THE 2 ALPHA-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 alph a subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase of subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) e lements of promoter DNA, using the same protein surface, To identify p ossible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orienta tion (''oriented alpha-heterodimer''). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl r adical-based DNA cleavage mediated bf (p-bromoacetamidobenzyl)-EDTA . Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits, The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-d istal region.