Structure probing of tmRNA in distinct stages of trans-translation
2007 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 13, no 5, 713-722 p.Article in journal (Refereed) Published
Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was d dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
Place, publisher, year, edition, pages
2007. Vol. 13, no 5, 713-722 p.
tmRNA, SmpB, structural probing, lead(II), trans-translation
IdentifiersURN: urn:nbn:se:uu:diva-92947DOI: 10.1261/rna.451507ISI: 000245882400010PubMedID: 17400816OAI: oai:DiVA.org:uu-92947DiVA: diva2:166275