During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.

中文翻译：

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（p）ppGpp通过阻止GTPase-核糖体相互作用来抑制金黄色葡萄球菌中70S核糖体的形成

在限制营养的过程中，细菌产生警报信号（p）ppGpp作为压力信号网络的效应物，称为严格反应。在金黄色葡萄球菌中筛选（p）ppGpp结合靶标，确定了四个核糖体相关的GTPases（RA-GTPases），RsgA，RbgA，Era和HflX，它们都是核糖体装配中的辅因子，它们在ON（GTP-约束）和OFF（受GDP约束）状态。GTP水解时会从ON状态进入OFF状态，而核糖体缔合时GTPase活性会大大增加。当与（p）ppGpp结合时，GTPase活性受到抑制，从而减少了70S核糖体装配。在这里，我们试图通过检查RA-GTPase和核糖体在各种核苷酸结合状态下的亲和力和动力学来确定（p）ppGpp如何影响RA-GTPase-核糖体相互作用。我们显示，RA-GTPases优先结合GTP上的含5'-二磷酸的核苷酸GDP和ppGpp，这很可能被利用为细胞内的调节机制。与体外和细菌细胞内的GTP结合状态相比，与（p）ppGpp的结合通过诱导OFF状态构象，降低了RA-GTP酶与核糖体亚单位的稳定缔合。我们建议，在这种构象中，G2 / switch I环采用与核糖体缔合不兼容的构象。总而言之，我们强调了（p）ppGpp介导的RA-GTPase抑制是严格的反应介导的生长控制的主要机制。与体外和细菌细胞内的GTP结合状态相比，与（p）ppGpp的结合通过诱导OFF状态构象，降低了RA-GTP酶与核糖体亚单位的稳定缔合。我们建议，在这种构象中，G2 / switch I环采用与核糖体缔合不兼容的构象。总而言之，我们强调了（p）ppGpp介导的RA-GTPase抑制是严格的反应介导的生长控制的主要机制。与体外和细菌细胞内的GTP结合状态相比，与（p）ppGpp的结合降低了RA-GTP酶与核糖体亚基的稳定缔合，这是通过诱导OFF状态构象实现的。我们建议，在这种构象中，G2 / switch I环采用与核糖体缔合不兼容的构象。总而言之，我们强调了（p）ppGpp介导的RA-GTPase抑制是严格的反应介导的生长控制的主要机制。