Dataset Information

Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase.

ABSTRACT: The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogen Staphylococcus aureus and most other bacteria, hibernation-promoting factor (HPF) homodimerizes the 70S ribosomes to form a translationally silent 100S complex. Conversely, the 100S ribosomes dissociate into subunits and are presumably recycled for new rounds of translation. The regulation and disassembly of the 100S ribosome are largely unknown because the temporal abundance of the 100S ribosome varies considerably among different bacterial phyla. Here, we identify a universally conserved GTPase (HflX) as a bona fide dissociation factor of the S. aureus 100S ribosome. The expression levels hpf and hflX are coregulated by general stress and stringent responses in a temperature-dependent manner. While all tested guanosine analogs stimulate the splitting activity of HflX on the 70S ribosome, only GTP can completely dissociate the 100S ribosome. Our results reveal the antagonistic relationship of HPF and HflX and uncover the key regulators of 70S and 100S ribosome homeostasis that are intimately associated with bacterial survival.

SUBMITTER: Basu A

PROVIDER: S-EPMC5625922 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Disassembly of the <i>Staphylococcus aureus</i> hibernating 100S ribosome by an evolutionarily conserved GTPase.

Basu Arnab A Yap Mee-Ngan F MN

Proceedings of the National Academy of Sciences of the United States of America 20170911 39

The bacterial hibernating 100S ribosome is a poorly understood form of the dimeric 70S particle that has been linked to pathogenesis, translational repression, starvation responses, and ribosome turnover. In the opportunistic pathogen <i>Staphylococcus aureus</i> and most other bacteria, hibernation-promoting factor (HPF) homodimerizes the 70S ribosomes to form a translationally silent 100S complex. Conversely, the 100S ribosomes dissociate into subunits and are presumably recycled for new round ...[more]

PMID: 28894000

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Project description: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. IMPORTANCE 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 in vitro 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.

Dataset Information

Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase.

Publications

Disassembly of the <i>Staphylococcus aureus</i> hibernating 100S ribosome by an evolutionarily conserved GTPase.

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