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