Transcription activation by Escherichia coli Rob at class II promoters: protein-protein interactions between Rob's N-terminal domain and the σ(70) subunit of RNA polymerase.

Bacterial transcription activators regulate transcription by making essential protein-protein interactions with RNA polymerase, for example, with region 4 of the σ(70) subunit (σ(70) R4). Rob, SoxS, and MarA comprise a closely related subset of members of the AraC/XylS family of transcription factors that activate transcription of both class I and class II promoters. Recently, we showed that interactions between SoxS and σ(70) R4 occlude the binding of σ(70) R4 to the -35 promoter element of class II promoters.

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the σ(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

Escherichia coli SoxS activates transcription of the genes of the soxRS regulon, which provide the cell's defense against oxidative stress. In response to this stress, SoxS is synthesized de novo. Because the DNA binding site of SoxS is highly degenerate, SoxS efficiently activates transcription by the mechanism of prerecruitment.

Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters.

Two functions of the C-terminal domain of Escherichia coli Rob: mediating "sequestration-dispersal" as a novel off-on switch for regulating Rob's activity as a transcription activator and preventing degradation of Rob by Lon protease.

In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in three to four immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Rob's C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase.

Inhibition of Lon-dependent degradation of the Escherichia coli transcription activator SoxS by interaction with 'soxbox' DNA or RNA polymerase.

Escherichia coli SoxS, the direct transcription activator of the SoxRS (superoxide) regulon, is intrinsically unstable with an in vivo half-life of approximately 2 min. Overexpression of SoxS is lethal, but mutations interfering with DNA binding relieve the toxicity. Here, we determined the effects on the half-life of SoxS of alanine substitutions that confer defects in positive control, i.

Sequence requirements for Lon-dependent degradation of the Escherichia coli transcription activator SoxS: identification of the SoxS residues critical to proteolysis and specific inhibition of in vitro degradation by a peptide comprised of the N-terminal 21 amino acid residues.

When Escherichia coli encounter redox-cycling compounds that endogenously generate superoxide, the cell's defense response is initiated by the de novo synthesis of SoxS, which then activates transcription of the genes of the SoxRS regulon. Recently, we showed that after the oxidative stress is relieved, the SoxRS system resets by an active process wherein SoxS synthesis ceases and the intrinsically unstable SoxS protein is rapidly degraded, primarily by Lon protease. Here, we use deletion mutants and a library of alanine-stretch mutants of the entire protein to identify the SoxS features responsible for Lon-dependent proteolysis in vivo.

Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon.

In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing approximately 42% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox-cycling compounds and antibiotics. On the basis of its approximately 43% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded by transposon Tn10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown.

Genetic evidence for pre-recruitment as the mechanism of transcription activation by SoxS of Escherichia coli: the dominance of DNA binding mutations of SoxS.

SoxS, the direct transcriptional activator of the Escherichia coli superoxide (SoxRS) regulon, displays several unusual characteristics which suggest that it is unlikely to activate transcription by the ususal recruitment mechanism. Thus, agents that generate superoxide endogenously and thereby provoke the defense response elicit the de novo synthesis of SoxS, and with the SoxS binding site being highly degenerate, the number of SoxS binding sites per cell far exceeds the number of SoxS molecules per cell. To account for these distinctive features of the SoxRS system, we proposed "pre-recruitment" as the mechanism by which SoxS activates transcription of the regulon's genes.

Novel protein--protein interaction between Escherichia coli SoxS and the DNA binding determinant of the RNA polymerase alpha subunit: SoxS functions as a co-sigma factor and redeploys RNA polymerase from UP-element-containing promoters to SoxS-dependent promoters during oxidative stress.

SoxS is the transcription activator of the SoxRS regulon. Despite being synthesized de novo in response to oxidative stress and despite the large disparity between the number of SoxS binding sites and the number of SoxS molecules per cell, SoxS-dependent promoters are rapidly activated after the onset of the stress. With the usual recruitment/post-recruitment mechanisms being unsuitable for activating gene expression under these conditions, we previously proposed that SoxS functions by "pre-recruitment".

Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons.

In Escherichia coli, the SoxRS regulon confers resistance to redox-cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut-off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately.

A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation.

SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate "soxbox" DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs.

Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell.

In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of approximately 100 genes. The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites. To estimate the number of soxboxes in the cell, we searched the E.

Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.

We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J.