We have used time-resolved x-ray-generated hydroxyl radical footprinting to directly characterize, at single-nucleotide resolution, several intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase on the T7A1 promoter at 37°C. Three sets of intermediates, corresponding to two major conformational changes, are resolved as a function of time; multiple conformations equilibrate amongst each other before the next large structural change. Analysis of these data in the context of published structural models indicates that initial recognition involves interaction of the UP element with the ␣-subunit Cterminal domain and binding of the subunit to the ؊35 sequence. In the subsequent isomerization step, two complexes with footprints extending into the ؊10 region can be differentiated as the DNA becomes distorted during nucleation of strand separation. During the final isomerization step, the downstream double helix becomes embedded in the ␤͞␤ jaws, leading to a transcriptionally active complex.DNA-protein interactions ͉ time-resolved footprinting ͉ transcription ͉ hydroxyl radicals D e novo RNA synthesis during DNA transcription is a highly regulated cellular process carried out by RNA polymerase. RNA polymerase binds to DNA and diffuses one-dimensionally to the promoter region (1-4); it then must make a number of interactions within the promoter as a prerequisite to forming a relatively stable complex. This complex is then in a state that favors isomerization, leading to strand separation from approximately Ϫ12 to ϩ3 with respect to the site of transcription initiation (5). Each of these steps is a possible site for regulation of the transcription levels. Extensive studies have been conducted on the structure of RNA polymerase-DNA complexes at equilibrium resulting in the characterization of the interaction of each of the enzyme's subunits with specific promoter elements (6, 7). However a direct characterization of the interactions formed at each step of the recognition process is still lacking.The T7A1 promoter used for the studies described here is one of the strongest known prokaryotic promoters (8). Its Ϫ35 sequence, TTGACT, is very close to the consensus (TTGACA), and its Ϫ10 sequence, GATACT, deviates from consensus (TATAAT). In addition, this promoter contains, from Ϫ42 to Ϫ80, a sequence rich in adenine and thymine residues, also known as an UP element (9-11). On other promoters this sequence has been shown to both increase the rate of promoter binding and stimulate isomerization to the open complex (12)(13)(14)(15). Kinetic studies on the formation of an RNA polymerasepromoter complex revealed the presence of a sequence of isomerization steps leading to the formation of an open complex (16-18). Furthermore, by decreasing the isomerization rates at lower temperatures, one or more intermediates in the pathway from the initial closed complex to the final open complex were isolated and characterized (19-22). However, the large, and sometimes nonlinear, temperature dependence of some ...

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO4 and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild-type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus –10 region. Finally, the comparison of the results obtained at 37°C with those at 20°C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.

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