Effect of Altered Spacing between uhpTPromoter Elements on Transcription Activation

ABSTRACT Many bacterial promoters possess multiple sites for binding of transcriptional activator proteins. The uhpT promoter, which controls expression of the sugar phosphate transport system inEscherichia coli, possesses multiple sites for its specific activator protein, UhpA, and a single site for binding of the global regulator, the catabolite gene activator protein (CAP). The binding of UhpA to the uhpT promoter was determined by DNase protection assays; UhpA displayed different affinities for the target sites. The upstream or strong sites, between positions −80 and −50, exhibited a higher affinity for UhpA than did the downstream or weak sites, between positions −50 and −32, adjoining the RNA polymerase-binding site. Phosphorylation of UhpA strongly increased its affinity for both sites. To examine the possible roles of the two sets of UhpA-binding sites, a series of insertion and deletion mutations were introduced at the boundary between them, as suggested from the positions that were protected by UhpA against hydroxyl radical cleavage. Deletions extended in the direction of the weak sites. The insertion or deletion of one helical turn of DNA resulted in the loss of promoter activity and of occupancy by UhpA of the remaining weak-site sequences but was accompanied by normal occupancy of the strong site and no change in the gel retardation behavior of the promoter fragments. However, the deletion of two helical turns of DNA, i.e., 20, 21, or 22 bp, resulted in the novel appearance of UhpA-independent expression and in an additional level of expression that was dependent on UhpA but independent of an inducing signal. The UhpA-independent promoter activity was shown to result from activation by CAP at its more proximal position. UhpA-dependent activity under noninducing conditions appears to result from the binding of unphosphorylated UhpA to the strong sites, which are now in the position normally occupied by the weak sites. Thus, regulated phosphorylation of the response regulator UhpA enhances its occupancy of the weak sites where favorable contacts can allow the binding of RNA polymerase to the promoter.

Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences in gut and papOperons

ABSTRACT Most of the adenine residues in GATC sequences in theEscherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepap GATC-I and GATC-II sites.