Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA.
Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.
Garza-Sánchez
Fernando
F
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA.
Janssen
Brian D
BD
Hayes
Christopher S
CS
D001665:Binding Sites; D015152:Blotting, Northern; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010441:Peptide Chain Elongation, Translational; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; C497347:SecM protein, E coli; D014157:Transcription Factors; C096222:tmRNA