TRANSCRIPTION ACTIVATION BY THE ESCHERICHIA-COLI CYCLIC-AMP RECEPTOR PROTEIN - RECEPTORS BOUND IN TANDEM AT PROMOTERS CAN INTERACT SYNERGISTICALLY

Starting with a semi-synthetic Escherichia coli promoter with a bindin g site for the cyclic AMP receptor protein (CRP) centred between base- pairs 41 and 42 upstream from the transcription start site, a second u pstream CRP-binding site, centred between base-pairs 90 and 91, was in troduced. CRP binding to this second upstream site results in a severa l-fold greater stimulation of CRP-dependent transcription initiation, compared to activation at the starting promoter with just one CRP-bind ing site. Activation of transcription by the upstream CRP molecule is blocked by the HL159 substitution, suggesting that the upstream-bound CRP makes a direct contact with RNA polymerase. Footprinting experimen ts suggest that RNA polymerase contacts the promoter DNA between the t wo CRP-binding sites, most likely due to interactions involving the C- terminal part of the alpha subunit. Synergy between tandem bound CR mo lecules in transcription activation requires that the two CRP-binding sites be separated by around 40 or 50 base-pairs, but is not found at intermediate spacings. An experiment in which the upstream CRP-binding site is replaced by a site for the related transcription factor, FNR, shows that heterologous synergistic interactions between FNR and CRP are possible