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The phage Mu middle promoter Pm contains a partial UP element.

Ma J, Howe MM - G3 (Bethesda) (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.

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Representative results for gel retardation analysis of Mor binding to Pm DNA fragments with upstream mutations. Radiolabeled WT or mutant Pm probes containing Pm sequence −98 to +10 (at ~0.4 nM) were incubated with 0 (−), 68 nM (+), or 340 nM (++) purified Mor protein in 20 μL of binding buffer (Ma and Howe 2004) for 15 min at 30°. Binding mixtures were loaded onto 8% native acrylamide gels (29:1) in 0.5x Tris/Borate/EDTA buffer and subjected to electrophoresis for 3 hr at 4° and 15V/cm. The positions of free probe and bound complexes are indicated by F and B, respectively.
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fig4: Representative results for gel retardation analysis of Mor binding to Pm DNA fragments with upstream mutations. Radiolabeled WT or mutant Pm probes containing Pm sequence −98 to +10 (at ~0.4 nM) were incubated with 0 (−), 68 nM (+), or 340 nM (++) purified Mor protein in 20 μL of binding buffer (Ma and Howe 2004) for 15 min at 30°. Binding mixtures were loaded onto 8% native acrylamide gels (29:1) in 0.5x Tris/Borate/EDTA buffer and subjected to electrophoresis for 3 hr at 4° and 15V/cm. The positions of free probe and bound complexes are indicated by F and B, respectively.

Mentions: Because mutations close to the Mor binding site (−51 to −36) could affect Pm activity by altering Mor binding, gel mobility shift assays were performed to examine Mor binding to mutant Pm DNA fragments. All of the mutants had substantial Mor binding as shown in the representative gels presented in Figure 4.


The phage Mu middle promoter Pm contains a partial UP element.

Ma J, Howe MM - G3 (Bethesda) (2015)

Representative results for gel retardation analysis of Mor binding to Pm DNA fragments with upstream mutations. Radiolabeled WT or mutant Pm probes containing Pm sequence −98 to +10 (at ~0.4 nM) were incubated with 0 (−), 68 nM (+), or 340 nM (++) purified Mor protein in 20 μL of binding buffer (Ma and Howe 2004) for 15 min at 30°. Binding mixtures were loaded onto 8% native acrylamide gels (29:1) in 0.5x Tris/Borate/EDTA buffer and subjected to electrophoresis for 3 hr at 4° and 15V/cm. The positions of free probe and bound complexes are indicated by F and B, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390567&req=5

fig4: Representative results for gel retardation analysis of Mor binding to Pm DNA fragments with upstream mutations. Radiolabeled WT or mutant Pm probes containing Pm sequence −98 to +10 (at ~0.4 nM) were incubated with 0 (−), 68 nM (+), or 340 nM (++) purified Mor protein in 20 μL of binding buffer (Ma and Howe 2004) for 15 min at 30°. Binding mixtures were loaded onto 8% native acrylamide gels (29:1) in 0.5x Tris/Borate/EDTA buffer and subjected to electrophoresis for 3 hr at 4° and 15V/cm. The positions of free probe and bound complexes are indicated by F and B, respectively.
Mentions: Because mutations close to the Mor binding site (−51 to −36) could affect Pm activity by altering Mor binding, gel mobility shift assays were performed to examine Mor binding to mutant Pm DNA fragments. All of the mutants had substantial Mor binding as shown in the representative gels presented in Figure 4.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.

Show MeSH
Related in: MedlinePlus