A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging.
In Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to func...
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doaj-685610642ba04a14965ca761b229b7bd2020-11-24T22:25:57ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01811e8131910.1371/journal.pone.0081319A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging.Brian D JanssenFernando Garza-SánchezChristopher S HayesIn Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to function in translation quality control because stalled ribosomes are recycled from A-site truncated transcripts by the tmRNA-SmpB "ribosome rescue" system. During rescue, the tmRNA-encoded ssrA peptide is added to the nascent chain, thereby targeting the tagged protein for degradation after release from the ribosome. Here, we examine the influence of A-site mRNA cleavage upon tmRNA-SmpB activity. Using a model transcript that undergoes stop-codon cleavage in response to inefficient translation termination, we quantify ssrA-peptide tagging of the encoded protein in cells that contain (rnb(+)) or lack (Δrnb) RNase II. A-site mRNA cleavage is reduced approximately three-fold in Δrnb backgrounds, but the efficiency of ssrA-tagging is identical to that of rnb(+) cells. Additionally, pulse-chase analysis demonstrates that paused ribosomes recycle from the test transcripts at similar rates in rnb(+) and Δrnb cells. Together, these results indicate that A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue and suggest that A-site mRNA cleavage process may play a role in other recycling pathways.http://europepmc.org/articles/PMC3834316?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Brian D Janssen Fernando Garza-Sánchez Christopher S Hayes |
spellingShingle |
Brian D Janssen Fernando Garza-Sánchez Christopher S Hayes A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. PLoS ONE |
author_facet |
Brian D Janssen Fernando Garza-Sánchez Christopher S Hayes |
author_sort |
Brian D Janssen |
title |
A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. |
title_short |
A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. |
title_full |
A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. |
title_fullStr |
A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. |
title_full_unstemmed |
A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. |
title_sort |
a-site mrna cleavage is not required for tmrna-mediated ssra-peptide tagging. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2013-01-01 |
description |
In Escherichia coli, prolonged translational arrest allows mRNA degradation into the A site of stalled ribosomes. The enzyme that cleaves the A-site codon is not known, but its activity requires RNase II to degrade mRNA downstream of the ribosome. This A-site mRNA cleavage process is thought to function in translation quality control because stalled ribosomes are recycled from A-site truncated transcripts by the tmRNA-SmpB "ribosome rescue" system. During rescue, the tmRNA-encoded ssrA peptide is added to the nascent chain, thereby targeting the tagged protein for degradation after release from the ribosome. Here, we examine the influence of A-site mRNA cleavage upon tmRNA-SmpB activity. Using a model transcript that undergoes stop-codon cleavage in response to inefficient translation termination, we quantify ssrA-peptide tagging of the encoded protein in cells that contain (rnb(+)) or lack (Δrnb) RNase II. A-site mRNA cleavage is reduced approximately three-fold in Δrnb backgrounds, but the efficiency of ssrA-tagging is identical to that of rnb(+) cells. Additionally, pulse-chase analysis demonstrates that paused ribosomes recycle from the test transcripts at similar rates in rnb(+) and Δrnb cells. Together, these results indicate that A-site truncated transcripts are not required for tmRNA-SmpB-mediated ribosome rescue and suggest that A-site mRNA cleavage process may play a role in other recycling pathways. |
url |
http://europepmc.org/articles/PMC3834316?pdf=render |
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