REAL-TIME FLUORESCENCE ASSAY SYSTEM FOR GENE-TRANSCRIPTION - SIMULTANEOUS OBSERVATION OF PROTEIN DNA BINDING, LOCALIZED DNA MELTING, AND MESSENGER-RNA PRODUCTION/

This article describes the development of an in vitro multicolor fluor escence assay system for studying protein/DNA complex formation, trans cription bubble formation, and mRNA production. These studies were acc omplished using three different fluorescent spectroscopic probes: rhod amine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-amino-purine in place of adenine to monitor transcription bubble formation, and gam ma-fluorophore-labeled UTP nucleotide to measure mRNA transcription ra tes. Combining these three assay systems allows the simultaneous deter mination of protein/DNA binding, localized DNA melting transitions, an d mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution. The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (bindi ng, open complex formation, and mRNA production) demonstrates the impo rtance of kinetically coupled events in gene transcription. (C) 1996 A cademic Press, Inc