SmpB:  A Protein that Binds to Double-Stranded Segments in tmRNA and tRNA

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(Delta90-299), 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(Delta90-299) and tRNA molecule. Irradiation of E. coli tmRNA and yeast tRNA(Phe) bound to a single SmpB molecule with UV light induced cross-links to residues C343 and m(1)A48, 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.

Global discovery of small RNAs in Yersinia pseudotuberculosis identifies Yersinia -specific small, noncoding RNAs required for virulence

“…Our finding that the majority of sRNAs tested in Y. pseudotuberculosis do not require Hfq for their expression and/or stability suggests the possibility of an alternative sRNA chaperone. Y. pseudotuberculosis may rely on SmpB, an Sm-like chaperone of tmRNA (42), or an as yet unidentified protein to stabilize sRNAs and promote interaction with mRNAs.…”

SmpB as the handyman of tmRNA during trans-translation

“…52 This result agrees with the distribution of the protections induced by the SmpB protein onto tmRNA as well as with biochemical data suggesting that the protein binds to the opposite sites of helix H2 from tmRNA. 19,53 Why needing a second SmpB? During standard translation elongation, codon recognition leads to a series of conformational changes that position EF-Tu for GTP hydrolysis.…”

A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation

“…Functional interaction of SmpB with tmRNA www.rnajournal.org 1725 been shown in Aquifex aeolicus tmRNA by a chemical and enzymatic footprinting study (Metzinger et al 2005), although it has not yet been detected in E. coli tmRNA by studies of cross-linking or probing with lead(II) (Barends et al 2001;Wower et al 2002;Ivanova et al 2007). Protection was also found at G 14 , U 16 , U 17 , and G 19 in TLD.…”