title: A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging.
abstract: 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.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Janssen
authors.forename: Fernando
authors.initials: F
authors.lastname: Garza-Sánchez
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Hayes
mesh_terms: D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D014176:Protein Biosynthesis; D059367:RNA Cleavage; D020871:RNA Stability; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA