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Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

ABSTRACT: The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP sigma(A) subunit and the -10 promoter sequence of trxA and B, and contact of the betabeta' subunits with core promoter DNA. No Spx-DNA contact was detected. Spx mutants, Spx(C10A) and Spx(G52R.), or RNAP alpha C-terminal domain mutants that impair productive Spx-RNAP interaction did not induce heightened sigma and betabeta' contact with the core promoter. Deletion analysis and the activity of hybrid promoter constructs having upstream trxB DNA fused at positions -31, -36 and -41 of the srf (surfactin synthetase) promoter indicated that a cis-acting site between -50 and -36 was required for Spx activity. Mutations at -43 and -44 of trxB abolished Spx-dependent transcription and Spx-induced cross-linking between the sigma subunit and the -10 region. These data are consistent with a model that Spx activation requires contact between the Spx/RNAP complex and upstream promoter DNA, which allows Spx-induced engagement of the sigma and large subunits with the core promoter.

SUBMITTER: Reyes DY

PROVIDER: S-EPMC2758557 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

Reyes Dindo Y DY Zuber Peter P

Molecular microbiology 20080801 3

The Spx protein of Bacillus subtilis interacts with RNA polymerase (RNAP) to activate transcription initiation in response to thiol-oxidative stress. Protein-DNA cross-linking analysis of reactions containing RNAP, Spx and trxA (thioredoxin) or trxB (thioredoxin reductase) promoter DNA was undertaken to uncover the organization of the Spx-activated transcription initiation complex. Spx induced contact between the RNAP sigma(A) subunit and the -10 promoter sequence of trxA and B, and contact of t ...[more]

PMID: 18687074

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Project description:The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.

Dataset Information

Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

Publications

Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation.

Similar Datasets

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