W0308

X-Ray Crystal Structures of Bacterial RNA Polymerase Holoenzyme and Holoenzyme/Promoter Complex. Katsuhiko S. Murakami, Shoko Masuda, Elizabeth A. Campbell, Oriana Muzzin, Seth A. Darst. Laboratory of Molecular Biophysics, The Rockefeller Univ., 1230 York Ave., New York, NY 10021.

The X-ray crystal structure of the initiating form of bacterial RNA polymerase (RNAP), containing core RNAP (α₂ββ’ω) and the promoter specificity σ subunit, has been determined at 4 Å resolution. Important structural features of the RNAP and their roles in positioning σ within the initiation complex are delineated, as well as the role σ region 1.1 plays in modulating the opening of the RNAP active site channel. The two C-terminal domains of σ are separated by 45 Å on the surface of the RNAP, but are linked by an extended loop that winds through the RNAP near the active site, where it may play a role in initiating nucleotide substrate binding, and out through the RNA exit channel. The advancing RNA transcript must displace the linker, leading to abortive initiation and ultimately to σ release.

The X-ray crystal structure of RNAP holoenzyme complexed with a fork-junction promoter DNA fragment has been determined at 6.5 Å resolution. The DNA lies across one face of the holoenzyme, completely outside the RNAP active site channel. All of the sequence-specific promoter contacts are mediated by the σ subunit via the DNA major groove. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. The structure explains how holoenzyme recognizes promoters containing variably spaced –10 and –35 elements, and provides the basis for models of the closed and open promoter complexes.