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Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis.

ABSTRACT: In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.

SUBMITTER: Takada H

PROVIDER: S-EPMC7797070 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis.

Takada Hiraku H Roghanian Mohammad M Caballero-Montes Julien J Van Nerom Katleen K Jimmy Steffi S Kudrin Pavel P Trebini Fabio F Murayama Rikinori R Akanuma Genki G Garcia-Pino Abel A Hauryliuk Vasili V

Nucleic acids research 20210101 1

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'clos ...[more]

PMID: 33330919

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Project description:BackgroundThe stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum.ResultsThe transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent strain.ConclusionIn C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occurring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins.

Dataset Information

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis.

Publications

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis.

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