title: Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.
abstract: Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kurita
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
authors.forename: Mickey R
authors.initials: MR
authors.lastname: Miller
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Muto
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA himeno@cc.hirosaki-u.ac.jp buskirk@chem.byu.edu.
authors.forename: Allen R
authors.initials: AR
authors.lastname: Buskirk
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan himeno@cc.hirosaki-u.ac.jp buskirk@chem.byu.edu.
authors.forename: Hyouta
authors.initials: H
authors.lastname: Himeno
mesh_terms: D020413:3' Untranslated Regions; D001426:Bacterial Proteins / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C096222:tmRNA; D010444:Peptide Elongation Factor Tu
keywords: SmpB; ribosome; tmRNA; trans-translation; translation