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Characterization of DNA structural elements and conformational changes accompanying the binding of T7 RNA polymerase to its promoter
The first step of transcription initiation, promoter binding, is characterized in the T7 RNA polymerase model system. A previous two-domain model for the T7 RNA polymerase promoter is tested by the use of different fluorescence methods. The increase in the fluorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations (best fit value of Kd is calculated to be 4.0 nM), and is the first quantitative assay to reliably measure both increases and decreases in the dissociation constant. The assay is used to follow polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence, and results show that the duplex region of the promoter upstream of and including position −5 is both necessary and sufficient for tight binding, and represents the core binding element of the promoter. In separate assays, 2-aminopurine, a fluorescent nucleotide analog was placed at different locations within the upstream and downstream domains of the promoter. The fluorescence intensity of 2-aminopurine reports on local base stacking and is used both to measure binding and also to provide direct evidence for local melting. Results with this probe confirm that the promoter region upstream of position −5 is recognized as a duplex, in contrast to the downstream promoter region, which appears to bind in a distorted or melted conformation, in the static polymerase-promoter complex. Our kinetic experiments also indicate that polymerase binding to its promoter and the structural transition in the DNA near the transcription start site is very fast, with binding close to the diffusion limit. Finally, gel mobility shift assays detect a small intrinsic (∼9°) and a larger (40–60°) protein-induced DNA bend for the T7 RNA polymerase. The protein-induced bend is estimated to be centered near the transcription start site. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. Furthermore, the protein-induced bend may provide the mechanism which drives melting near the start site.
Ujvari, Andrea, "Characterization of DNA structural elements and conformational changes accompanying the binding of T7 RNA polymerase to its promoter" (1999). Doctoral Dissertations Available from Proquest. AAI9920662.