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Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

ABSTRACT: During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed.

SUBMITTER: Lloyd GS

PROVIDER: S-EPMC1219155 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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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.

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Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

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