The phage Mu middle promoter Pm contains a partial UP element.
Bottom Line: The results demonstrated that mutations upstream of -57 had no effect on Pm activity in vivo, assayed by expression of lacZ fused downstream of a wild-type or mutant Pm.In DNase I footprinting and gel mobility shift assays, paired mutations at positions -55 and -54 did not affect Mor binding but decreased the synergistic binding of Mor with histidine tagged α (His-α), indicating that His-α binds to Pm in a sequence- and/or structure-specific manner.Taken together, these results demonstrate that Pm has a strong proximal UP element subsite, but lacks a distal subsite.
Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.Show MeSH
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Mentions: The footprinting patterns of Mor alone with wild-type or mutant DNA probes (Figure 5) were the same as observed previously (Artsimovitch et al. 1996; Ma and Howe 2004). Reactions with RNA polymerase alone produced little or no protection but did exhibit hypersensitive bands at positions −51 and −12, demonstrating the presence of unstable or weak interactions between RNAP and Pm DNA, as observed previously by Mo and Howe (2014). The upstream mutations had no effect on the −12 hypersensitivity, presumably because it reflects interaction of RNAP with the −10 hexamer. In contrast, none of the mutant probes showed the same high degree of hypersensitivity at band −51. There are two possible explanations: first, the interactions between RNAP and promoter DNA was affected by the mutations, leading to differences in the footprint patterns or second, the local structure of the DNA was changed by the mutations, affecting the accessibility of DNase I to the DNA, thereby altering the footprint pattern. When both Mor and RNA polymerase were present, the −61 to −59 region of Pm was protected. With increasing concentrations of RNA polymerase, this protection became more complete. At the same time, a very weak protection was observed extending downstream to −4. Protection to −4 is characteristic of a closed complex in which RNAP binding is unstable.
Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.