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 a 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 a subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct a 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 a 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.
|Original language||English (US)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Mar 4 1997|
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