6 research outputs found

    Characterization of the DNA binding sites of Rv2034.

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    <p>DNaseI footprinting assays of the coding strand (<b>A</b>) and the non-coding strand (<b>B</b>). The ability of Rv2034 to protect the Rv2034 promoter DNA against DNaseI digestion was investigated in the presence of increasing amounts of Rv2034 (lanes 1–4). The sequencing ladders are shown on the side and the corresponding protected regions are indicated by black bars. The DNA sequence of protected regions were read from the sequencing ladder and shown. (<b>C</b>) Sequence and structural characteristics of the protected Rv2034 promoter regions. The regions protected are underlined and the 20 bp sequences containing the inverted repeats (IR) are shown in bold. The translation start codon of Rv2034 is also indicated in bold.</p

    The collection of genes regulated by Rv2034.

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    <p>These genes have been characterized by one or more methods (B1H, EMSA, ChIP, ChIP-seq, etc.). Some of them were critical transcriptional regulators.</p>*<p>indicates by which encoded gene is a transcriptional regulator. Abbreviates: B1H, bacterial one-hybrid assay; EMSA, electrophoretic mobility shift assay; ChIP, Chromatin immunize precipitation assay; ChIP-seq, Chromatin immunize precipitation following DNA sequencing; IHR, initial hypoxic response; EHR, enduring hypoxic response;</p

    Regulation of the <i>dosR</i> gene by Rv2034.

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    <p>(<b>A</b>) Alignment of the promoter DNAs of Rv2034p and Rv3133cp. The numbers indicate the distance from the start codon of each gene. The inverted repeats (IR) are shown in bold and mismatches are highlighted in red. (<b>B</b>) EMSA assays for the DNA-binding activity of Rv2034 on the promoter DNA of Rv3133cp. The DNA substrate was co-incubated with 0, 0.2, 0.4, and 0.8 µM of Rv2034 proteins. (<b>C</b>) The effect of Rv2034 on the <i>dosR</i> promoter region was assayed by constructing a series of promoter-lacZ fusion plasmids. Null promoter-lacZ (pMZ0) and hsp60-lacZ (pMZ+) were used as controls. The names of lacZ-fused plasmids are listed in the left panel, and the elements differing among them are shown in the middle panel. Right panel: The activity of β-galactosidase for the corresponding reporter plasmids were measured in the <i>M.sm</i> wild type strains (hollow bar) or the <i>M.sm</i>/<i>attB</i>::Rv2034 strains (solid bar). β-galactosidase activities (Miller units) are reported as averages from three independent experiments. Errors bars represent standard deviations. For statistical analyses, independent two-sample student's t-tests were performed. Significant differences (P<0.05) are indicated by asterisks (*).</p

    The effects of metal ions on the DNA-binding activity of Rv2034.

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    <p>(<b>A</b>) EMSA assays with or without metal ions. The Rv2034p DNA substrate was incubated with various amounts of the Rv2034 protein (0, 0.1, 0.2, 0.3, 0.4, and 0.5 µM) with or without 1 mM metal ions, as indicted on the top. (<b>B</b>) β-galactosidase activity was measured in <i>M. smegmatis</i> mc<sup>2</sup> 155 containing the pMZ1 and pMZ2 reporter plasmids. Cells were cultured with no metal supplement or maximum permissive concentrations of Zn(II) (70 µM), Ni(II) (35 µM), Co(II) (15 µM), Cd(II) (75 µM), Pb(II) (10 µM), Cu(II) (50 µM), or Mn(II) (0.75 µM).</p

    Rv2034 forms dimer <i>in vitro</i>.

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    <p>Rv2034 wild type and mutant proteins were boiled in the sample buffer in the absence or presence of 10 mM dithiothreitol (DTT) as indicated on the top. The protein size markers are labeled on the left.</p

    DNA-binding activities of Rv2034 wild type and mutant proteins.

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    <p>(<b>A</b>) The effects of Rv2034 and its mutant variants on gene expression were measured by β-galactosidase activity assays. The names of <i>lacZ</i>-fused plasmids are listed in the left panel, and the elements differing among the plasmids are shown in the middle panel. β-galactosidase activities are presented as Miller units in the right panel. The values reported are the averages of three independent experiments. Error bars represent standard deviations. (<b>B</b>) Diagram illustrating Rv2034 variants tested in our assays. The dimerization interface and putative zinc binding sites are illustrated. The Rv2034AE (A32G, E35G) and Rv2034C61A variants were produced by site-directed mutagenesis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036255#s4" target="_blank">Materials and Methods</a>. The Rv2034ΔN28 variant was produced by deleting the first 28 amino acid residues from the N-terminal of Rv2034. (<b>C</b>) EMSA assays for the binding of Rv2034 variants to the Rv2034p promoter DNA. The mutant variants of Rv2034 were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036255#s4" target="_blank">Materials and Methods</a> and co-incubated with the DNA substrate labeled with [γ- <sup>32</sup>P]. The final concentrations of the proteins were 0, 0.1, 0.2, and 0.4 µM in lanes 1, 2, 3 and 4, respectively.</p