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In addition, the propagation of this torsional stress (DNA unwinding), accompanied by a shift of the DNA curvature apex towards the TSS, would also facilitate DNA melting and the recruitment of the RNA Pol II at the TATA box.
The resulting materials were analyzed on native polyacrylamide gels, in a nucleosome gel-shift whereby a shift of the DNA band indicates mononucleosome formation.
As a positive control we used standard conditions to detect the PdhR-dependent shift of the DNA fragment of the previously reported PdhR binding site within the promoter region of the pdhR- aceEF- ldpA operon (Fig. 4A).
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The shifting of the DNA was concentration dependent on Sryα lysate.
When an unrelated transcriptional regulator VicR was used instead of MbrC in the binding reaction as a control to test the specificity of the assay, no shifting of the DNA band was found while with increasing MbrC concentrations, a clear shift was observed (upper band around 350 bp).
The decrease in the dye's DNA binding affinity due to salt effect, as suggested by the shifting of the DNA binding curves to the right, was also observed when the 100 mM KCl in the buffer was replaced with 100 mM MgCl2 (data not shown).
There is great homogeneity in the shift of all the DNA-B points relative to the DNA-A points, and the general trend is a change roughly parallel to the diagonal (indicated by the white arrows).
However, the further increase of the GO concentration did not continually increase the shift distances of the DNA fragment.
The chemical shift perturbations of the DNA nonexchangeable protons of the major conformer relative to its unmodified control duplex are depicted in Figure 4B.
Anti-Sp1 and anti-Sp3 antibodies could bring about a super-shift of the DNA-protein complex (Figure 6A, lanes 2, 3) demonstrating that PMA does not affect the Sp1- Sp3 interaction.
To obtain a model that was free of clashes, only a minor shift of the upstream DNA was required.
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