Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the alpha 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 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.

FIG. 1

rhaSR-rhaBAD intergenic region. The top line shows a schematic representation of the regulatory region between rhaBAD and rhaSR. The relative positions of the two RNA polymerases and the activator proteins RhaS, CRP, and RhaR are shown. The bottom lines show the DNA sequence upstream of rhaBAD extending back to position −239 (the rhaSR transcription start site). The positions of the RhaS and RhaR-binding sites are shown by everted arrows, and the position of the CRP-binding site is shown by inverted arrows. The −10 and −35 regions of the two promoters are marked. The upstream endpoints of rhaBAD promoter fusions are identified.

FIG. 2

Effect of single alanine substitutions within α-CTD on activation from Φ(rhaB-lacZ)Δ110 (SME1035) and Φ(rhaB-lacZ)Δ84 (SME1036). Activities are expressed as a percentage of the average activity measured from cells transformed with plasmids encoding the wild-type α subunit. Values shown are the averages of at least three independent experiments. Each α-CTD alanine substitution that significantly lowered expression compared to wild-type α-CTD as determined by analysis of variance statistical analysis is indicated by an asterisk above the bar.

FIG. 3

Space-filling model of predicted α-CTD structure. The model was based on the atomic coordinates of Jeon et al. (15). Colored residues are those identified as important at the Φ(rhaB-lacZ)Δ84 promoter fusion. Orange residues are those that may be involved in interaction with DNA (DNA-binding category), while violet residues are the residues that are unlikely to be involved in interaction with DNA (Other category). Residue numbers for some of the important residues are shown. The two models are related to one another by a 90° rotation around the vertical axis.