The cell’s armoury of exonucleases

Question 1

What are the steps of translation termination? (4)

Answer 1

• Translation termination factors recognise the stop codon, not tRNAs
• Binding of release factor (RF1/RF2 prokaryotes, eRF1 in eukaryotes) triggers peptide hydrolysis to release peptide
• RF3 (eRF3) GTPase binds, GTP to GDP hydrolysis which drives release of RF1/RF2 from the ribosome
• Set of recycling factors including EF-G dissociate the ribosomal subunits and mRNA after termination

Question 2

What are translational road-blocks? (3)

Answer 2

• Elongating ribosomes can stall due to secondary structures, mRNA damage or local sequence
• In early stalling there is no termination codon to facilitate ribosome recycling
• Pathways required to degrade truncated protein product and the faulty mRNA transcript

Question 3

What is trans-translation? (5)

Answer 3

• System to recover ribosomes IN BACTERIA that have stopped translating when they get to the end of a truncated mRNA so accumulate without being recycled
• tmRNA binds to the vacant A site and functions initially as an Ala-tRNA so forms a peptide bond with the peptide
• Then the ribosome translates a short ORF within the tmRNA
• Adds a particular sequence to the end of the truncated peptide (C-terminal tag) which targets it for degradation by tail-specific protease (Tsp)
• The truncated mRNA is degraded by the 3’ exoribonuclease RNase R

Question 4

What are the comparable quality control pathways to trans-translation in eukaryotes? (2)

Answer 4

• No-go decay (NGD)
• Nonstop decay (NSD)

Question 5

What is NGD? (2)

Answer 5

• No-go decay
• Translation-coupled mRNA degradation due to stalled elongating ribosomes

Question 6

What is NSD? (2)

Answer 6

• Nonstop decay
• Translation-coupled mRNA degradation due to truncated mRNAs with no stop codon

Question 7

What is the result of NGD/NSD? (2)

Answer 7

• Stalled mRNAs are cleaved endonucleolytically on the ribosome which produces 5’ phosphate and 3’ hydroxyl which are the substrates for exonucleases, then degradation
• Protein product is degraded by ribosome-associated quality control (RQC) pathways

Question 8

How does the NSD pathway work? (3)

Answer 8

• Dependent on the exosome and Ski complex
• C-terminal domain of Ski7 has homology with translation factors that bind to the empty ribosomal A site so proposed to bind to the stalled ribosome
• Ski2 helicase binds to the small ribosomal subunit at the mRNA exit site so grabs hold of the mRNA as it leaves the ribosome and feeds it into the exosome for degradation

Question 9

How does translation speed trigger mRNA and protein degradation?

Answer 9

Cell doesn’t directly notice slow ribosomes but it detects ribosome collision events

Question 10

What is a disome?

Answer 10

Formed by collision of 2 ribosomes

Question 11

What happens in the event of a ribosome collision? (5)

Answer 11

• Disome interface is largely on the small subunit, the mRNA between them gets pulled tighter and tighter
• RQC is dependent on Hel2 which ubiquitinates S3 and S10 ribosomal proteins
• RACK1 kinase and Slh1 (Ski2-like helicase) are recruited
• Rqc2 adds “CAT tails” (uses tRNAs but not codon-dependent) and E3 ligase Ltn1 ubiquitinates the polypeptide
• Cue2 endonuclease cleaves the mRNA at the A site of the colliding ribosome

Question 12

What are CAT tails? (2)

Answer 12

• C-terminal alanine and threonine tails
• Modification added to incomplete polypeptides in stalled translation

Question 13

How does mRNA stability relate to translation? (3)

Answer 13

• mRNAs have distinct intrinsic stabilities with more stable mRNAs being translated more efficiently so more protein coming from these
• Rate of elongation in translation is dependent on availability of charged tRNAs for the codons within the mRNAs (codon optimality)
• Rare codons = less efficient translation = potential trigger for ribosome stalling and mRNA degradation

Question 14

What is codon optimality? (2)

Answer 14

• How efficiently a codon is translated based on the available tRNA pool
• Slightly different but linked to codon bias which is the unequal usage of synonymous codons which is influenced by tRNA availability and genome composition

Question 15

What is the role of Dhh1? (4)

Answer 15

• Dhh1 is a helicase which is recruited to the ribosome during elongation and promotes mRNA turnover
• Stimulates deadenylation and decapping by destabilising binding of PABP to mRNA
• High codon optimality = fast translation elongation = weak Dhh1 association (less time)
• Low codon optimality = slow translation elongation = strong Dhh1 association

Question 16

What is NMD? (5)

Answer 16

• Nonsense mediated decay which degrades mRNAs with an early stop codon
• Context of termination is judged to be correct/incorrect based on interaction between the ribosome and factors within the 3’ UTR
• RF3 GTPase (involved in translation termination) makes contact with PABP in 3’ UTR which is critical for normal efficient translation termination
• If not close enough to the 3’ end, RF3 instead interacts with Upf1 (key NMD factor) RNA helicase
• If Upf1 binds and hydrolyses ATP before interaction with PABP, the transcript undergoes NMD (kinetic proofreading function for delayed termination)

Question 17

What can generate transcripts that will be targeted by NMD? (3)

Answer 17

• uORFs can generate early stop codons
• Genomic rearrangements e.g. in antibody genes generate NMD substrates
• Alternative splicing produces the most NMD substrates