RESUMEN
Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking - involving rapid-quench-flow mixing and pulsed-laser irradiation - permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/efectos de la radiación , ADN/metabolismo , Escherichia coli/genética , Biología Molecular/métodos , Proteínas/metabolismo , Transcripción Genética/genética , Animales , ADN/química , ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Cinética , Luz , Proteínas/químicaRESUMEN
We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.