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The stringent response inhibits 70S ribosome formation in staphylococcus aureus by impeding GTPase-ribosome interactions

DOI: 10.1128/mBio.02679-21 DOI Help

Authors: Daniel J. Bennison (University of Sheffield) , Jose A. Nakamoto (Universidad Peruana de Ciencias Aplicadas (UPC)) , Timothy D. Craggs (University of Sheffield) , Pohl Milón (Universidad Peruana de Ciencias Aplicadas (UPC)) , John B. Rafferty (University of Sheffield) , Rebecca M. Corrigan (University of Sheffield)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Mbio

State: Published (Approved)
Published: November 2021
Diamond Proposal Number(s): 17773

Open Access Open Access

Abstract: 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.

Diamond Keywords: Bacteria; Enzyme

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-Macromolecular Crystallography

Added On: 15/11/2021 11:36


Discipline Tags:

Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)