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

Griffith, Kevin L.; Shah, Ishita M.; Myers, Todd E.; O'Neill, Michael C.; Wolf, Richard M.

doi:10.1006/bbrc.2002.6559

Cited by 56 publications

(108 citation statements)

References 42 publications

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“…Our results argue that MelR binds to the melAB promoter and, when triggered by melibiose, activates transcription by recruitment of RNA polymerase. This is in contrast to the prerecruitment mechanism recently suggested for the related SoxS and MarA activators that appear to bind to free RNA polymerase before binding to target promoters (6,10).…”

Section: Resultscontrasting

confidence: 95%

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

et al. 2004

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Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.Expression of the Escherichia coli melAB genes, which encode proteins necessary for transport and metabolism of the disaccharide melibiose, is dependent on the transcription activator, MelR, encoded by the adjacent melR gene (13). Previous studies have shown that transcription from the melAB promoter is activated by MelR and have focused on using biochemistry to understand the mechanism of activation (1,4,7,12). Recent work has shown that MelR activates transcription by direct interaction with the RNA polymerase subunit via residue D261 (5). Although activation requires the inducer melibiose, in vitro studies have shown that MelR can bind to the melAB promoter both in the presence and absence of melibiose (1). In the experiments presented here, we have exploited novel chromatin immunoprecipitation (ChIP) and microarray technologies to study the interactions of MelR in vivo. ChIP has been used to investigate MelR and RNA polymerase binding to the melAB regulatory region in vivo, while microarrays have been used to show that the melAB promoter is the principal target for MelR in E. coli. MATERIALS AND METHODSE. coli strains, plasmids, and oligonucleotides. Bacterial strains, plasmids, and oligonucleotides used in this work are listed in Table 1. In all experiments, E. coli strains WAM131, WAM132, or MG1655, carrying plasmids as appropriate, were grown to mid-exponential phase (optical density at 650 nm of 0.4 to 0.6) in minimal M63 medium, supplemented with fructose and amino acids, either with or without melibiose, according to the same method used previously in studies of the regulation of the E. coli mel operon (13).ChIP. In all experiments, in vivo cross-linking of bacterial nucleoprotein was initiated by the addition of formaldehyde (final concentration of 1%) to cultures. After 20 min, cross-linking was quenched by the addition of glycine (final concentration of 0.5 M). Typically, cells were then harvested from 10 ml of culture by centrifugation, washed twice with Tris-buffered-saline (pH 7.5), resuspended in 1 ml of lysis buffer (10 mM Tris [pH 8.0], 20% sucrose, 50 mM NaCl, 10 mM EDTA, 10 mg of lysozyme per ml) and incubated at 37°C for 30 min. Following lysis, 4 ml of immuno...

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Section: Resultscontrasting

confidence: 95%

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

et al. 2004

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“…2A). Assuming a cell cytoplasm volume of 6.7 ϫ 10 Ϫ16 liters (69; http://redpoll.pharmacy.ualberta.ca/CCDB /cgi-bin/STAT_NEW.cgi), we could estimate that XylS concentration in the cell was in the range of 50 nM, which corresponds to about 200 molecules per cell, in agreement with reported SoxS basal levels of 100 molecules per cell (21 Pm245 is a mutant promoter with five point mutations in the XylS binding site (Fig. 3B) and accordingly produces no detectable XylS-dependent transcriptional activity in vivo (16).…”

Section: Xyls Specifically Binds Directs Repeats In Pm Sequencesupporting

confidence: 84%

Roles of Effectors in XylS-Dependent Transcription Activation: Intramolecular Domain Derepression and DNA Binding

et al. 2008

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XylS, an AraC family protein, activates transcription from the benzoate degradation pathway Pm promoter in the presence of a substrate effector such as 3-methylbenzoate (3MB). We developed a procedure to obtain XylS-enriched preparations which proved suitable to analyze its activation mechanism. XylS showed specific 3MB-independent binding to its target operator, which became strictly 3MB dependent in a dimerizationdefective mutant. We demonstrated that the N-terminal domain of the protein can make linker-independent interactions with the C-terminal domain and inhibit its capacity to bind DNA. Interactions are hampered in the presence of 3MB effector. We propose two independent roles for 3MB in XylS activation: in addition to its known influence favoring protein dimerization, the effector is able to modify XylS conformation to trigger N-terminal domain intramolecular derepression. We also show that activation by XylS involves RNA polymerase recruitment to the Pm promoter as demonstrated by chromatin immunoprecipitation assays. RNA polymerase switching in Pm transcription was reproduced in in vitro transcription assays. All 32 -, 38 -, and 70 -dependent RNA polymerases were able to carry out Pm transcription in a rigorous XylS-dependent manner, as demonstrated by the formation of open complexes only in the presence of the regulator.

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“…Indeed, it has been observed that overexpression of MarA and SoxS or activation of Rob can lead to severe growth inhibition. 13,14 Commensurate activation is likely relevant to transcriptional regulation of systems other than the marA/soxS/rob regulon. For example, it has previously been suggested that different promoters of the CRP modulon are activated at different levels of cAMP-CRP, leading to an observed hierarchy of response to cAMP concentration 15,16 although parallel measurements of promoter activity and cAMP-CRP concentration were not measured in those studies.…”

Section: Discussionmentioning

confidence: 99%

Activation of the Escherichia coli marA/soxS/rob Regulon in Response to Transcriptional Activator Concentration

Martin

Bartlett

Rosner

et al. 2008

Journal of Molecular Biology

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SummaryThe paralogous transcriptional activators, MarA, SoxS and Rob, activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between MarA concentration needed for half-maximal promoter activity in vivo with marbox binding affinity in vitro was poor and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS implying that the two activators interact with RNAP in different ways at the different promoters. Thus, the concentration and nature of activator determines which regulon promoters are activated and the extent of their activation.

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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

Cited by 56 publications

References 42 publications

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Roles of Effectors in XylS-Dependent Transcription Activation: Intramolecular Domain Derepression and DNA Binding

Activation of the Escherichia coli marA/soxS/rob Regulon in Response to Transcriptional Activator Concentration

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