The ribosome-associated quality control pathway supports survival in the absence of non-stop ribosome rescue factors

Abstract

In bacteria, if a ribosome translates an mRNA lacking a stop codon it becomes stalled at the 3' end of the message. These ribosomes must be rescued by trans-translation or the alternative rescue factors (ArfA or ArfB). However, mounting evidence suggests that the ribosome quality control (RQC) pathway may also rescue non-stop ribosomes. Here, we surveyed the conservation of ribosome rescue pathways in >15,000 bacterial genomes. We found that trans-translation is conserved in >97% of bacterial genomes, while the other rescue pathways are restricted to particular phyla. We did not detect the gene encoding RqcH, the major mediator of RQC, in Proteobacteria (Pseudomonadota). In all Proteobacteria investigated to date, trans-translation is essential in the absence of the Arf proteins. Therefore, we tested whether expression of RQC components from Bacillus subtilis could rescue viability in the absence of trans-translation and ArfA in Escherichia coli. We found that the RQC pathway indeed functions in E. coli and rescues the well-documented synthetic lethal phenotype of ∆ssrA∆arfA. Moreover, we show that the RQC pathway in B. subtilis is essential in the absence of trans-translation and ArfA, further supporting a role for the RQC pathway in the rescue of non-stop ribosomes. Finally, we report a strong co-occurrence between RqcH and the ribosome splitting factor MutS2, but present experimental evidence that there are likely additional ribosome splitting factors beyond MutS2 in B. subtilis. Altogether, our work supports a role for RQC in non-stop ribosome rescue and provides a broad survey of ribosome rescue pathways in diverse bacteria.

Importance: In bacteria, it is estimated that 2%-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes are essential for viability. We determined the functional overlap between the ribosome quality control pathway and the classical non-stop rescue systems [alternative rescue factor (ArfA) and trans-translation] in a representative Firmicute and Proteobacterium, phyla that are evolutionarily distinct. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >15,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.

Keywords: RNA binding proteins; bacteria; molecular genetics; ribosomes; trans-translation; translation.

Fig 1

Conservation of ribosome rescue pathways across >15,000 bacterial genomes. Heatmap showing the percentage of genomes per phyla containing smpB, ssrA, arfA, arfB, smrB, rqcH, and mutS2. The tree was built using 16S rRNA genes from a single representative species for each phylum (Table 2). NCBI accession numbers of all surveyed genomes are given in Table S1, and the query sequences used to search for each gene are given in Table S2. Bootstrap percentages are shown as darkened circles at each node.

Fig 2

E. coli can survive the synthetic lethality of ∆ssrA::cat^R∆arfA::kan^R when provided with B. subtilis rqcH and rqcP. (A) Strains depicted in the schematic were grown on plates with or without 1% arabinose to induce expression of RqcH/RqcP. (B) Growth curves in lysogeny broth (LB) of E. coli ∆arfA::kan^R, ∆ssrA::cat^R, and ∆ssrA::cat^R∆arfA::kan^R strain harboring the plasmid expressing RqcH and RqcP. Error bars represent the standard deviation of three independent growth curves performed in parallel from individual colonies.

Fig 3

Bacillus subtilis ∆smpB∆rqcH and ∆smpB∆brfA cells exhibit defects in maximum growth rate and delayed entry into the exponential phase. Cells were grown under aerobic conditions in LB at 37°C. (Left) Maximum growth rate. (Right) Time to exit lag phase (measured as time at which optical density exceeds 0.125 when starting from 0.05 OD diluted from overnight culture). Error bars represent the standard deviation of four independent replicates performed on different days. P values represent the results of an unpaired t-test with Welch’s correction.

Fig 4

Impact of SmpB depletion from cells lacking brfA and rqcH in B. subtilis. (A) Schematic showing depletion of smpB using CRISPRi. Guide RNA targeting smpB (sgRNA^smpB) under the vegetative promoter in B. subtilis is co-expressed with deactivated Cas9 (dCas9) under the control of a xylose inducible promoter resulting in transcriptional repression. (B) Strains harboring sgRNA^smpB and dCas9 were serially diluted and spot plated on LB agar with or without 1% xylose at 37°C and 30°C. Images are representative of three independent experiments.

Fig 5

The RQC pathway can still function in the absence of MutS2. (Top) Co-occurrence matrices of mutS2 vs rqcH and smpB vs ssrA. The values represent the number of genomes with or without these genes. (Bottom) Strains harboring sgRNA^smpB and dCas9 were serially diluted and spot plated on LB agar with or without 1% xylose at 37°C and 30°C. Image shows representative from triplicate experiments.

Fig 6

Summary of ribosome rescue pathways that are essential for viability. trans-Translation and ArfA are present in E. coli. Although ArfB is present in E. coli, it is not sufficiently expressed from the native locus to rescue viability and is therefore not shown. B. subtilis additionally uses the RQC pathway. Stalled ribosomes are recognized by a ribosome splitting factor such as MutS2 when collision occurs with the trailing ribosome. After being split from the mRNA, the large subunit remains obstructed with peptidyl-tRNA. RqcH and RqcP add an alanine tag to the stalled peptide, exposing the aminoacyl bond to the cytoplasm where it is cleaved by PTH.