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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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Related in: MedlinePlus

DNase I footprinting of Pm at 15°. Linear 5′ end-labeled probes containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences were made by PCR using labeled vector primer IRI 21, unlabeled vector primer IRI 22, and plasmids containing WT or mutant Pm as templates. Probe (~0.8 nM) was incubated with 860 nM Mor (+) alone, 50 nM RNAP (+++) alone, or with Mor at 860 nM plus RNAP at 10 nM (+), 20 nM (++), or 50 nM (+++) at 15° before DNase I digestion. The region protected by Mor is indicated with a vertical black line, and the upstream footprint is shown by a black rectangle. The numbers on the right correspond to positions in the Pm sequence relative to the start site +1. The names for the Pm mutant probes are indicated at the top of each panel; (A) probes containing WT Pm and Pm mutants JM6-5, JM6-7, and JM6-10 and (B) probes containing WT and mutant JM6-1.
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fig5: DNase I footprinting of Pm at 15°. Linear 5′ end-labeled probes containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences were made by PCR using labeled vector primer IRI 21, unlabeled vector primer IRI 22, and plasmids containing WT or mutant Pm as templates. Probe (~0.8 nM) was incubated with 860 nM Mor (+) alone, 50 nM RNAP (+++) alone, or with Mor at 860 nM plus RNAP at 10 nM (+), 20 nM (++), or 50 nM (+++) at 15° before DNase I digestion. The region protected by Mor is indicated with a vertical black line, and the upstream footprint is shown by a black rectangle. The numbers on the right correspond to positions in the Pm sequence relative to the start site +1. The names for the Pm mutant probes are indicated at the top of each panel; (A) probes containing WT Pm and Pm mutants JM6-5, JM6-7, and JM6-10 and (B) probes containing WT and mutant JM6-1.

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.


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

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

DNase I footprinting of Pm at 15°. Linear 5′ end-labeled probes containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences were made by PCR using labeled vector primer IRI 21, unlabeled vector primer IRI 22, and plasmids containing WT or mutant Pm as templates. Probe (~0.8 nM) was incubated with 860 nM Mor (+) alone, 50 nM RNAP (+++) alone, or with Mor at 860 nM plus RNAP at 10 nM (+), 20 nM (++), or 50 nM (+++) at 15° before DNase I digestion. The region protected by Mor is indicated with a vertical black line, and the upstream footprint is shown by a black rectangle. The numbers on the right correspond to positions in the Pm sequence relative to the start site +1. The names for the Pm mutant probes are indicated at the top of each panel; (A) probes containing WT Pm and Pm mutants JM6-5, JM6-7, and JM6-10 and (B) probes containing WT and mutant JM6-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: DNase I footprinting of Pm at 15°. Linear 5′ end-labeled probes containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences were made by PCR using labeled vector primer IRI 21, unlabeled vector primer IRI 22, and plasmids containing WT or mutant Pm as templates. Probe (~0.8 nM) was incubated with 860 nM Mor (+) alone, 50 nM RNAP (+++) alone, or with Mor at 860 nM plus RNAP at 10 nM (+), 20 nM (++), or 50 nM (+++) at 15° before DNase I digestion. The region protected by Mor is indicated with a vertical black line, and the upstream footprint is shown by a black rectangle. The numbers on the right correspond to positions in the Pm sequence relative to the start site +1. The names for the Pm mutant probes are indicated at the top of each panel; (A) probes containing WT Pm and Pm mutants JM6-5, JM6-7, and JM6-10 and (B) probes containing WT and mutant JM6-1.
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.

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