The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.
The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.
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DOI：
10.1128/mbio.02679-21
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发表时间：
2021-12-21
期刊：
影响因子：
6.4
通讯作者：
Corrigan RM
中科院分区：
文献类型：
--
作者：
Bennison DJ;Nakamoto JA;Craggs TD;Milón P;Rafferty JB;Corrigan RM
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。RsgA、RbgA、Era和HflX是与金黄色葡萄球菌中的（p）ppGpp结合的四种核糖体相关GTP酶（RA-GTP酶）。这些酶是核糖体组装中的辅因子，在核糖体组装中它们在ON（GTP结合）和OFF（GDP结合）核糖体相关状态之间循环。进入OFF状态发生在GTP水解时，其中GTP酶活性在核糖体缔合时显著增加。当与（p）ppGpp结合时，GTdR活性被抑制，减少70 S核糖体组装和生长。在这里，我们确定了（p）ppGpp如何影响RA-GT-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，这可能是利用细胞内的调节机制，在应激期间关闭核糖体生物合成。停流荧光和缔合测定揭示，当与（p）ppGpp结合时，RA-GTP酶与核糖体亚基的缔合在体外和细菌细胞内都是不稳定的。一致地，ppGpp结合的RA-GTTR RsgA的结构分析揭示了类似于GDP结合状态的OFF状态构象，其中G2/开关I环采用与核糖体缔合不相容的构象。总之，我们强调（p）ppGpp介导的抑制RA-GTP酶作为一个主要的机制，严格的反应介导的核糖体组装和生长控制。
During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF 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 and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. 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 to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control.