Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit

We demonstrate here that the previously described bacterial promoter upstre am element (UP element) consists of two distinct subsites, each of which, b y itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-term inal domain (RNAP alpha CTD) and stimulate transcription. Using binding-sit e-selection experiments, we identify the consensus sequence for each subsit e. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAA ARNR-3') stimulate transcription up to 170-fold, and the selected distal su bsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transc ription up to 16-fold. RNAP has subunit composition alpha(2)beta beta'sigma and thus contains two copies of alpha CTD. Experiments with RNAP derivativ es containing only one copy of alpha CTD indicate, in contrast to a previou s report, that the two alpha CTDs function interchangeably With respect to UP element recognition, furthermore, function of the consensus proximal sub site requires only one copy of alpha CTD, whereas function of the consensus distal subsite requires both copies of alpha CTD. We propose that each sub site constitutes a binding site for a copy of alpha CTD, and that binding o f an alpha CTD to the proximal subsite region (through specific interaction s with a consensus proximal subsite or through nonspecific interactions wit h a nonconsensus proximal subsite) is a prerequisite for binding of the oth er alpha CTD to the distal subsite.