TRANSCRIPTION ACTIVATION AT ESCHERICHIA-COLI PROMOTERS DEPENDENT ON THE CYCLIC-AMP RECEPTOR PROTEIN - EFFECTS OF BINDING SEQUENCES FOR THE RNA-POLYMERASE ALPHA-SUBUNIT

Transcription activation at two semi-synthetic Escherichia coli promot ers, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream fr om the respective transcription startpoints. An UP-element that can bi nd the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit wa s cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent prom oter activity was increased by the UP-element, but CRP-independent act ivity was not increased. DNase I footprinting was exploited to investi gate the juxtaposition of bound CRP and RNAP alpha-subunits. In both c ases, CRP and RNAP alpha-subunits occupy their cognate binding sites i n ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the alpha-element in the absence of CRP, but this is not sufficient fo r open complex formation. The positive effects of binding RNAP alpha-s ubunits upstream of the DNA site for CRP at -41.5 are suppressed if th e UP-element is incorrectly positioned.