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

We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alpha CTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)beta beta'sigma and thus contains two copies of alpha CTD. Experiments with RNAP derivatives containing only one copy of alpha CTD indicate, in contrast to a previous report, that the two alpha CTDs function interchangeably With respect to UP element recognition, furthermore, function of the consensus proximal subsite requires only one copy of alpha CTD, whereas function of the consensus distal subsite requires both copies of alpha CTD. We propose that each subsite constitutes a binding site for a copy of alpha CTD, and that binding of an alpha CTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alpha CTD to the distal subsite.