Quantitative parameters of productive transcription on T5 N25–based promoters are modulated by the initial transcribed sequence and template supercoiling

Productive initiation on the escape rate-limited T5 phage N25 promoter is subject to substantial modulation by the initial transcribed sequence (ITS). It is further compromised by the formation of two classes of open complexes-productive and unproductive. To decipher their roles, we performed single-cycle transcription assays under RNA polymerase (RNAP)-limiting conditions to quantitatively determine the rate of promoter escape and the productive fraction of RNAP open complexes formed at four N25-ITS variant promoters. Each promoter variant was transcribed from three different template conformations, that is, two fragment templates of different lengths and a supercoiled plasmid DNA template. In addition, each time-course transcription reaction was performed in parallel without or with GreB. Our results indicate that ITS variation greatly impacts both parameters, which together determine the extent of productive RNA synthesis from a promoter. Further, both parameters are highly stimulated by template supercoiling, which yields a higher fraction of productive complexes that undergo promoter escape at a faster rate. In contrast, the effect of GreB is selective, showing little effect on RNAP partitioning but increasing the escape rate of N25 variants bearing non-native ITSs. Analysis of the abortive RNA synthesis kinetics on the highly abortive N25anti(-A) promoter reveals the existence of an unproductive ITC making a 7-nt abortive RNA continuously. Based on our new kinetic data and recently published structural information on promoter complexes, we propose for T5 N25 promoters a mechanism of transcription initiation-promoter escape consistent with the roles of the ITS and template supercoiling.