Analysis of RNA polymerase-promoter complex formation

Ross, Wilma; Gourse, Richard L.

doi:10.1016/j.ymeth.2008.10.018

Cited by 58 publications

(51 citation statements)

References 85 publications

(138 reference statements)

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“…This assay enables investigation of promoter melting at the onset of transcription initiation. Potassium permanganate reacts specifically with thymine residues in singlestranded DNA regions formed during the formation of the RNAPopen promoter complex (28). We observed that RNAP Eh alone is able to form an open promoter complex at P vraSR ( Fig.…”

Section: Figmentioning

confidence: 66%

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Belcheva

Verma

Korenevsky

et al. 2011

Journal of Bacteriology

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Cell wall damage in Staphylococcus aureus induces a rapid genome-wide response, referred to as the cell wall stress stimulon. This response is mediated by a two-component system, the vancomycin resistance-associated sensor/regulator (VraSR). The response regulator protein VraR is a transcription factor. Here, we demonstrate that two VraR binding sites in the vraSR operon control region are involved in the regulation of the vraSR operon. The sites are centered at the ؊60 and ؊35 nucleotide positions and are referred to as R1 and R2, respectively. DNase I footprinting and lux operon reporter vector studies showed that both of these sites communicate intimately with each other to fine-tune the activity of the vraSR operon. Mutagenesis of the VraR binding sites showed that dimerization of unphosphorylated VraR at R1 is driven by a hierarchy in VraR binding and by the proximity of the two tandem VraR binding sequences at this site. On the other hand, these studies show that the lack of sequence conservation and the distance between the VraR binding sequences in R2 ensure that VraR is recruited to this site only when phosphorylated (hence, under stress conditions). Furthermore, we demonstrate that sigma A (SigA) factor is involved in the regulation of the vraSR operon. Our study shows that sigma A factor does not bind to the vraSR operon control region in the absence of VraR, suggesting that VraR may interact directly with this factor.

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Section: Figmentioning

confidence: 66%

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Belcheva

Verma

Korenevsky

et al. 2011

Journal of Bacteriology

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“…RNA was extracted using a hot-phenol method from wild-type and ⌬dksA cells containing pBR322 or pJK537, and transcription in vivo was analyzed by primer extension as described previously (31). Cells were grown in morpholinepropanesulfonic acid (MOPS) minimal medium supplemented with 0.4% glucose, 0.4% Casamino Acids, 40 g/ml tryptophan, and 10 g/ml thiamine at 30°C to an OD 600 of ϳ0.3.…”

Section: Methodsmentioning

confidence: 99%

Suppression of a dnaKJ Deletion by Multicopy dksA Results from Non-Feedback-Regulated Transcripts That Originate Upstream of the Major dksA Promoter

et al. 2012

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cDksA is an RNA polymerase (RNAP) binding transcription factor that controls expression of a large number of genes in concert with the small-molecule "alarmone" ppGpp. DksA also aids in the resolution of conflicts between RNAP and DNA polymerase (DNAP) during genome replication. DksA was originally identified as a multicopy suppressor of the temperature sensitivity caused by deletion of the genes coding for the DnaKJ chaperone system. Here, we address a longstanding question regarding the role of DksA in ⌬dnaKJ suppression. We demonstrate that DksA expression from a multicopy plasmid is necessary and sufficient for suppression, that overexpression occurs despite the fact that the major dksA promoter is feedback regulated in wildtype cells, and that weak, non-feedback-regulated transcription originating upstream of the major promoter for the dksA gene accounts for overexpression. We tentatively rule out three potential explanations for suppression related to known functions of DnaKJ. Because a determinant in DksA needed for the regulation of transcription initiation, but not for resolution of RNAP-DNAP conflicts, is needed to bypass the need for DnaKJ, we suggest that suppression results from an unidentified product whose promoter is directly or indirectly regulated by DksA.

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“…although it varies from one transcription event to another, measurements on an unrepressed Lac promoter (Lutz et al, 2001) suggest that it follows a Gaussian distribution with a mean of 40 s and std. of 4 s, but this varies widely from gene to gene (Ross and Gourse, 2009). We tested for the present case and in previous works (Ribeiro, 2007) (Ribeiro, 2008) the effects of introducing variable delays following a normal distribution with the same mean values and with standard deviations small compared to the mean values, and the dynamics of the toggle switch was not significantly different to the dynamics of model with fixed delays, i.e., qualitatively, our conclusions hold when using a model with variable delays.…”

Section: Model Of the Toggle Switchmentioning

confidence: 99%

Variability of the distribution of differentiation pathway choices regulated by a multipotent delayed stochastic switch

Ribeiro

Dai

Yli‐Harja

2009

Journal of Theoretical Biology

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We study the plasticity of a delayed stochastic model of a genetic toggle switch as a multipotent differentiation pathway switch, at the single cell and cell population levels, by observing distributions of differentiation pathways choices of genetically homogeneous cell populations. Assuming a model of stochastic pathway determination of cell differentiation that is regulated by the proteins of the switch, we vary the proteins' expression level and degradation rates, which cells are known to be able to regulate, to vary mean level, noise, and bias of the proteins' expression levels. It is shown that small changes in each of these dynamical features significantly and distinctively affects the dynamics of the switch at the single cell level and thus, the cell differentiation patterns. The regulation of these features allows cells to regulate their pluripotency and cell populations' distribution of lineage choice, suggesting that the stochastic switch has high plasticity regarding differentiation pathway choice regulation, thus providing adaptability to environmental stresses and changes.

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Analysis of RNA polymerase-promoter complex formation

Cited by 58 publications

References 85 publications

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Roles of DNA Sequence and Sigma A Factor in Transcription of the vraSR Operon

Suppression of a dnaKJ Deletion by Multicopy dksA Results from Non-Feedback-Regulated Transcripts That Originate Upstream of the Major dksA Promoter

Variability of the distribution of differentiation pathway choices regulated by a multipotent delayed stochastic switch

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