The kinetic mechanism of bacterial ribosome recycling

Abstract

Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine–Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.

Roles of specific aminoglycoside–ribosome interactions in the inhibition of translation

“…5A). Although quite slow, the observed rate of this reaction is comparable to rates seen previously for analogous POST complexes in which SD-ASD pairing occurs (Peske et al 2005;Chen et al 2017). As expected, Neo and Tob inhibited recycling of the control ribosome, with IC 50 values of 1.9 ± 0.3 µM and 23 ± 3 µM, respectively (Fig.…”

Translation in Prokaryotes

“…In the latter case, GTP hydrolysis by EF-G was suggested to promote mRNA release, which would be a novel, unexpected function for EF-G. This is followed by tRNA dissociation and finally subunit splitting (Chen et al 2017). Future experiments will show which order of events is correct or whether multiple different dissociation pathways are possible.…”

“…However, in all these experiments, model mRNAs were used that contained an SD sequence that could stabilize the mRNA binding. An alternative pathway was recently suggested for mRNAs that do not contain an SD sequence in the vicinity of the stop codon (which is a normal case after termination) (Chen et al 2017). In the latter case, GTP hydrolysis by EF-G was suggested to promote mRNA release, which would be a novel, unexpected function for EF-G.…”

The Mechanisms of Action of Ribosome-Targeting Peptide Antibiotics

“…Stabilization of tRNAs in the A-site by viomycin has also been shown to promote back-translocation (Shoji et al, 2006 ). Viomycin also inhibits mRNA and tRNA release and splitting of ribosomal subunits (Hirokawa et al, 2002 ; Savelsbergh et al, 2009 ; Chen et al, 2017 ) that is normally mediated by RRF and EF-G during ribosome recycling. Additionally, viomycin has been reported to interfere with the canonical translation termination as well as the ArfA-RF2-dependent rescue system (Zeng and Jin, 2016 ).…”