RqcH and RqcP catalyze processive poly-alanine synthesis
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ABSTRACT:
PROVIDER: PRJNA728973 | ENA |
REPOSITORIES: ENA
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Project description: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
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Project description:In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here 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. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.
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