Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha subunit in transcription activation of the Escherichia coli rhaBAD operon

The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD. Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.     

Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.     

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.     

Genetic evidence for interaction of sigma A with two promoters in Bacillus subtilis.

The specificity of promoter binding by RNA polymerase is governed by the sigma subunit. Recent studies, in which single-amino-acid substitutions in sigma factors have been found to suppress the effects of specific base pair substitutions in promoters, support the model that these sigma factors make sequence-specific contacts with nucleotides at the -10 and -35 regions of promoters. We found that single-amino-acid substitutions in the putative -35 region and -10 region recognition domains of sigma A specifically suppressed the effects of mutations in the -35 and -10 regions, respectively, of two promoters that are expressed in exponentially growing Bacillus subtilis. These mutations change the specificity of sigma A, the primary sigma factor in growing B. subtilis, and demonstrate that this sigma factor interacts with promoters in a manner similar to that of its homolog in Escherichia coli, sigma 70. These mutant derivatives of sigma A also provide a tool that may be useful for determining whether sigma A uses specific promoters in vivo.