Translation 5- Termination and Antibiotics

Question 1

What are the release factors involved in termination

Answer 1

1. RF1 recognizes UAG and UAA- Stop codons
2. RF2 recognizes UGA and UAA- Stop codons
3. RF3 (GTP), stimulates the binding of RF1 and RF2.
4. RRF- together with EF-G induces ribosomal dissociation to small and large subunits

Question 2

What is eukaryotic release factor

Answer 2

1. eRF recognises all 3 termination codons
2. Requires GTP to bind
3. Hydrolysis probably promotes dissociation of eRF

Question 3

What is needed for RF to act

Answer 3

1. Need peptidyl tRNA in P site and termination codon in A site for RFs to act
2. Signal for release factors to come in
3. Stop codon needs to arrive in A site and no tRNA which recognises stop codon- open for recognition by release factors which can recognise stop codons
4. Molecular mimicry

Question 4

What is the first step of prokaryotic termination

Answer 4

1. The termination codons are recognised by class I release factors- RF1 and RF2
2. Swapping a conserved PXT tripeptide in RF-1 with a conserved SPF tripeptide in RF-2 interchanges their stop codon specificities - suggests the mimic anticodons

Question 5

What is the second step of prokaryotic termination

Answer 5

1. On binding to their corresponding stop codon, RF1 and RF2 induce the transfer of the peptidyl group from tRNA to water rather than to an aminoacyl-tRNA - releasing the completed polypeptide
2. Act at site A
3. Compete with suppressor tRNAs for termination codons
4. cannot bind to the ribosome simultaneously with EF-G

Question 6

What is the third step of termination

Answer 6

1. Once newly synthesised polypeptide has been released from the ribosome, RF3 in complex with GDP binds to the ribosome at the same site as do EF-Tu and EF-G
2. On binding it exchanges GDP for GTP
3. Resulting conformational change of RF-3 causes it to bind more tightly to the ribosome and expel the RF-1/2

Question 7

What is step foUr of termination

Answer 7

1. Interaction of RF-3 and GTP with the ribosome stimulates it to hydrolyse its bound GTP as occurs with EF-Tu.GTP and EFG.GTP
2. Resulting RF-3.GDP then dissociates from the ribosome
3. RRF then binds in a ribosomal A site followed by EF-G

Question 8

What is step 5 of termination

Answer 8

1. EF-G hydrolyses its bound GTP which causes RRF to be translocated to the P site and the tRNAs previously in the P and E sites to be released
2. The large and small ribosomal subunits separate, process is facilitated by the binding of IF-3 and RRF
3. EF-G.GDP and mRNA are released
4. Ribosomal subunits can then participate in a new round of initiation

Question 9

Give example of molecular mimicry with EF-Tu

Answer 9

1. EF-Tu and charged tRNA
2. Makes ribosome think tRNA is being delivered but is not- frees up ribosome

Question 10

Describe mimicry of RF2 and RRF

Answer 10

1. Mimic tRNA in shape and function so can bind
2. RRF has a few amino acids at bottom which mimic anticodon of tRNA- Functional mimicry

Question 11

Desribe how eRF2 mimics

Answer 11

1. Mimics tRNA
2. Participates in the hydrolysis of the ester bond between the peptide and the tRNA in the neighbouring peptidyl-tRNA in the P site of the ribosome

Question 12

How do RF1 and RF2 mimic

Answer 12

1. A tripeptide recognises the stop codon
2. RF1 and RF2 have 3 amino acids which bind to the stop codon

Question 13

What effect do antibiotics have on protein synthesis

Answer 13

1. Antibiotics are bacterially, fungally or synthetically produced substances that inhibit growth of microorganisms
2. The majority of known antibiotics block translation- probs cus so complex

Question 14

What are natural protein synthesis inhibitors

Answer 14

1. E. cold- cold shock factor
2. Blocks A and P sites
3. Protein production v costly
4. Natural factors inhibit protein synthesis to conserve energy

Question 15

What are ribosome modulating factor and hibernation modulating factors

Answer 15

1. Eubacteria inactivate their ribosomes as 100S dimers or 70S monomers upon entry into stationary phase.
2. In Escherichia coli, 100S dimer formation is mediated by ribosome modulation factor (RMF) and hibernation promoting factor (HPF), or alternatively, the YfiA protein inactivates ribosomes as 70S monomers.
3. The binding site of RMF overlaps with that of the messenger RNA (mRNA) Shine-Dalgarno sequence, which prevents the interaction between the mRNA and the 16S ribosomal RNA.
4. The nearly identical binding sites of HPF and YfiA overlap with those of the mRNA, transfer RNA, and initiation factors, which prevents translation initiation.
5. The binding of RMF and HPF, but not YfiA, to the ribosome induces a conformational change of the 30S head domain that promotes 100S dimer formation.

Question 16

What does RMF do

Answer 16

1. RMF - The binding site of RMF overlaps with that of mRNA Shine-Dalgarno sequence, which prevents the interaction between the mRNA and the 16S ribosomal RNA

Question 17

What do HPF-YfiA

Answer 17

1. HPF-YfiA- The binding sites of HPF and YfiA overlap with those of the mRNA, transfer RNA, and initiation factors, which prevents translation initiation

Question 18

What are 6 antibiotics that inhibit translation

Answer 18

1. Edeine- hinders mRNA progression
2. Tetracyclin - prevents tRNA binding in A-site
3. Puromycin- mimics tRNA in A-site
4. Clindamycin - obstructs peptide bond formation
5. Erythromycin - blocks protein exit channel
6. Troleandomycin - barricades tunnel passage

Question 19

What does edeine do

Answer 19

1. pentapeptide‐amide antibiotic elaborated by a strain of Bacillus brevis and effective against Gram‐positive and Gram‐negative bacteria, some fungi and other eukaryotic cells
2. inhibits the initiation of protein synthesis by linking critical features for tRNA, IF3 and mRNA binding, thus imposing constraints on ribosomal mobility that accompany the translation process

Question 20

What does tetracycline do

Answer 20

1. binding the 30S ribosomal subunit, and, through an interaction with 16S rRNA, they prevent the docking of amino-acylated tRNA.
2. Doesn’t block messenger but blocks trnase
3. Blcoks after EF-Tu catalysed GTP hydrolysis has occurred, resulting in the release of tRNA
4. Also causes the the unproductive hydrolysis of GTP- drains cell of energy

Question 21

What does puromycin do

Answer 21

1. Puromycin terminates translation by mimicking a tRNA in the A site – mimics amino acid – functional over shape mimicry
2. Puromycin binds in the A site and participates in peptide bond formation
3. Once completed, puromycin and any associated polypeptide diffuses out of the ribosome
4. Leaves the ribosome arrested in elongation (immobilised)- blocks elongation

Question 22

What does clindamycin do

Answer 22

1. Obstructs peptide bond formation
2. Clindamycin interacts with 23 S rRNA through an extensive hydrogen bond network – stops catalysis by ribosome
3. Extraction of proton by amino group is being blocked

Question 23

What does Azythromycin do

Answer 23

1. Does same as erythromycin
2. Has different molecular structure

Question 24

What does Troleandomycin do

Answer 24

1. Doesn’t block exit channel itself
2. Sits close to exit channel and binds to protein that lines the exit channel and changes the conformation so protein blocks exit channel

Question 25

What does Paromomycin do

Answer 25

1. not protein synthesis inhibitor
   Induces errors in sequence
2. Not blocker just increases error rate
3. Binds to region and flips out bases- tRNA cant tell where to bind so binds to wrong place

Question 26

What are other translation inhibitors

Answer 26

1. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis.