Location of the C-Terminal Domain of the RNA Polymerase α Subunit in Different Open Complexes at the Escherichia Coli Galactose Operon Regulatory Region

• Published in: Nucleic acids research Vol. 24; no. 12; pp. 2243 - 2251
• Main Authors: Belyaeva, Tamara A., Bown, Jon A., Fujita, Nobuyuki, Ishihama, Akira, Busby, Stephen J. W.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.06.1996
• Subjects: Base Sequence; Binding Sites; DNA; DNA Footprinting; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - metabolism; Galactose - genetics; Molecular Sequence Data; Operon; Promoter Regions, Genetic; Receptors, Cyclic AMP - metabolism; Structure-Activity Relationship
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/24.12.2243

Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1- mutant. We have identified the precise location of a binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase α subunit: α binds to different sites at the upstream end of each complex. Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of α, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex. Thus, the C-terminal domain of α can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of α in ternary CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and α but do not affect the position of α binding to its site upstream of bound CRP.