title: An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue.
abstract: In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.
authors.forename: Mickey R
authors.initials: MR
authors.lastname: Miller
authors.forename: Allen R
authors.initials: AR
authors.lastname: Buskirk
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010444:Peptide Elongation Factor Tu / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding; D011728:Pyridones / Q000494:pharmacology; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D011728:Pyridones; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; C008536:mocimycin
keywords: EF-Tu; SmpB; decoding; ribosome; tmRNA