Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C-terminal domain of the RNA polymerase alpha subunit.

We have investigated the role of the RNA polymerase alpha subunit during MelR-dependent activation of transcription at the Escherichia coli melAB promoter. To do this, we used a simplified melAB promoter derivative that is dependent on MelR binding at two 18 bp sites, located from position -34 to -51 and from position -54 to -71, upstream of the transcription start site. Results from experiments with hydroxyl radical footprinting, and with RNA polymerase, carrying alpha subunits that were tagged with a chemical nuclease, show that the C-terminal domains of the RNA polymerase alpha subunits are located near position -52 and near position -72 during transcription activation. We demonstrate that the C-terminal domain of the RNA polymerase alpha subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase alpha subunit can interact with MelR. Finally, we used alanine scanning to identify determinants in the C-terminal domain of the RNA polymerase alpha subunit that are important for MelR-dependent activation of the melAB promoter.