VL 26 (Isabel Bäurle)

Question 1

Ribosome structure

Answer 1

• large + small SU
  –> small: mRNA interaction (35 bp)
  –> large: aminoacyl-tRNA interaction
  many interactions at SU interface
• rRNA provides “backbone” that touches most r-proteins
• organelles with different ribosomes

The Ribosome has 3 tRNA binding sites:
1. A site: aminoacyl-tRNA enters
2. P site: peptidyl-tRNA is bound
3. E site: deacylated tRNA

• The ribosome has two sites for binding charged tRNA
• An amino acid is added to the polypeptide chain by
  transferring the polypeptide from peptidyl-tRNA in the P site to aminoacyl-tRNA in the A site (translocation).

Question 2

Translation stages

Answer 2

initiation:
translation stages up to synthesis of first polypeptide bond; rate-limiting step
elongation:
translation stages where polypeptide chain is extended by addition of individual SU
termination:
separate reaction that ends translation by stopping SU-addition
→ disassembly of synthetic apparatus

• Trasnlation occurs by Initiation, Elongation, and Termination
• Initiation is usually the rate-limiting step of translation

* https://www.youtube.com/watch?v=5bLEDd-PSTQ

Question 3

Initiation in Bacteria Needs 30S
Subunits and Accessory Factors

Answer 3

Initiation involves base pairing between mRNA – rRNA:
* initiation site on bacterial mRNA: AUG initiation codon preceded by Shine-Dalgarno polypurine hexamer (AGGAGG; ~ 10 bp upstream; bacterial mRNA)
* 16S rRNA of 30S bacterial ribosomal SU with complementary Shine-Dalgarno sequence (3 ́)
→base pairing

Translation initiation in bacteria needs 30S SU and accessory factors:
* ribosome-binding site
–> sequence on bacterial mRNA
–> includes initiation codon bound by 30S SU in initiation phase in initiation phase of polypeptide translation
* requires separate 30S, 50S ribosome SU + initiation factors (IF-1/2/3), which bind 30S SU
* 30S SU carrying initiation factors binds to mRNA initiation site
→initiation complex
* IF-3 released
→50S SU joins 30S-mRNA complex

Question 4

A Special Initiator tRNA Starts the Polypeptide Chain

Answer 4

• tRNAmMet – The bacterial tRNA that inserts
  methionine at internal AUG codons.
• Met (AUG) = first aa
• Different Met-tRNAs involved in initiation + elongation
• Bacteria, Organelles: N-formyl-methionyl-tRNA (tRNAfMet): aminoacyl-tRNA; initiates bacterial translation; Met amino-group = formylated

Question 5

Use of fMet-tRNAf controlled by IF-2 and ribosome:

Answer 5

IF-2 binds initiator fMet-tRNAf → allows it to enter partial P site on 30S SU

Question 6

Elongation Factor Tu Loads
Aminoacyl-tRNA into the A Site

Answer 6

• EF-Tu (an elongation factor): monomeric G protein; active form (GTP-bound) binds to aminoacyl-tRNA
• EF-Tu-GTP-aminoacyl-tRNA complex binds A site

Polypeptide chain is transferred to aminoacyl-tRNA:
* 50S SU has peptidyl transferase activity, as provided by rRNA ribozyme
* nascent polypeptide chain transferred from peptidyl-tRNA in P site to aminoacyl-tRNA in A site
* peptide bond synthesis generates deacylated tRNA in P site, peptidyl-tRNA in A site

Question 6

Translocation Moves the Ribosome

Answer 6

• translocation moves mRNA through ribosome by 3 nucleotides
• translocation moves deacylated tRNA into E site, peptidyl-tRNA into P site
  → empties A site
• hybrid state model:
  translocation in 2 stages – 50S moves relative to 30S → 30S moves along mRNA
  → restore original conformation
• RFs bind ribosome A site
• Molecular mimicry: structurally analogous
  but sequence-wise different (RF/RFF/EF)

Question 7

Termination codons are recognized by protein release factors:

Answer 7

Termination codons recognized by protein release factors (RF), not by aminoacyl-tRNAs

RF1: recognizes UAA, UAG
→terminate polypeptide translation

RF2: recognizes UAA, UGA
→terminate polypeptide translation

–> Ribosome recycling factor (RRF)

Question 8

The Cycle of Bacterial Messenger RNA

Answer 8

• transcription, translation occur simultaneously in bacteria (called coupled transcription/translation) as ribosomes begin translating before completed synthesis
• unstable; half-life: ~2 min
• transcription: 40-50 nuc/s
• translation: 15 aa/s
• nascent RNA: ribonucleotide chain; still being synthesized→3 ́ end paired with DNA, where RNA Pol is elongating
• monocistronic mRNA:
  encodes one protein
• polycistronic mRNA:
  several coding regions = different cistrons; encodes >= 2 proteins

https://www.youtube.com/watch?v=qIwrhUrvX-k

Question 9

Codon–Anticodon Recognition Involves Wobbling

Answer 9

Multiple codons that encode the same amino acid most often differ at the third base position
(the wobble hypothesis)