The Florey Institute, Department of Molecular Biology and Biotechnology, University of Sheffieldgrid.11835.3e, Sheffield, United Kingdom.
Department of Molecular Biology and Biotechnology, University of Sheffieldgrid.11835.3e, Sheffield, United Kingdom.
mBio. 2021 Dec 21;12(6):e0267921. doi: 10.1128/mBio.02679-21. Epub 2021 Nov 9.
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 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. The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.
在营养限制期间，细菌会产生警报素（p）ppGpp，作为一种称为严格响应的应激信号网络的效应物。RsgA、RbgA、Era 和 HflX 是与 Staphylococcus aureus 中的（p）ppGpp 结合的四种核糖体相关 GTPase（RA-GTPase）。这些酶是核糖体组装的辅助因子，在核糖体组装过程中，它们在 ON（GTP 结合）和 OFF（GDP 结合）核糖体相关状态之间循环。GTP 的水解导致进入 OFF 状态，与核糖体结合后 GTPase 活性大大增加。当与（p）ppGpp 结合时，GTPase 活性受到抑制，从而减少 70S 核糖体的组装和生长。在这里，我们确定了（p）ppGpp 如何影响 RA-GTPase-核糖体相互作用。我们表明，RA-GTPase 优先结合 5'-二磷酸核苷酸 GDP 和 ppGpp 而不是 GTP，这可能是细胞内作为一种调节机制，在应激期间关闭核糖体生物发生。停止流动荧光和结合测定表明，当与（p）ppGpp 结合时，RA-GTPase 与核糖体亚基的结合不稳定，无论是在 还是在细菌细胞内。一致地，与 ppGpp 结合的 RA-GTPase RsgA 的结构分析揭示了一种类似于 GDP 结合状态的 OFF 状态构象，其中 G2/开关 I 环采用与核糖体结合不兼容的构象。总之，我们强调了（p）ppGpp 介导的 RA-GTPase 抑制作为严格响应介导的核糖体组装和生长控制的主要机制。严格响应是一种细菌信号网络，利用核苷酸 pppGpp 和 ppGpp 来重新编程细胞以在营养压力下存活。然而，关于这些重要核苷酸如何控制细胞重编程的知识还很有限。我们之前的工作表明，（p）ppGpp 可以结合并抑制四种核糖体相关 GTPase（RA-GTPase）的酶活性，这些酶有助于成熟 50S 和 30S 核糖体亚基。在这里，我们检查了这种情况的发生机制，并证明这种相互作用阻止了 RA-GTPase 在核糖体亚基上的容纳，无论是在 还是在细菌细胞内，ppGpp 结合状态在结构上模拟了酶的无活性 GDP 结合构象。我们还揭示了这些 GTPase 酶对诱导 OFF 状态的核苷酸具有更高的亲和力，这是一种可能在生长过程中控制核糖体组装的机制。有了这个，我们进一步了解了核糖体功能如何受（p）ppGpp 控制，使细菌能够在压力下存活。
