Binding of RNA polymerase and the catabolite gene activator protein within the cat promoter in Escherichia coli

1981 ◽  
Vol 150 (2) ◽  
pp. 185-196 ◽  
Author(s):  
Stuart F.J. Le Grice ◽  
Hans Matzura
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Activator Protein ◽  
Catabolite Gene Activator Protein

scholarly journals Effect of Altered Spacing between uhpTPromoter Elements on Transcription Activation

2000 ◽  
Vol 182 (16) ◽  
pp. 4430-4436 ◽  
Author(s):  
Qing Chen ◽  
Robert J. Kadner
Keyword(s):  
Rna Polymerase ◽  
Promoter Activity ◽  
Response Regulator ◽  
Phosphate Transport ◽  
Activator Proteins ◽  
Activator Protein ◽  
Radical Cleavage ◽  
Catabolite Gene Activator Protein ◽  
Polymerase Binding ◽  
Helical Turn

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.

scholarly journals Catabolite Gene Activator Protein Mutations Affecting Activity of the araBAD Promoter

1998 ◽  
Vol 180 (2) ◽  
pp. 195-200 ◽  
Author(s):  
Xin Zhang ◽  
Robert Schleif
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Dna Looping ◽  
Activator Protein ◽  
Induced Folding ◽  
Activation Mechanisms ◽  
Dna Binding Affinity ◽  
Catabolite Gene Activator Protein ◽  
Indirect Activation

ABSTRACT We have studied catabolite gene activator protein (CAP) activation at the araBAD promoter, pBAD , in the absence of DNA looping. We ruled out the two most plausible indirect activation mechanisms: CAP-induced folding of upstream DNA back upon RNA polymerase, and CAP-induced stabilization of AraC binding to DNA. Therefore, a direct CAP-RNA polymerase interaction seemed likely. We sought and found CAP mutants defective in transcription activation at pBAD that retained normal DNA binding affinity. Some mutations altered residues in the interval from positions 150 to 164 that includes CAP activating region 1 (AR1), which has been shown to contact RNA polymerase at a number of promoters. Unexpectedly, additional mutations were found that altered residues in the region between positions 46 and 68 and at position 133. This includes the region known as activating region 3 (AR3). Mutations from both groups also affect the araFGH and rhaBADpromoters.

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