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Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Bacteriophage T7 RNA polymerase undergoes major conformational changes as transcription proceeds from initiation to elongation. Using limited trypsin digestion and stopped-flow fluorescence kinetic methods, we have monitored promoter release, initial bubble collapse, and refolding of the 152–205 region (subdomain H), the latter being important for RNA channel formation. The kinetic studies show that the conformational changes are temporally coupled, commencing at the synthesis of 9 nt and completing by the synthesis of 12 nt of RNA. The temporal coupling of initial bubble collapse and RNA channel formation is proposed to facilitate proper binding of the RNA dissociated from the late initiation complexes into the RNA channel. Using promoter mutations, we have determined that promoter contacts are broken sequentially during transition from initiation to elongation. The specificity loop interactions are broken after synthesis of 8 nt or 9 nt of RNA, whereas the upstream promoter contacts persists up to synthesis of 12 nt of RNA. Both promoter contacts need to be broken for transition into elongation. The A-15C mutation resulted in efficient transition to elongation by synthesis of 9 nt of RNA, whereas the C-9A mutation resulted in early transition to elongation by synthesis of 7–8 nt of RNA. The effect of early promoter clearance in the mutant promoters was observed as reduced production of long abortive products. [Copyright &y& Elsevier]