DNA-Dependent Renaturation of an Insoluble DNA Binding Protein

Egan, Susan M.; Schleif, Robert

doi:10.1006/jmbi.1994.1684

Cited by 65 publications

(75 citation statements)

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“…In the presence of L-rhamnose, the AraC family activator RhaS (reviewed in reference 12) binds to a site that spans from position Ϫ32 to position Ϫ81 relative to the rhaBAD transcription start site (9,10). This RhaS-binding site consists of two 17-bp inverted repeat half-sites that are separated by 16 bp of DNA not contacted by RhaS (9). RhaS alone can activate rhaBAD expression approximately 1,000-fold above the extremely low basal level (10).…”

mentioning

confidence: 99%

Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon

Holcroft

Egan

2000

J Bacteriol

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The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the ؊35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at ؊92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the ␣ subunit of RNA polymerase deleted for the entire C-terminal domain (␣-⌬235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the ␣ subunit (␣-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on ␣-CTD. Overall, our results suggest that CRP interaction with ␣-CTD may not be required for rhaBAD activation; however, ␣-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.Regulation of the Escherichia coli rhaBAD operon responds to both availability of L-rhamnose and catabolite repression. In the presence of L-rhamnose, the AraC family activator RhaS (reviewed in reference 12) binds to a site that spans from position Ϫ32 to position Ϫ81 relative to the rhaBAD transcription start site (9, 10). This RhaS-binding site consists of two 17-bp inverted repeat half-sites that are separated by 16 bp of DNA not contacted by RhaS (9). RhaS alone can activate rhaBAD expression approximately 1,000-fold above the extremely low basal level (10). The cyclic AMP receptor protein (CRP) mediates catabolite repression at rhaBAD by binding to a site immediately upstream of RhaS that is centered at position Ϫ92.5 relative to the rhaBAD transcription start site (10). CRP alone does not activate rhaBAD expression, but in the presence of RhaS CRP can contribute 30-to 50-fold additional activation (10).CRP is a global regulator of catabolite repression in E. coli (reviewed in reference 6). Interactions between CRP and RNA polymerase (RNAP) that are required for transcription activation have been well defined for promoters where CRP is the only activator. These simple CRP-dependent promoters are categorized according to the location of the CRP-binding site. At class I CRP-dependent promoters CRP binds upstream but not adjacent to RNAP, with sites f...

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confidence: 99%

Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon

Holcroft

Egan

2000

J Bacteriol

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“…With respect to the other inverted repeat potentially acting as RhaS binding site in rhaT, a significant base-pair matching is also found with that of rhaBAD. It has to be pointed out that in rbaBAD promoter this inverted repeat has been experimentally proved to be recognized by RhaS (Egan & Schleif, 1994). Both regulatory proteins, RhaR and RhaS, recognize and bind L-rhamnose, becoming in this way activated to induce the corresponding elements of the rhamnose regulon.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional regulation of the Escherichia coli rhaT gene

et al. 1996

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Transcriptional regulation of the rhaT gene, one of the operons forming the rhamnose regulon in Escherichia coli, was studied by fusing its complete or deleted promoter to the reporter gene lacZ. Analysis of p-galactosidase activities induced in these constructions grown under different conditions predicted the presence of two putative control elements: one for the RhaS regulatory protein and activating the gene not only by L-rhamnose but also by L-lyxose or L-mannose, the other for CAMP-catabolite repression protein and activating this gene in the absence of glucose. Anaerobiosis increased the promoter function two-to threefold with respect to the aerobic condition. Experiments involving complementation of strains containing the rhaTpromoter fusion and carrying a deletion in the rhaS and/or rhaR genes with plasmids bearing the rhamnose regulatory genes showed that rhaT is controlled by a regulatory cascade, in which RhaR induces rhaSR and the accumulated RhaS directly activates rhaT.

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“…The column was washed with urea buffer (8 M urea, 0.5 M NaCl, 20 mM Tris-HCl buffer, pH 8.0) at pH 6.6, and the protein eluted with urea buffer (8 M urea, 0.5 M NaCl, 20 mM Tris-HCl buffer, pH 8.0) at pH 4.5. Fractions were first analyzed on gels, then gathered and dialyzed in the presence of 0.05% salmon sperm DNA against phosphate-buffered saline (57). The protein was then centrifuged at 12,000 ϫ g, and the soluble fraction was analyzed: after dialysis, the fractions that had been eluted at pH Յ 5.5 appeared as a single band in a Coomassie Blue-stained polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

“…This domain (Rv1395Cter, amino acids 229 -344) was His-tagged, overproduced in E. coli, and purified from inclusion bodies in denaturing conditions. Rv1395Cter was then resolubilized and kept in solution in the presence of nonspecific DNA using the method of Egan et al (57). The soluble protein was tested in a binding assay.…”

Section: Rv1395 Binds To the Intergenic Region Of Rv1395-rv1394c By Imentioning

confidence: 99%

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

Recchi

Sclavi

Rauzier

et al. 2003

Journal of Biological Chemistry

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Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing Cterminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.

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DNA-Dependent Renaturation of an Insoluble DNA Binding Protein

Cited by 65 publications

References 0 publications

Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon

Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon

Transcriptional regulation of the Escherichia coli rhaT gene

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

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