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RqcH and RqcP catalyze processive poly-alanine synthesis

ABSTRACT: In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. We demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway. the synthesis of C-terminal poly-Alanine ‘tail’. While it is well-established that RqcP and RqcH are essential for poly-Alanine-tailing in living cells, here, by reconstituting the RQC elongation biochemically we provide a direct proof that the two factors are sufficient. We demonstrate that while RqcH is responsible for tRNAAla selection during RQC elongation, RqcP lacks the tRNA specificity. Through mutational mapping we show that stable interaction of RqcP with 23S rRNA – but not with tRNA – is crucial for RQC, supporting the role of the E-site bound RqcP as a pawl of RQC elongation ratchet. We show that ribosomal protein uL11 is crucial for RqcH – but nor RqcP – recruitment to 50S, and B. subtilis lacking uL11 is RQC-deficient. Finally, we show that proteins encoded by non-stop mRNAs are not universally efficiently targeted by RQC, and that simultaneous disruption of RQC and trans-translation results in bacterial filamentation

ORGANISM(S): Enterococcus faecalis Bacillus subtilis subsp. subtilis str. 168

PROVIDER: GSE174254 | GEO | 2021/08/19

REPOSITORIES: GEO

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Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.

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RqcH and RqcP catalyze processive poly-alanine synthesis

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