TRANSCRIPTION ACTIVATION AT PROMOTERS CARRYING TANDEM DNA SITES FOR THE ESCHERICHIA-COLI CYCLIC-AMP RECEPTOR PROTEIN - ORGANIZATION OF THE RNA-POLYMERASE ALPHA-SUBUNITS

We have constructed a family of promoters carrying tandem DNA sites fo r the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the trans cription start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters i s completely dependent on CRP and that, depending on the precise locat ion, CRP bound at the upstream site increases transcription activation . Hydroxyl radical footprinting was exploited to investigate the bindi ng of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP alpha subunits bi nd adjacent to the upstream CRP and that their Precise positioning dep ends on the location of upstream-bound CRP. The C-terminal domains of the RNAP alpha subunits interact with both the upstream and downstream -bound CRP via activating region 1 of CRP. (C) 1998 Academic Press Lim ited .