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Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C‐terminal domain of the RNA polymerase α subunit
Author(s) -
Grainger David C.,
Belyaeva Tamara A.,
Lee David J.,
Hyde Eva I.,
Busby Stephen J. W.
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2003.03930.x
Subject(s) - biology , rna polymerase , microbiology and biotechnology , specificity factor , transcription (linguistics) , rna polymerase i , rna polymerase ii holoenzyme , polymerase , transcription factor ii d , transcription bubble , rna polymerase ii , protein subunit , rna , promoter , biochemistry , gene , gene expression , linguistics , philosophy
Summary We have investigated the role of the RNA polymerase α 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.