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 upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme α subunit carboxy-terminal domain (RNAP αCTD) 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 α2ββ′ς and thus contains two copies of αCTD. Experiments with RNAP derivatives containing only one copy of αCTD indicate, in contrast to a previous report, that the two αCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of αCTD, whereas function of the consensus distal subsite requires both copies of αCTD. We propose that each subsite constitutes a binding site for a copy of αCTD, and that binding of an αCTD 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 αCTD to the distal subsite.