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 transtranslation. 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.

ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue

“…We used two kinds of mRNAs having 0 and 21 nucleotides extending from the P-site in consideration of the span of the downstream mRNA path from the P-site (Figure 5A ). Ribosome was incubated with tRNA fMet and an mRNA having a Shine-Dalgarno (SD) sequence, an AUG start codon and 0 or 21 nucleotides of 3′ extension (mRNA + 0 or mRNA + 21) to produce a stalled ribosome as previously reported ( 17 ). This stalled ribosome was incubated with 5-TAMRA-labeled ArfA.…”

“…Note that ArfA binds to the ribosome regardless of the length of 3′ extension of mRNA (Figure 5A ). Recently, we found that tmRNA-SmpB enters the ribosome stalled even in the middle of mRNA to trigger GTP hydrolysis by EF-Tu, whereas subsequent peptidyl-transfer occurs only in the ribosome stalled near the 3′ end of a truncated mRNA ( 17 ). Taken together, either ArfA or SmpB initially binds to the region around the decoding center for which the C-terminal region would not compete with the 3′ extension of mRNA.…”

Mechanism of ribosome rescue by alternative ribosome-rescue factor B

“…In the context of ribosome rescue in E. coli, the specific role of ArfB remains unclear, as ArfB has the same preference for ribosomes stalled on short mRNAs as tmRNA-SmpB and ArfA [11][12][13] . As suggested earlier, ArfB may be simply not as crucial in E. coli as in other species that lack one or both of the other rescue systems 5 .…”

“…Under normal conditions in E. coli, ArfB appears redundant with tmRNA-SmpB and ArfA 6 . While tmRNA-SmpB and ArfA preferentially target ribosomes stalled on truncated mRNAs [11][12][13] , ArfB has been suggested to act on ribosomes stalled on mRNAs extending downstream past the P site 7,14 . If so, one potential function of ArfB could be to rescue ribosomes pausing on rare codon clusters 7 .…”

Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii

“…However, the alternative pathways have evolved to compensate for the deficiency of trans-translation. For example, the alternative rescue factor A (ArfA) acts to release the stocked mRNA in Proteobacteria and mycobacteria, and the alternative rescue factor B (ArfB) appears in 34% of bacteria (Kurita et al, 2014; Huter et al, 2017; Fiedler et al, 2018). Therefore, superbacteria might have alternate ways for SmpB defects, while no reports indicate that other functions of SmpB are maintained by small RNA (sRNA).…”