A real-time fluorescence method to monitor the melting of duplex DNA during transcription initiation by RNA polymerase

The melting of duplex DNA in the vicinity of the transcription start site i s an essential step of transcription initiation, Here we describe a fluores cent promoter technique which allows the melting of promoter DNA to be obse rved in a real-time manner with high sensitivity. We have constructed a 114 -bp lacUV5 promoter fragment (-89 to +25) which contains a fluorescence pro be in the region between the -10 consensus hexamer and the transcription st art site, This region was chosen to incorporate a fluorescence probe as it undergoes strand separation subsequent to binding RNA polymerase (RNAP) (i. e., open complex formation). Upon mixing RNAP and fluorochrome-labeled prom oter a time-dependent biphasic change in fluorescence was observed, The sec ond slower component was shown to be due to the open complex by comparing t he fluorescence data with the kinetics of open complex formation as measure d by using alternative methods of open complex detection. The rate constant s for open complex formation and dissociation were determined and found to be in excellent agreement with previously reported values, The techniques p resented herein can generally be applied to other systems. Furthermore, thi s method will serve as an important research tool as well as it could be us ed in designing high-throughput assays involving transcription complexes. ( C) 1999 Academic Press.