The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Anurag Kumar Sinha,Kristoffer Skovbo Winther

doi:10.1038/s42003-021-01963-z

Anurag Kumar Sinha, Kristoffer Skovbo Winther

Open Access

https://doi.org/10.1038/s42003-021-01963-z

Copy DOI

Abstract

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

Highlights

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p) ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance
RelA activation occurs when RelA binds with an uncharged tRNA at an empty A-site of a stalled ribosome, which leads to induction of (p)ppGpp synthesis[18]
Cryo-EM structures of RelA in complex with uncharged tRNA and the ribosome have revealed that the C-terminal Zinc-finger domain (ZFD) and RNA recognition motif (RRM) of RelA are responsible for ribosome binding at helix 38, the A-site finger of 23 S ribosomal RNA in the 50 S

Summary

Introduction

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p) ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p) ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudohydrolase domain. Weak SpoT-dependent (p)ppGpp synthesis has been reported under multiple starvation conditions; RelA exclusively synthesizes (p)ppGpp in response to amino acid starvation[10,11]. These metabolic cues are not mutually exclusive and accumulating evidence suggest that diverse starvation signals including glucose and fatty acid starvation can indirectly lead to conditions that trigger the RelA-dependent stringent response[4,7,16,17] RelA is thought to bind with tRNA at ribosomal A-sites during amino acid starvation, when EF-TuGTPtRNA ternary complexes are scarce[18]

Methods

Results

Conclusion