Open complex stability regulates transcription initiation by E. coli RNA polymerase from the rrnB P1 promoter

Abstract

Initiation of transcription by E. coli RNA polymerase (RNAP) begins with specific binding to promoter DNA and ends with promoter escape. For the four T7A1 and λPR promoter/discriminator combinations, we found that the discriminator determined the lifetime and stability of the open complex and the escape point of RNAP in initiation from productive promoter complexes (1). Quantitative initiation studies with these promoter/discriminator combinations reveal that the RNA‐DNA hybrid length for promoter escape and the length distribution of short (abortive) RNA released before escape both increase with the lifetime or stability of the initiation‐competent open complex (OC). The escape point for productive complexes increases from the 7‐mer to 8‐mer step for T7A1 discriminators to the 10‐mer to 11‐mer step for λPR discriminators. Nonproductive complexes make a short RNA smaller than the escape length and stall, slowly releasing it and reinitiating. These findings led us to predict that escape from a promoter like the ribosomal rrnB P1 promoter, with an unstable OC, should occur at a very short RNA‐DNA hybrid length and without release of short RNAs (1). Currently, to obtain the experimental data to test this prediction, we are performing single round transcription assays for the rrnB P1 promoter and other promoters with the rrnB P1 discriminator. In preliminary studies, we have found NTP concentrations where a significant fraction of promoters initiate rapidly. For these NTP concentrations, we find that only the shortest RNAs (e.g. 3‐mer) are made by nonproductive complexes during the time period required for productive complexes to escape and begin elongation. In addition, initiation of λPR promoter with rrnB P1 discriminator shows similar pattern comparing to initiation of rrnB P1 promoter with its own discriminator. We conclude that the current results are consistent with the proposal of early escape of RNAP from promoters with unstable OC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.