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Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

ABSTRACT: Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase (RNAP) holoenzyme ? subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (Spx?CHA) bearing a 12-amino-acid deletion of the C terminus (Spx?C) and a hemagglutinin (HA) epitope tag and Spxc-Myc, a full-length Spx with a C-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-tagged variants were transcriptionally active in vivo and in vitro. Reaction mixtures containing Spx?CHA and Spxc-Myc combined with RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of the epitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionally active. In vivo production of Spx?C and Spx?CHA followed by anti-HA affinity column chromatography of a cleared lysate resulted in retrieval of Spx/RNAP with only the Spx?CHA derivative. Binding reactions that combined active Spxc-Myc, inactive Spx(R60E)?CHA, and RNAP, when applied to the anti-HA affinity column, yielded only inactive Spx(R60E)?CHA/RNAP complexes. The results strongly argue for a model in which a single Spx monomer engages RNAP to generate an active transcriptional complex.

SUBMITTER: Lin AA

PROVIDER: S-EPMC3302468 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

Lin Ann A AA Zuber Peter P

Journal of bacteriology 20120203 7

Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase (RNAP) holoenzyme α subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (SpxΔCHA) bearing a 12-amino-ac ...[more]

PMID: 22307755

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Project description:Bacillus subtilis Spx is a global transcriptional regulator that is conserved among Gram-positive bacteria, in which Spx is required for preventing oxidatively induced proteotoxicity. Upon stress induction, Spx engages RNA polymerase (RNAP) through interaction with the C-terminal domain of the rpoA-encoded RNAP ? subunit (?CTD). Previous mutational analysis of rpoA revealed that substitutions of Y263 in ?CTD severely impaired Spx-activated transcription. Attempts to substitute alanine for ?CTD R261, R268, R289, E255, E298, and K294 were unsuccessful, suggesting that these residues are essential. To determine whether these RpoA residues were required for productive Spx-RNAP interaction, we ectopically expressed the putatively lethal rpoA mutant alleles in the rpoAY263C mutant, where "Y263C" indicates the amino acid change that results from mutation of the allele. By complementation analysis, we show that Spx-bound ?CTD amino acid residues are not essential for Spx-activated transcription in vivo but that R261A, E298A, and E255A mutants confer a partial defect in NaCl-stress induction of Spx-controlled genes. In addition, strains expressing rpoAE255A are defective in disulfide stress resistance and produce RNAP having a reduced affinity for Spx. The E255 residue corresponds to Escherichia coli ?D259, which has been implicated in ?CTD-?70 interaction (?70 R603, corresponding to R362 of B. subtilis ?A). However, the combined rpoAE255A and sigAR362A mutations have an additive negative effect on Spx-dependent expression, suggesting the residues' differing roles in Spx-activated transcription. Our findings suggest that, while ?CTD is essential for Spx-activated transcription, Spx is the primary DNA-binding determinant of the Spx-?CTD complex.IMPORTANCE Though extensively studied in Escherichia coli, the role of ?CTD in activator-stimulated transcription is largely uncharacterized in Bacillus subtilis Here, we conduct phenotypic analyses of putatively lethal ?CTD alanine codon substitution mutants to determine whether these residues function in specific DNA binding at the Spx-?CTD-DNA interface. Our findings suggest that multisubunit RNAP contact to Spx is optimal for activation while Spx fulfills the most stringent requirement of upstream promoter binding. Furthermore, several ?CTD residues targeted for mutagenesis in this study are conserved among many bacterial species and thus insights on their function in other regulatory systems may be suggested herein.

Project description:In Bacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression of trxB, an Spx-dependent gene. Inactivation of ctsR, encoding the regulator of the Clp proteases, reduced trxB expression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted the ctsR phenotype and increased trxB expression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reduced trxB expression. Finally, we show that Spx is itself a regulator of the ctsR operon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity in B. subtilisIMPORTANCE In Bacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of the ctsR operon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.

Dataset Information

Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

Publications

Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

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