[10] Footprinting protein-DNA complexes in Vivo

Sasse-Dwight, Selina; Gralla, Jay D.

doi:10.1016/0076-6879(91)08012-7

Cited by 167 publications

(179 citation statements)

References 20 publications

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“…The in vivo footprinting reactions were performed essentially as described by Sasse-Dwight and Gralla (1991). Briefly, the overnight cultures were diluted in fresh medium and allowed to grow for 1 h at 37ЊC.…”

Section: Dms Footprintingmentioning

confidence: 99%

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

Schneider

Travers

Muskhelishvili

1997

Molecular Microbiology

114

104

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SummaryThe Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.

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Section: Dms Footprintingmentioning

confidence: 99%

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

Schneider

Travers

Muskhelishvili

1997

Molecular Microbiology

114

104

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“…The procedure used for chemical probing for secondary structure was similar to that of Sasse-Dwight and Gralla (1991). Specifically for in vitro probing, the DNA template was prepared using the alkaline lysis procedure (Sambrook et al, 1989), suspended in 10 mM Tris, 1 mM EDTA, pH 8.0, and treated with 1 mM KMnO 4 at 20ЊC for 15 min.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H‐NS, and CRP–cAMP in regulation

Mukerji

Mahadevan

1997

Molecular Microbiology

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SummaryThe bgl operon of Escherichia coli is rendered cryptic and uninducible in wild-type cells by the presence of DNA structural elements that negatively regulate transcription. We have carried out a detailed analysis of the sequences implicated in negative regulation. Finestructure deletion analysis of the upstream sequences showed the presence of at least two elements involved in silencing the promoter. Chemical probing of genomic DNA in vivo showed that a region of dyad symmetry, present upstream of the promoter, is hypersensitive to KMnO 4 . The hypersensitive region detected corresponds to the potential cruciform structure implicated earlier in negative regulation. Enhancement of transcription from the wild-type promoter, observed in the presence of the gyrase inhibitor novobiocin, was absent in a mutant that carried point mutations in the inverted repeat. This observation suggests that the activation seen in a gyrase mutant is mediated by destabilization of the cruciform because of reduced supercoiling. Deletion of sequences downstream of the potential cruciform also resulted in an increase in transcription, indicating the presence of a second regulatory element. Measurement of transcription from the bgl promoter carrying the deletion, in a strain that has a mutation in the hns gene, indicated that this region is likely to be involved in binding to H-NS or a protein regulated by H-NS, which acts as a non-specific repressor. We also provide evidence which suggests that transcriptional activation by mutations at the cAMP receptor protein (CRP)-binding site is mediated partly by antagonization of the negative effect of H-NS by CRP-cAMP as a result of its increased affinity for the mutant site.

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“…This assay takes advantage of KMnO 4 reactivity with thymine residues in single-stranded DNA, thus allowing us to probe open complex formation by RNA polymerase (42). As expected, E S is favored in promoter opening at the wild type aidB promoter; E S -induced KMnO 4 reactivity extends to a T nucleotide at position ϩ2 in the template strand.…”

Section: Promoter Elements Determinant For Selective Recognition Of Tmentioning

confidence: 99%

Nucleotides from –16 to –12 Determine Specific Promoter Recognition by Bacterial σS-RNA Polymerase

Lacour

Kolb

Landini

2003

Journal of Biological Chemistry

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The alternative sigma factor S , mainly active in stationary phase of growth, recognizes in vitro a ؊10 promoter sequence almost identical to the one for the main sigma factor, 70 , thus raising the problem of how specific promoter recognition by S -RNA polymerase (E S ) is achieved in vivo. We investigated the promoter features involved in selective recognition by E S at the strictly S -dependent aidB promoter. We show that the presence of a C nucleotide as first residue of the aidB ؊10 sequence (؊12C), instead of the T nucleotide canonical for 70 -dependent promoters, is the major determinant for selective recognition by E S . The presence of the ؊12C does not allow formation of an open complex fully proficient in transcription initiation by E 70 . The role of ؊12C as specific determinant for promoter recognition by E S was confirmed by sequence analysis of known E S -dependent promoters as well as site-directed mutagenesis at the promoters of the csgB and sprE genes. We propose that E S , unlike E 70 , can recognize both C and T as the first nucleotide in the ؊10 sequence. Additional promoter features such as the presence of a C nucleotide at position ؊13, contributing to open complex formation by E S , and a TG motif found at the unusual ؊16/؊15 location, possibly contributing to initial binding to the promoter, also represent important factors for S -dependent transcription. We propose a new sequence, TG(N) 0 -2 CCATA(c/a)T, as consensus ؊10 sequence for promoters exclusively recognized by E S .Bacterial cells adapt to changing environmental and physiological conditions by modulating gene expression. Sigma ( ) factors of RNA polymerase, as the subunits responsible for promoter recognition, play a major role in programming gene expression. At least seven different subunits have been identified in Escherichia coli; 70 is the main subunit and can carry out transcription from the majority of E. coli promoters. The alternative subunits can direct transcription toward specific sets of genes (i.e. heat-shock, extracellular proteins, etc.) whose transcription is directed by -specific promoter sequences. A partial exception to the typical role for alternative subunits is represented by S , the product of the rpoS gene, mainly expressed in the stationary phase of growth (1-3). Unlike the other subunits, S -RNA polymerase (E S ) 1 can initiate transcription from several promoters also recognized by E 70 , suggesting that they recognize similar promoter sequences (4). The recognition of similar promoter sequences by S and 70 is reflected by their strong similarity in the DNA binding domains (5). The alignment of E S -dependent promoters and the search for an optimal promoter for E S in vitro using the systematic evolution of ligands by exponential enrichment (SELEX) procedure have pointed to a Ϫ10 consensus sequence for E S , CTATA(c/a)T that is very similar to the canonical TATAAT sequence for 70 (6 -10). These results suggest that promoter selectivity between 70 and S might be determined by factors other than promote...

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[10] Footprinting protein-DNA complexes in Vivo

Cited by 167 publications

References 20 publications

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H‐NS, and CRP–cAMP in regulation

Nucleotides from –16 to –12 Determine Specific Promoter Recognition by Bacterial σS-RNA Polymerase

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