DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Jordi, Bart J.A.M.; Owen-Hughes, Tom; Hulton, C. S. J.; Higgins, Claire A.

doi:10.1002/j.1460-2075.1995.tb00256.x

Cited by 44 publications

(40 citation statements)

References 57 publications

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“…One possible mechanism by which the DRE might act as an effector is by undergoing specific topological changes in response to the interaction of H-NS. This is consistent with the findings reported here that, even in the absence of H-NS, the nature of the downstream sequence influences promoter activity, with published data showing that the proU promoter is sensitive to topological changes (6,13,41) and with observations that H-NS influences DNA topology (5-9). The simplest model consistent with available data is that, in the absence of H-NS, the DRE allows the promoter to adopt an architecture leading to maximum promoter activity.…”

Section: Discussionsupporting

confidence: 93%

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Jordi¹,

Fielder

Burns

et al. 1997

Journal of Biological Chemistry

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H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has beenshown to exhibit a binding preference for certain ATrich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A K d of 1-2 M was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

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Section: Discussionsupporting

confidence: 93%

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Jordi¹,

Fielder

Burns

et al. 1997

Journal of Biological Chemistry

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“…Another model includes a direct competition between H-NS and the RNA polymerase for overlapping binding sites on or near the promoter (10,40). Alternatively, H-NS would exert its repression through modifications of DNA supercoiling (17,20,39).…”

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confidence: 99%

Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing Escherichia coli Hemolysin

et al. 2002

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Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli. We have been able to identify two H-NS binding sites in the hly regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription. Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

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“…Mutations in the hns gene are highly pleiotropic, affecting genome stability (16), recombination-related events (5, 11), and transcription from a variety of promoters (9,10,13,21). Many H-NS-dependent promoters are sensitive to factors which alter DNA supercoiling, and it has been suggested that H-NS may influence transcription through changes in DNA topology (10,11,14,26). Consistent with this hypothesis, H-NS has been shown to constrain DNA supercoils in vitro (42), and hns mutants show changes in the linking number of plasmid DNA isolated from cells (10,12,13).…”

mentioning

confidence: 99%

In vivo supercoiling of plasmid and chromosomal DNA in an Escherichia coli hns mutant

Mojica

Higgins

1997

J Bacteriol

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We have used trimethylpsoralen to measure localized levels of unconstrained DNA supercoiling in vivo. The data provide direct evidence that plasmid and chromosomal DNA supercoiling is altered in vivo in an hns mutant. This increase in supercoiling is independent of transcription or changes in the activity of topoisomerase I. These data have implications for the mechanisms by which the chromatin-associated protein H-NS may influence chromosome organization and gene expression.The Escherichia coli chromosome is organized as a highly condensed structure, the nucleoid. The two most abundant architectural proteins in the nucleoid are HU and H-NS (7). H-NS is a 16.5-kDa polypeptide which binds DNA relatively nonspecifically, although it exhibits a preference for curved DNA (15,27,44). Mutations in the hns gene are highly pleiotropic, affecting genome stability (16), recombination-related events (5, 11), and transcription from a variety of promoters (9,10,13,21). Many H-NS-dependent promoters are sensitive to factors which alter DNA supercoiling, and it has been suggested that H-NS may influence transcription through changes in DNA topology (10,11,14,26). Consistent with this hypothesis, H-NS has been shown to constrain DNA supercoils in vitro (42), and hns mutants show changes in the linking number of plasmid DNA isolated from cells (10,12,13).Although changes in plasmid linking number provide an indication of the level of DNA supercoiling in vivo, this method of assessment suffers from several limitations. First, linking number reports the level of supercoiling of naked DNA after purification from the cell. Because of the constraining influence of proteins bound to DNA in vivo, changes in linking number do not necessarily correspond to changes in unconstrained supercoiling in vivo (1, 17). Second, linking number can report only the mean level of supercoiling throughout an entire DNA molecule; localized domains of supercoiling have been shown to exist within a plasmid (25,33,45). Finally, of course, changes in plasmid linking number cannot be used to determine levels of chromosomal supercoiling (8,30).To circumvent these limitations, and to measure plasmid and chromosomal DNA supercoiling directly in vivo, we have used the DNA cross-linking reagent trimethylpsoralen. Psoralen derivatives are able to penetrate into living cells and intercalate into DNA in a supercoiling-dependent fashion (38). Upon irradiation with long-wavelength UV light, psoralen forms covalent cross-links. The rate of formation of these cross-links is proportional to the level of supercoiling of the DNA in the cell (4, 25) and can detect changes in superhelical density of as small as 12% (25). Using this approach, we have shown that hns mutants have increased negative supercoiling of plasmid DNA in vivo. Furthermore, we demonstrate that chromosomal supercoiling is similarly altered. These studies provide a direct demonstration that the net level of negative supercoiling of both plasmid and chromosomal DNA in vivo is increased in hns mutants. These ...

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DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Cited by 44 publications

References 57 publications

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

DNA Binding Is Not Sufficient for H-NS-mediated Repression ofproU Expression

Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing Escherichia coli Hemolysin

In vivo supercoiling of plasmid and chromosomal DNA in an Escherichia coli hns mutant

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