzes the reaction:
- (aa)n-tRNA + aa-tRNA’ → (aa)n+1-tRNA’ + tRNA
What evidence suggests that the ribosome is a ribozyme? (2)
1) No ribosomal proteins are located in the peptidyl-transferase center (PTC)
- Nearest ones are 15-18Å away – too far to take part
2) Ribosomes depleted of protein still exhibit peptidyl-transferase activity
How do ribosomes effectively work? (2)
- Ribosome helps to position substrates and water molecules which aids proton transfer and stabilisation of intermediates
- Ribosome does not work by chemical catalysis, but instead decreases activation energy needed for peptide bond formation
What occurs in Elongation with reference to elongation factors (EFs/eEFs) ? (3)
- Ensures correct amino acid is added sequentially to the growing protein chain by base pairing of transfer RNA (tRNA) with mRNA
- Decodes 10 – 40 aa per sec with only 1 in 10,000 error rate
- Already seen that accuracy of charging tRNAs with the correct amino acid is key to this, but elongation factors do check fidelity of codon:anticodon pairings
Process of Elongation - Prokaryotes (6)
1) The next aminoacyl-tRNA binds to A-site aided by elongation factor EFTuGTP
2) If codon:anticodon is correct, EFTuGTP is hydrolysed to EFTuGDP
3) EFTuGDP is then released and recycled to EFTuGTP by EFTs
- EFTs is a guanine nucleotide-exchange factor (GEF)
- EFTuGDP + GTP –> EFTuGTP + GDP
4) Peptide bond -CO-NH- formation occurs in the PTC
5) “Translocation” then occurs:
- peptidyl-tRNA moves to the P site
- The spent tRNA moves to the E-site and exits the ribosome
- This requires EFGGTP which is hydrolysed to EFGGDP
6) The next aminoacyl-tRNA binds at A-site; cycle is repeated
Costs 2x GTP molecules
Why is tRNA always base-paired with mRNA? (3)
- Prevents frameshifting
- Ensures accuracy
- Shows why code is read triplet by triplet
How do nascent (new) proteins exit? (3)
- Nascent chain passes through the ribosome exit tunnel
- Protects new chain from inappropriate interactions
- Allows it to sample multiple conformations
How do proteins fold during biosynthesis?
The N-terminal portion will start to fold before the C- terminal region is completed
What are similarities in the components of prokaryotic and eukaryotic elongation? (4)
- eEF1A equivalent to EFTu
- eEF1B = EFTs (GEF for recycling eEF1A)
- eEF2 = EFG
Phosphorylation of eEF2 (in response to rise in AMP, rise in Ca2+) reduces elongation rate
How do antibiotics affect bacterial ribosomes? (3)
Most antibiotics bind specifically to bacterial ribosomes because of small structural differences
- Chloramphenicol - blocks peptidyl transferase
- Erythromycin - blocks elongation by binding to the 23S rRNA tunnel
- Tetracycline - prevents amino acyl tRNA binding
Action of aminoglycoside antibiotics
Aminoglycoside antibiotics paromomycin and streptomycin bind to decoding centre and induce errors in translation
- In the presence of paromomycin, even ‘near-cognate’ AA-tRNAs can induce this conformational change, so that mistakes occur more easily
