Abstract

ABSTRACTDuring 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.

Highlights

  • 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

  • To examine the nucleotide binding affinities of these RA-GTPases for GDP in comparison to ppGpp, pppGpp, and GTP, and in the absence of a large tag, we used a differential radial capillary action of ligand assay (DRaCALA) with recombinant RsgA, RbgA, Era, and HflX fused to a smaller 6Â His tag (Fig. 1B; see Fig. S2A to C in the supplemental material) [36]

  • With the rapid accumulation and high affinity of interaction, it is likely that inhibition of RA-GTPases by (p)ppGpp could occur extremely early during the stringent response, rapidly halting the de novo production of ribosomal subunits [19]

Read more

Summary

Introduction

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. The concentration of (p)ppGpp within the cell rises to reach between 1 and 2 mM with a concurrent drop in GTP levels [17, 18] This results in a plethora of downstream effects during what is thought to be a highly prioritized process [19], including alterations to (i) transcription through derepression of the CodY regulon [20]; (ii) translation through the binding and inhibition of several translation factors, including elongation factor G (EF-G), elongation factor Tu (EF-Tu), and bacterial initiation factor 2 (IF2) [21,22,23]; and (iii) DNA replication, as well as regulating late-stage growth phases such as sporulation or biofilm formation [24,25,26]. Era is a highly conserved protein that interacts with the antiShine-Dalgarno sequence toward the 39 ends of 16S rRNA and pre-16S rRNA [3] in

Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call