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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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DNase I footprinting analysis of WT and mutant Pm DNA using purified His-α. Linear end-labeled probe containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences was made by PCR using labeled primer IRI 21, unlabeled primer IRI 22, and plasmid pMM1 as template. Probe (~0.8 nM) was incubated with either 850 nM Mor or 2.6 μM (++) His-α for 10 min at 30°. For the synergistic binding assays, Mor was incubated with probe for 5 min before the addition of 1.3 μM (+) or 2.6 μM (++) His-α. The mixture was incubated for another 10 min at 30° before DNase I digestion. The region protected by Mor is indicated with a long vertical black line, and the upstream footprint is indicated by a black rectangle. The numbers on the left correspond to positions in the Pm sequence relative to the start site +1. Plasmid names and altered sequence are labeled at the top of each panel as follows: the number indicates the position of the mutagenized base, the first letter indicates the WT base and the second letter indicates the mutant base. (A) Probes were made from WT or mutant plasmid pJM7. (B) Probes were made from WT or mutant plasmids pJM8-pJM11.
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fig6: DNase I footprinting analysis of WT and mutant Pm DNA using purified His-α. Linear end-labeled probe containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences was made by PCR using labeled primer IRI 21, unlabeled primer IRI 22, and plasmid pMM1 as template. Probe (~0.8 nM) was incubated with either 850 nM Mor or 2.6 μM (++) His-α for 10 min at 30°. For the synergistic binding assays, Mor was incubated with probe for 5 min before the addition of 1.3 μM (+) or 2.6 μM (++) His-α. The mixture was incubated for another 10 min at 30° before DNase I digestion. The region protected by Mor is indicated with a long vertical black line, and the upstream footprint is indicated by a black rectangle. The numbers on the left correspond to positions in the Pm sequence relative to the start site +1. Plasmid names and altered sequence are labeled at the top of each panel as follows: the number indicates the position of the mutagenized base, the first letter indicates the WT base and the second letter indicates the mutant base. (A) Probes were made from WT or mutant plasmid pJM7. (B) Probes were made from WT or mutant plasmids pJM8-pJM11.

Mentions: The mutations in pJM7-pJM11 were designed not to affect Mor binding. As expected, gel mobility shift assays with these mutants showed wild-type levels of Mor binding (data not shown), allowing DNase I footprinting assays to be carried out to examine His-α binding without different levels of Mor binding as a confounding variable. The DNase I footprinting pattern of the pJM7 probe, containing mutations −57GA and −56TA and making almost wild-type levels of β-galactosidase was very similar to that of the wild-type probe (Figure 6A); a slight footprint at position −59 and a more pronounced footprint at −61 were generated when Mor and His-α were both added. The pJM8 and pJM10 probes containing −55AG and −54AC mutations (pJM8) or −55AC and −54AC mutations (pJM10) showed little or no footprint at −59 and −61, indicating a substantial reduction in His-α binding (Figure 6B). These results are consistent with the substantially lower β-galactosidase activities of these mutant plasmids. The probe pJM9 containing −55AG and −54AG mutations showed a His-α binding ability intermediate between that of the wild-type probe and that of pJM8 and JM10, consistent with its in vivo transactivation assay which was slightly less than half of the wild-type β-galactosidase activity. When pJM11 was assayed, it had no −61 band, presumably due to the multiple mutations it carries, and showed no changes in band pattern beyond those caused by Mor binding, providing no evidence for or against His-α interaction with the DNA.


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

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

DNase I footprinting analysis of WT and mutant Pm DNA using purified His-α. Linear end-labeled probe containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences was made by PCR using labeled primer IRI 21, unlabeled primer IRI 22, and plasmid pMM1 as template. Probe (~0.8 nM) was incubated with either 850 nM Mor or 2.6 μM (++) His-α for 10 min at 30°. For the synergistic binding assays, Mor was incubated with probe for 5 min before the addition of 1.3 μM (+) or 2.6 μM (++) His-α. The mixture was incubated for another 10 min at 30° before DNase I digestion. The region protected by Mor is indicated with a long vertical black line, and the upstream footprint is indicated by a black rectangle. The numbers on the left correspond to positions in the Pm sequence relative to the start site +1. Plasmid names and altered sequence are labeled at the top of each panel as follows: the number indicates the position of the mutagenized base, the first letter indicates the WT base and the second letter indicates the mutant base. (A) Probes were made from WT or mutant plasmid pJM7. (B) Probes were made from WT or mutant plasmids pJM8-pJM11.
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Related In: Results  -  Collection

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fig6: DNase I footprinting analysis of WT and mutant Pm DNA using purified His-α. Linear end-labeled probe containing Pm sequence from −98 to +10 flanked by pIA12 vector sequences was made by PCR using labeled primer IRI 21, unlabeled primer IRI 22, and plasmid pMM1 as template. Probe (~0.8 nM) was incubated with either 850 nM Mor or 2.6 μM (++) His-α for 10 min at 30°. For the synergistic binding assays, Mor was incubated with probe for 5 min before the addition of 1.3 μM (+) or 2.6 μM (++) His-α. The mixture was incubated for another 10 min at 30° before DNase I digestion. The region protected by Mor is indicated with a long vertical black line, and the upstream footprint is indicated by a black rectangle. The numbers on the left correspond to positions in the Pm sequence relative to the start site +1. Plasmid names and altered sequence are labeled at the top of each panel as follows: the number indicates the position of the mutagenized base, the first letter indicates the WT base and the second letter indicates the mutant base. (A) Probes were made from WT or mutant plasmid pJM7. (B) Probes were made from WT or mutant plasmids pJM8-pJM11.
Mentions: The mutations in pJM7-pJM11 were designed not to affect Mor binding. As expected, gel mobility shift assays with these mutants showed wild-type levels of Mor binding (data not shown), allowing DNase I footprinting assays to be carried out to examine His-α binding without different levels of Mor binding as a confounding variable. The DNase I footprinting pattern of the pJM7 probe, containing mutations −57GA and −56TA and making almost wild-type levels of β-galactosidase was very similar to that of the wild-type probe (Figure 6A); a slight footprint at position −59 and a more pronounced footprint at −61 were generated when Mor and His-α were both added. The pJM8 and pJM10 probes containing −55AG and −54AC mutations (pJM8) or −55AC and −54AC mutations (pJM10) showed little or no footprint at −59 and −61, indicating a substantial reduction in His-α binding (Figure 6B). These results are consistent with the substantially lower β-galactosidase activities of these mutant plasmids. The probe pJM9 containing −55AG and −54AG mutations showed a His-α binding ability intermediate between that of the wild-type probe and that of pJM8 and JM10, consistent with its in vivo transactivation assay which was slightly less than half of the wild-type β-galactosidase activity. When pJM11 was assayed, it had no −61 band, presumably due to the multiple mutations it carries, and showed no changes in band pattern beyond those caused by Mor binding, providing no evidence for or against His-α interaction with the DNA.

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