Fluorescence-based assay to measure the real-time kinetics of nucleotide incorporation during transcription elongation.

Understanding the mechanism and fidelity of transcription by the RNA polymerase (RNAP) requires measurement of the dissociation constant (K(d)) of correct and incorrect NTPs and their incorporation rate constants (k(pol)). Currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. Here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. The fluorescent adenine analogue 2-aminopurine was incorporated at various single positions in the template or the nontemplate strand of the promoter-free elongation substrate. On addition of the correct NTP to the T7 RNAP-DNA, 2-aminopurine fluorescence increased rapidly and exponentially with a rate constant similar to the RNA extension rate obtained from the radiometric assay. The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. Using this assay, we report the k(pol) and K(d) of all four correct NTP additions by T7 RNAP, which showed a range of values of 145-190 s(-1) and 28-124 μM, respectively. The fluorescent elongation substrates were used to determine the misincorporation kinetics as well, which showed that T7 RNAP discriminates against incorrect NTP both at the nucleotide binding and incorporation steps. The fluorescence-based assay should be generally applicable to all DNA-dependent RNAPs, as they use similar elongation substrates. It can be used to elucidate the mechanism, fidelity, and sequence dependency of transcription and is a rapid means to screen for inhibitors of RNAPs for therapeutic purposes.