Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the Escherichia coli rhaBAD Operon

Abstract

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 ␣ subunit of RNA polymerase deleted for the entire C-terminal domain (␣-⌬235) 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 ␣ subunit (␣-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 ␣-CTD. Overall, our results suggest that CRP interaction with ␣-CTD may not be required for rhaBAD activation; however, ␣-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.Regulation of the Escherichia coli rhaBAD operon responds to both availability of L-rhamnose and catabolite repression. In the presence of L-rhamnose, the AraC family activator RhaS (reviewed in reference 12) binds to a site that spans from position Ϫ32 to position Ϫ81 relative to the rhaBAD transcription start site (9, 10). This RhaS-binding site consists of two 17-bp inverted repeat half-sites that are separated by 16 bp of DNA not contacted by RhaS (9). RhaS alone can activate rhaBAD expression approximately 1,000-fold above the extremely low basal level (10). The cyclic AMP receptor protein (CRP) mediates catabolite repression at rhaBAD by binding to a site immediately upstream of RhaS that is centered at position Ϫ92.5 relative to the rhaBAD transcription start site (10). CRP alone does not activate rhaBAD expression, but in the presence of RhaS CRP can contribute 30-to 50-fold additional activation (10).CRP is a global regulator of catabolite repression in E. coli (reviewed in reference 6). Interactions between CRP and RNA polymerase (RNAP) that are required for transcription activation have been well defined for promoters where CRP is the only activator. These simple CRP-dependent promoters are categorized according to the location of the CRP-binding site. At class I CRP-dependent promoters CRP binds upstream but not adjacent to RNAP, with sites f...

The TodS–TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins

“…We first tested activation in a mutant background in which ␣-C-terminal domain (CTD) lacked 235 residues and found that activity from P todX was Ϸ50% of that in the wild type. This observation is consistent with data obtained for other promoters, such as mtr regulated by TyrR (18), the TOL plasmid P m promoter (17), and the rha promoter of E. coli (15). Subsequently, we used a series of point mutants exhibiting alanine substitutions (19,20).…”

“…The consensus sequence between the two TodT boxes (AAACNNT-NGTTT) can be considered a specific TodT-binding motif. (15,16). To test whether this interaction occurs in the case for TodT, experiments were carried out in Escherichia coli with defects in the ␣-subunits bearing pMIR66 and pMIR77.…”

“…To test whether this interaction occurs in the case for TodT, experiments were carried out in Escherichia coli with defects in the ␣-subunits bearing pMIR66 and pMIR77. This approach was based on an experimental set-up in which most of the RNA polymerase pool contained a mutant subunit (15)(16)(17). The assays were designed to primarily detect a negative effect on transcription.…”

The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

“…It is likely relevant that although the RhaS and RhaR binding sites at rhaBAD and rhaSR are identically positioned (Fig. 1), the CRP (TGTGA motifs [4]) and RhaS sites at rhaBAD are separated by 3 bp, while the CRP and RhaR sites at rhaSR are separated by 21 bp (12,16). In both cases, there is little or no activation by CRP in the absence of RhaS or RhaR (Table 2) (16); presumably, CRP requires DNA bending by the primary activator to enable contacts with ␣-CTD (8).…”

Escherichia coli mhpR gene expression is regulated by catabolite repression mediated by the cAMP–CRP complex

“…Taking our results together with previous data (Torres et al, 2003) we have demonstrated that the glucose effect on mhp expression is exerted at the level of both catabolic and regulatory promoters. This simultaneous CRP control is not unique to the mhp pathway, since it has been also described for the genes for propionate catabolism in E. coli and Salmonella enterica (Lee et al, 2005) and for some regulons from E. coli such as those for L-rhamnose (Holcroft & Egan, 2000;Wickstrum et al, 2005), melibiose (Webster et al, 1988;Belyaeva et al, 2000) and maltose (Chapon & Kolb, 1983;Richet & Sogaard-Andersen, 1994;Richet, 2000). While the expression of the mhpR, melR and malT regulators depends only on the presence of CRP (Webster et al, 1988;Chapon & Kolb, 1983), the expression of rhaSR and prpR depends on the presence of CRP and the transcription activators RhaR and PrpR, respectively (Tobin & Schleif, 1990a, b;Wickstrum et al, 2005;Lee et al, 2005).…”