E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.

A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.     

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.