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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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Gel retardation analysis of His-α and Mor binding to WT and mutant Pm DNA. Radiolabeled DNA probes (~0.4 nM) containing Pm sequence −98 to +10 were incubated with 136 nM Mor or 2.7 μM (++) His-α for 10 min at 30°. When both proteins were used, Mor was incubated with the probe for 10 min, His-α was added to a final concentration of 1.3 μM (+) or 2.7 μM (++), and a second incubation for 10 min at 30° was performed. Binding mixtures were then loaded onto 4% acrylamide native gels containing 5% glycerol and 0.5x Tris/Borate/EDTA (TBE) buffer and subjected to electrophoresis in 0.5x TBE buffer containing 2% glycerol at 3°. The name of each mutant plasmid template and the mutations in each is given above each bracket. The positions of free probe, Mor-DNA binary complex, and Mor-His-α-DNA ternary complexes are indicated with F, B, and T, respectively. Note the unusual behavior of the ternary complex: the greater the His-α concentration, the slower the mobility of the complex. This behavior is characteristic of α whether or not it is tagged. In fact, when multiple concentrations are used, each increased concentration yields a slower band (Savery et al. 1998). An explanation for this behavior has not been determined.
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fig7: Gel retardation analysis of His-α and Mor binding to WT and mutant Pm DNA. Radiolabeled DNA probes (~0.4 nM) containing Pm sequence −98 to +10 were incubated with 136 nM Mor or 2.7 μM (++) His-α for 10 min at 30°. When both proteins were used, Mor was incubated with the probe for 10 min, His-α was added to a final concentration of 1.3 μM (+) or 2.7 μM (++), and a second incubation for 10 min at 30° was performed. Binding mixtures were then loaded onto 4% acrylamide native gels containing 5% glycerol and 0.5x Tris/Borate/EDTA (TBE) buffer and subjected to electrophoresis in 0.5x TBE buffer containing 2% glycerol at 3°. The name of each mutant plasmid template and the mutations in each is given above each bracket. The positions of free probe, Mor-DNA binary complex, and Mor-His-α-DNA ternary complexes are indicated with F, B, and T, respectively. Note the unusual behavior of the ternary complex: the greater the His-α concentration, the slower the mobility of the complex. This behavior is characteristic of α whether or not it is tagged. In fact, when multiple concentrations are used, each increased concentration yields a slower band (Savery et al. 1998). An explanation for this behavior has not been determined.

Mentions: To examine the effects of the pJM7-pJM11 mutations on synergistic binding of His-α and Mor to Pm, we carried out gel mobility shift assays with purified His-α and Mor (Figure 7). When wild-type probes were used, no shift was observed for His-α alone even at a high concentration of His-α (2.7 μM), and only one-third to one-half of the probe was shifted with Mor alone. In contrast, when both proteins were added, the wild-type DNA probes were almost completely found in a supershifted species corresponding to a Mor-His-α-DNA ternary complex, even at a low concentration of His-α (1.3 μM). When mutant probes were used, different shifting behaviors were observed. Probe containing mutations at positions −57 and −56 (pJM7) gave a shift similar to the wild-type probe, consistent with its wild-type β-galactosidase activity. When probes containing −55AC and −54AC mutations (pJM10 and pJM11) were used, only a binary complex of Mor and probe was observed, even at a greater concentration of His-α (2.7 μM), suggesting that these mutations disrupted interactions between the αCTD and DNA, which is consistent with their very low β-galactosidase activities. The probe containing −55AG and −54AC mutations (pJM8) showed reduced synergistic binding, relative to the wild-type probe. At the lower concentration of His-α (1.3 μM), there was no supershifted complex, and when more His-α (2.7 μM) was used, only a small fraction of the probe was seen in a supershifted complex. The probe containing −55AG and −54AG mutations (pJM9) showed a shifting ability intermediate between the wild-type probe and the pJM10 and pJM11 probes containing A to C substitutions at these positions. For pJM9, at 1.3 μM His-α, both binary and ternary complexes were observed and when a higher concentration of His-α (2.7 μM) was used, more than half of the probe was seen in the supershifted species.


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

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

Gel retardation analysis of His-α and Mor binding to WT and mutant Pm DNA. Radiolabeled DNA probes (~0.4 nM) containing Pm sequence −98 to +10 were incubated with 136 nM Mor or 2.7 μM (++) His-α for 10 min at 30°. When both proteins were used, Mor was incubated with the probe for 10 min, His-α was added to a final concentration of 1.3 μM (+) or 2.7 μM (++), and a second incubation for 10 min at 30° was performed. Binding mixtures were then loaded onto 4% acrylamide native gels containing 5% glycerol and 0.5x Tris/Borate/EDTA (TBE) buffer and subjected to electrophoresis in 0.5x TBE buffer containing 2% glycerol at 3°. The name of each mutant plasmid template and the mutations in each is given above each bracket. The positions of free probe, Mor-DNA binary complex, and Mor-His-α-DNA ternary complexes are indicated with F, B, and T, respectively. Note the unusual behavior of the ternary complex: the greater the His-α concentration, the slower the mobility of the complex. This behavior is characteristic of α whether or not it is tagged. In fact, when multiple concentrations are used, each increased concentration yields a slower band (Savery et al. 1998). An explanation for this behavior has not been determined.
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Related In: Results  -  Collection

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fig7: Gel retardation analysis of His-α and Mor binding to WT and mutant Pm DNA. Radiolabeled DNA probes (~0.4 nM) containing Pm sequence −98 to +10 were incubated with 136 nM Mor or 2.7 μM (++) His-α for 10 min at 30°. When both proteins were used, Mor was incubated with the probe for 10 min, His-α was added to a final concentration of 1.3 μM (+) or 2.7 μM (++), and a second incubation for 10 min at 30° was performed. Binding mixtures were then loaded onto 4% acrylamide native gels containing 5% glycerol and 0.5x Tris/Borate/EDTA (TBE) buffer and subjected to electrophoresis in 0.5x TBE buffer containing 2% glycerol at 3°. The name of each mutant plasmid template and the mutations in each is given above each bracket. The positions of free probe, Mor-DNA binary complex, and Mor-His-α-DNA ternary complexes are indicated with F, B, and T, respectively. Note the unusual behavior of the ternary complex: the greater the His-α concentration, the slower the mobility of the complex. This behavior is characteristic of α whether or not it is tagged. In fact, when multiple concentrations are used, each increased concentration yields a slower band (Savery et al. 1998). An explanation for this behavior has not been determined.
Mentions: To examine the effects of the pJM7-pJM11 mutations on synergistic binding of His-α and Mor to Pm, we carried out gel mobility shift assays with purified His-α and Mor (Figure 7). When wild-type probes were used, no shift was observed for His-α alone even at a high concentration of His-α (2.7 μM), and only one-third to one-half of the probe was shifted with Mor alone. In contrast, when both proteins were added, the wild-type DNA probes were almost completely found in a supershifted species corresponding to a Mor-His-α-DNA ternary complex, even at a low concentration of His-α (1.3 μM). When mutant probes were used, different shifting behaviors were observed. Probe containing mutations at positions −57 and −56 (pJM7) gave a shift similar to the wild-type probe, consistent with its wild-type β-galactosidase activity. When probes containing −55AC and −54AC mutations (pJM10 and pJM11) were used, only a binary complex of Mor and probe was observed, even at a greater concentration of His-α (2.7 μM), suggesting that these mutations disrupted interactions between the αCTD and DNA, which is consistent with their very low β-galactosidase activities. The probe containing −55AG and −54AC mutations (pJM8) showed reduced synergistic binding, relative to the wild-type probe. At the lower concentration of His-α (1.3 μM), there was no supershifted complex, and when more His-α (2.7 μM) was used, only a small fraction of the probe was seen in a supershifted complex. The probe containing −55AG and −54AG mutations (pJM9) showed a shifting ability intermediate between the wild-type probe and the pJM10 and pJM11 probes containing A to C substitutions at these positions. For pJM9, at 1.3 μM His-α, both binary and ternary complexes were observed and when a higher concentration of His-α (2.7 μM) was used, more than half of the probe was seen in the supershifted species.

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