The Two α Subunits of Escherichia coli RNA Polymerase are Asymmetrically Arranged and Contact Different Halves of the DNA Upstream Element

RNA polymerase core enzyme of Escherichia 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 the rrnBP1 UP element, and that the β ′-associated α subunit is bound to the promoter-distal region.