SmpB: A protein that binds to double-stranded segments in tmRNA and tRNA

Jacek Wower, Christian W. Zwieb, David W. Hoffman, Iwona K. Wower

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Binding of the SmpB protein to tmRNA is essential for trans- translation, a process that facilitates peptide tagging of incompletely synthesized proteins. We have used three experimental approaches to study these interactions in vitro. Gel mobility shift assays demonstrated that tmRNA(Δ90-299), a truncated tmRNA derivative lacking pseudoknots 2-4, has the same affinity for the Escherichia coli and Aquifex aeolicus SmpB proteins as the intact E. coli tmRNA. These interactions can be challenged by double-stranded RNAs such as tRNAs and 5S rRNA and are abolished by removal of 24 amino acids from the C-terminus of the A. aeolicus protein. A combination of enzymatic probing and UV-induced cross-linking showed that three SmpB molecules can bind to a single tmRNA(Δ90-299) and tRNA molecule. Irradiation of E. coli tmRNA and yeast tRNAPhe bound to a single SmpB molecule with UV light induced cross-links to residues C343 and m1A48, respectively, in their T-loops and to their 3′ terminal adenosines. These findings indicate that the acceptor-T arm constitutes the primary SmpB binding site in both tmRNA and tRNA. The remaining two SmpB molecules associate with the anticodon stem-like region of tmRNA and the anticodon arm of tRNAs. As the T and anticodon loops are dispensable for SmpB binding, it seems that SmpB recognizes double helical segments in both tmRNA and tRNA molecules. Although these interactions involve analogous elements in both molecules, their different effects on aminoacylation appear to reflect subtle structural differences between the tRNA-like domain of tmrna and tRNA.

Original languageEnglish (US)
Pages (from-to)8826-8836
Number of pages11
JournalBiochemistry
Volume41
Issue number28
DOIs
StatePublished - Jul 16 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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