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