(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions
(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions
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DOI：
10.1101/2021.01.19.427108
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发表时间：
2021-01
期刊：
影响因子：
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通讯作者：
D. J. Bennison;J. A. Nakamoto;Timothy D. Craggs;P. Milón;J. Rafferty;R. Corrigan
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文献类型：
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作者：
D. J. Bennison;J. A. Nakamoto;Timothy D. Craggs;P. Milón;J. Rafferty;R. Corrigan
在营养限制期间，细菌产生alarmones（p）ppGpp作为称为严格响应的应激信号网络的效应物。在金黄色葡萄球菌中筛选（p）ppGpp结合靶标鉴定了四种核糖体相关GTP酶（RA-GTP酶），RsgA、RbgA、Era和HflX，它们中的每一种都是核糖体组装中的辅因子，其中它们在ON（GTP结合）和OFF（GDP结合）状态之间循环。GTP水解后从ON状态进入OFF状态，GTP酶活性在核糖体结合后显著增加。当与（p）ppGpp结合时，GTdR活性被抑制，减少70 S核糖体组装。在这里，我们试图通过检查各种核苷酸结合状态下RA-GTP酶和核糖体之间的结合亲和力和动力学来确定（p）ppGpp如何影响RA-GTP酶-核糖体相互作用。我们发现RA-GTP酶优先结合5′-二磷酸核苷酸GDP和ppGpp超过GTP，这可能是利用作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状态构象而降低RA-GTP酶与核糖体亚基的稳定缔合。我们建议，在这种构象中，G2/开关I环采用与核糖体协会不相容的构象。总之，我们强调（p）ppGpp介导的RA-GTP酶抑制作为严格响应介导的生长控制的主要机制。
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.