A Real-Time Fluorescence Method to Monitor the Melting of Duplex DNA during Transcription Initiation by RNA Polymerase

Abstract

The melting of duplex DNA in the vicinity of the transcription start site is an essential step of transcription initiation. Here we describe a fluorescent promoter technique which allows the melting of promoter DNA to be observed in a real-time manner with high sensitivity. We have constructed a 114-bplacUV5 promoter fragment (−89 to +25) which contains a fluorescence probe in the region between the −10 consensus hexamer and the transcription start 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 promoter a time-dependent biphasic change in fluorescence was observed. The second slower component was shown to be due to the open complex by comparing the fluorescence data with the kinetics of open complex formation as measured by using alternative methods of open complex detection. The rate constants for open complex formation and dissociation were determined and found to be in excellent agreement with previously reported values. The techniques presented herein can generally be applied to other systems. Furthermore, this method will serve as an important research tool as well as it could be used in designing high-throughput assays involving transcription complexes.