Takada Hiraku, Roghanian Mohammad, Murina Victoriia, Dzhygyr Ievgen, Murayama Rikinori, Akanuma Genki, Atkinson Gemma C, Garcia-Pino Abel, Hauryliuk Vasili
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.
Front Microbiol. 2020 Feb 28;11:277. doi: 10.3389/fmicb.2020.00277. eCollection 2020.
The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized RelA and Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.
（p）ppGpp介导的严谨反应是一种细菌应激反应，与毒力和抗生素耐受性有关。（p）ppGpp警报素核苷酸的合成和降解均由RelA-SpoT同源物（RSH）酶介导，这些酶大致可分为两类：单结构域“短”RSH和多结构域“长”RSH。调节性ACT（天冬氨酸激酶、分支酸变位酶和TyrA）/RRM（RNA识别基序）结构域是长RSH几乎普遍存在的C末端结构域。在单功能（仅合成）RelA以及双功能（即能够降解和合成警报素）Rel中删除RRM会导致长RSH产生细胞毒性，原因是（p）ppGpp过度产生。为了探究这种效应背后的分子机制，我们对缺乏RRM的RelA和Rel RSH进行了表征。我们证明，首先，去除RRM引起的细胞毒性可被二级突变抵消，这些突变破坏了RSH与饥饿核糖体复合物的相互作用——RelA和Rel产生（p）ppGpp的最终诱导物，其次，Rel的水解活性在截短突变体中并未丧失。因此，我们得出结论，缺乏RRM结构域的RSH导致（p）ppGpp过度产生，并非由于缺乏RRM时的自抑制缺失和/或（p）ppGpp水解缺陷。相反，我们认为这是由核糖体对RSH激活的调控失调所致。
