An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

RNA
Mickey R Miller, Allen Buskirk

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 togethe...Continue Reading

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Citations

Aug 20, 2014·FEMS Microbiology Reviews·Agata L StarostaDaniel N Wilson
Dec 3, 2014·Biochimie·Hyouta HimenoTatsuhiko Abo
Apr 16, 2015·Nature Reviews. Microbiology·Kenneth C Keiler
Apr 30, 2014·Frontiers in Genetics·Hyouta HimenoAkira Muto
Aug 29, 2014·Frontiers in Microbiology·Corey M HudsonKelly P Williams
Apr 9, 2014·Frontiers in Microbiology·Emmanuel GiudiceReynald Gillet
Sep 18, 2014·Frontiers in Microbiology·Mickey R Miller, Allen R Buskirk
Jul 16, 2017·Molecular Microbiology·Samantha M PreziosoJoanna B Goldberg
Feb 16, 2019·Science·Christopher D RaeV Ramakrishnan
Jun 2, 2018·Protein Expression and Purification·Juanjuan YangDonghai Lin
Apr 6, 2021·Frontiers in Microbiology·Claudia MüllerDaniel N Wilson

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