Open AccessThe two α subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element
Author(s) -
Katsuhiko Murakami,
Makoto Kimura,
Jeffrey T. Owens,
Claude F. Meares,
Akira Ishihama
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.5.1709
Subject(s) - specificity factor , protein subunit , polymerase , rna polymerase , transcription (linguistics) , microbiology and biotechnology , transcription bubble , dna , biology , rna , chemistry , biochemistry , rna dependent rna polymerase , gene , linguistics , philosophy
RNA polymerase core enzyme ofEscherichia coli is composed of two α subunits and one each of the β and β′ subunits. The C-terminal domain of the RNA polymerase α subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two α subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct α subunit derivatives in a defined orientation (“oriented α-heterodimer”). The binding sites of two α C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p -bromoacetamidobenzyl)-EDTA·Fe, which was bound at Cys-269 on the UP recognition surface of one or both α subunits. The results clearly indicated that the two α subunits bind in tandem to two helix turns of therrnB P1 UP element, and that the β′-associated α subunit is bound to the promoter–distal region.