Kinetics of promoter escape varies as a function of KCl concentration

Abstract

The efficiency of promoter escape in E. coli transcription initiation is dependent on two factors: 1) the rate of escape, and 2) the extent of RNA polymerase (RNAP) partitioning into escape‐competent open complexes. Both parameters can be obtained from a single cycle assay performed under RNAP‐limiting conditions. For T5 N25 and N25anti promoters, these two factors are governed by the initial transcribed sequence (ITS). N25 and N25anti promoters differ only in +3 to +20 region of the ITS, but exhibit half‐lives of ∼3 min and ∼42 min, respectively, for full‐length RNA synthesis. Interestingly, the slower escaping N25anti promoter forms a higher fraction of productive open complexes and yields higher levels of productive RNA than N25; this result was unexpected. The above studies were performed in 200 mM KCl solution. We next examined the effect of KCl concentration and found that lowering [KCl] from 200 – 10 mM increased the rate of escape for N25 while also raising its productive fraction. For N25anti, the escape rate remained the same while lower [KCl] brought about higher productive synthesis. We conclude that KCl concentration affects the open complex formation and partitioning similarly in both promoters, but influences their open complex collapse and escape differently, confirming that the rate of promoter escape is ITS‐sequence dependent. Supported by an NSF grant (RUI‐0418316) to L. M. Hsu.