title: Fluorescence characterization of the transfer RNA-like domain of transfer messenger RNA in complex with small binding protein B.
abstract: Transfer messenger RNA (tmRNA) and small binding protein B (SmpB) are the main components of the trans-translation rescue machinery that releases stalled ribosomes from defective mRNAs. Little is known about how SmpB binding affects the conformation of the tRNA-like domain (TLD) of tmRNA. It has been previously hypothesized that the absence of a D stem in the TLD provides flexibility in the elbow region of tmRNA, which can be stabilized by its interaction with SmpB. Here, we have used Förster resonance energy transfer to characterize the global structure of the tRNA-like domain of tmRNA in the presence and absence of SmpB and as a function of Mg(2+) concentration. Our results show tight and specific binding of SmpB to tmRNA. Surprisingly, our data show that the global conformation and flexibility of tmRNA do not change upon SmpB binding. However, Mg(2+) ions induce an 11 Å compaction in the tmRNA structure, suggesting that the flexibility in the H2a stem may allow different conformations of tmRNA as the TLD and mRNA-like domain need to be positioned differently while moving through the ribosome.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States.
authors.forename: May
authors.initials: M
authors.lastname: Daher
authors.forename: David
authors.initials: D
authors.lastname: Rueda
mesh_terms: D001483:Base Sequence; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D012343:RNA, Transfer; D008274:Magnesium