A fluorescent assay to compare the effect of NTP concentration and temperature on transcript elongation by E. coli RNA polymerase.

Abstract

Traditional approaches to investigating the rate of RNA transcription have used reduced NTP concentrations and/or low temperature to decrease the transcription rate into the time scale in which it is feasible to conduct manual quenching (1–3). Since these conditions may have some intrinsic effect on transcription rates and the response of RNA polymerase to regulatory signals, we have investigated the efficacy of various fluorescently labeled nucleic acid probes, such as molecular beacons (4), to track the rate of RNA transcription between specific “check points” along the RNA transcript in real time. The kinetics of probe binding to complementary DNA or RNA oligonucleotides was analyzed by stopped-flow fluorescence spectroscopy and, for rapidly binding probes, was in the order of seconds within the concentration range feasible for transcription reactions. Probes with rapid binding kinetics were used to monitor the rate of RNA synthesis during multiple rounds of transcription. The fluorescent signal altered significantly with time in reactions containing RNA polymerase compared to those where the enzyme was absent or inhibited, and is consistent with previous work (5). These probes are being used to track the rate of RNA synthesis during a single round of transcription in stopped-flow fluorescence assays and analysis of the effect of temperature and NTP concentration on transcription elongation are ongoing.