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Regulation of an auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene via ribosome-mediated transcriptional attenuation.

ABSTRACT: cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5' leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs of trpRS1 that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of a trpRS1 ortholog (SAV4725) in Streptomyces avermitilis. We conclude that the trpRS1 locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyl-tRNA synthetase genes are regulated in bacteria.

SUBMITTER: Vecchione JJ

PROVIDER: S-EPMC2897330 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Regulation of an auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene via ribosome-mediated transcriptional attenuation.

Vecchione James J JJ Sello Jason K JK

Journal of bacteriology 20100507 14

cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5' leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, ...[more]

PMID: 20453096

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Project description:Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNA(Trp) molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNA(Trp) after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNA(Trp) recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual TrpRS from Giardia, these protozoan structures broaden our perspective on the extent of structural variation found in eukaryotic TrpRS homologs.

Project description:Type II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact. hipBA of Escherichia coli strain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of hipA inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed that trpS in high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT of E. coli O127 is a kinase that phosphorylates tryptophanyl-tRNA synthetase in vitro at a conserved serine residue. Consistently, induction of hipT inhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream from hipT, called hipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipS in vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additional hipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus, hipBST constitutes a novel family of tricomponent TA modules where hipA has been split into two genes, hipS and hipT, that function as a novel type of TA pair.IMPORTANCE Bacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified was hipA of Escherichia coli strain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. The hipA gene encodes the toxin of the hipBA TA module, while hipB encodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family, hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.

Dataset Information

Regulation of an auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene via ribosome-mediated transcriptional attenuation.

Publications

Regulation of an auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene via ribosome-mediated transcriptional attenuation.

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