Effects of Discriminator Length on Transcription Initiation by E. coli RNA Polymerase

Abstract

Transcription initiation is a highly regulated step of gene expression. Here we investigate how different discriminator sequence lengths affect initiation. The discriminator is the region between the −10 element and the transcription start site (TSS) of the promoter and does not have a consensus sequence or length. Previous studies exchanged 6 and 7 base pair(bp) discriminators between promoters and found that the discriminator largely determines the lifetime of the binary RNA polymerase (RNAP)‐promoter open complex as well as the RNA‐DNA hybrid length at which RNAP escapes the promoter 1. Promoters with the 7 bp T7A1 discriminator have shorter lifetimes and escape points. Here we compare open complex lifetimes, initiation kinetics and RNAP escape points of the gcdp1 promoter (5 bp discriminator, a common length for E. coli promoters) with those of promoters with longer discriminators. Transcription start sites and discriminator lengths were determined by a primer extension assay. Fast‐mixing (RQF) and benchtop initiation experiments with α‐32P‐UTP or α‐32P‐GTP in the NTP mix were used to quantify transient amounts of short and long RNA products from productive complexes and stalling and abortive synthesis by nonproductive complexes. Results to date with the gcdp1 promoter/discriminator reveal a similar escape point to the λPR promoter. RQF studies with the T7A1 promoter show similar initiation kinetics to the λPR variant with the T7A1 discriminator, and an earlier escape point than for these promoters with the λPR discriminator. These results add to our understanding of the significance of the discriminator as a determinant of efficient initiation and promoter escape by RNAP.