Dataset Information

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes.

ABSTRACT: The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.

SUBMITTER: Takada H

PROVIDER: S-EPMC7058999 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes.

Takada Hiraku H Roghanian Mohammad M Murina Victoriia V Dzhygyr Ievgen I Murayama Rikinori R Akanuma Genki G Atkinson Gemma C GC Garcia-Pino Abel A Hauryliuk Vasili V

Frontiers in microbiology 20200228

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM i ...[more]

PMID: 32184768

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Project description:RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.

Dataset Information

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes.

Publications

The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes.

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