Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter

Abstract

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 ...

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

“…It reacts rapidly and exhibits little if any sequence specificity, making it very useful for probing transient protein-DNA interactions (11). Additionally, the rapid rate of ⅐OH reaction with the DNA backbone means that fractional protection is approximately proportional to fractional occupancy for a short-lived, rapidly equilibrating intermediate like I 1 (10). By following the appearance of protection of the DNA backbone as a function of time (milliseconds to seconds) after mixing at 37°C, this study (10) circumvented potential issues raised by trapping promoter complexes at low temperature (11) and possible displacement of weak upstream interactions by DNase I (8,9).…”

Catching RNA polymerase in the act of binding: Intermediates in transcription illuminated by synchrotron footprinting

“…Chemical and radiolytic methods have been used to generate • OH for static, equilibrium, and time-resolved nucleic acid footprinting (6,(8)(9)(10)(11). A singular advantage to the use of a synchrotron x-ray beam [such as that used by Sclavi et al (1)] for radiolytic generation of • OH is that millisecond exposure yields sufficient concentrations of radicals for footprinting. When coupled to a rapid mixer, synchrotron footprinting can follow the evolution of changes in the solvent-accessible surface of nucleic acids with resolution as fine as single-nucleotide at times as short as milliseconds.…”

“…B. Sclavi (1) participated in the development of this technique, principally in its application to RNA folding (11)(12)(13). While synchrotron footprinting has also been applied to the kinetics of protein-DNA interactions (14), the study by Sclavi et al (1) breaks new ground in the complexity of the reaction being explored, the analyis approach being used, and the technical implementation of the experiment.…”

“…Sclavi et al (1) are the first to explicitly test footprinting progress curves against formal kinetic models. The development of the structural-kinetic model shown in figure 5 of ref.…”

“…The technique of nucleic acid footprinting at the heart of the approach used by Sclavi et al (1) is an ensemble approach that typically utilizes the inhibition of nuclease cleavage to report changes in the solvent accessibility of the backbone of DNA or RNA due to conformational changes and͞or protein binding. Nuclease cleavage products are sorted by size and separated by electrophoresis; the term ''footprint'' reflects the decreased intensity of the electrophoretic bands representing nucleotides whose backbone is protected from cleavage (Fig.…”