Transcription activation at the Escherichia coli melAB promoter: Interactions of MelR with the C-terminal domain of the RNA polymerase α subunit

Abstract

We have investigated the role of the RNA polymerase o. 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 α subunits that were tagged with a chemical nuclease, show that the C-terminal domains of the RNA polymerase α subunits are located near position -52 and near position -72 during transcription activation. We demonstrate that the C-terminal domain of the RNA polymerase α subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase α subunit can interact with MelR. Finally, we used alanine scanning to identify determinants in the C-terminal domain of the RNA polymerase α subunit that are important for MelR-dependent activation of the melAB promoter.