L10 - Prokaryotic Translation

Question 1

Activation of an AA to form amino acyl tRNA:

Answer 1

tRNA takes amino acid that is appropriate for its anticodon sequence.

AA + tRNA + ATP → aminoacyl-tRNA + PPi + AMP AMINOACYL-tRNA SYNTHETASE

ATP hydrolysed to provide energy. AA specific to enzyme. 20 different types of tRNA classed by their amino acid e.g. tRNA^Phe. If charged they are called activated amino acids and named as Phe-tRNA^Phe.

Question 2

Initiation:

Answer 2

1. 30S subunit binds IF1 and IF3
2. Binds mRNA to small subunit
3. First codon is met. (fmet for prokaryotes). fMet-tRNA^fmet binds to GTP-IF2 as it enters P-site.
4. fMet-tRNA^fmet binds to AUG.
5. Large subunit comes and binds to small subunit and uses hydrolysis of GTP to GDP to release energy.
6. First tRNA sits in P site.

Has shine-Dalgarno sequence upstream of initiation codon. Shows ribosome which met to start on and identifies reading frame. Sequence binds to 16s rRNA. Prokaryotes have many sequences as it codes for different proteins at same time by having different reading frames. In eukaryotes, they only have 1 sequence.

Question 3

Elongation:

Answer 3

1. fMet in P site.
2. 2nd codon in A site. tRNA with correct AA goes to A site (bound to EF-Tu and coupled with GTP).
3. 2 tRNA in ribosome where each have AA
4. Proofreading - correct tRNA bound to correct codon which require hydrolysis of GTP.
5. EF leaves (EF-Tu GDP).
6. fMet leaves tRNA and assembled on top of AA₂ (next AA) using peptidyl transferase.
7. One tRNA has 2 AA on top and one empty tRNA. Confirmation shift occurs.
8. Ribosome moves (translocates) one nucleotide so empty tRNA is in E site and dipeptide on P site and A site empty. EF-G-GTP → EF-G-GDP + P_i
9. Empty tRNA on E site is removed.
10. Deliver of AA₃ starts next round of peptide bond synthesis.

Question 4

Termination:

Answer 4

1. Stop codon on mRNA presented to A site
2. Release factor (RF1 or RF2) binds (RF2 binds to A site).
3. Hydrolysis of protein from tRNA (binding recognised by ribosome so termination occurs. Releases polypeptide).
4. Ribosome complex disassembles.
5. Requires IF3 and ribosomal recycling factor and GTP hydrolysis.

Transcription has a structural element telling it to stop (hairpin loop). Translation has a sequence element (stop codons UAG, UAA, UGA).

Question 5

Eukaryotes vs. Prokaryotes:

Answer 5

Prokaryotes have different anchor sequence (polycistronic). Same mRNA codes for different proteins.

Eukaryotes are monocistronic so one mRNA makes one protein. It doesn’t rely on anchor sequence but rely on cap. Always has 5’ cap to show where translation starts.

Question 6

Answer 6