The effects of the initially transcribed sequence on reiterative transcription in Escherichia coli.

摘要

Transcription, the synthesis of a ribonucleic acid transcript using a nucleic acid template, is the first step in gene expression for all life. In most organisms, high fidelity transcription is achieved through the formation of an RNA:DNA hybrid which allows the RNA polymerase to maintain the correct register. Slippage, which would lead to improper base pairing, is prevented by the heterogeneous sequence of the DNA template. However, when homogenous tracts of bases are present in the template, slippage can occur, allowing a single base in the template to encode multiple bases in the transcript in a process called reiterative transcription. Promoter proximal reiterative transcription only requires three identical bases in a row within the first five bases of the initially transcribed sequence in order for reiterative transcription to occur. Numerous examples of regulation by reiterative transcription have been described in the literature, however, the interactions between the RNA polymerase and the nucleic acids which leads to reiterative transcription are poorly understood. To date there have been no systematic studies looking at the effects of the initially transcribed sequence on outcomes of reiterative transcription. In this dissertation we examine the effects of the number of bases between the homopolymeric tract and the transcription start site on the ability of RNA polymerase to escape the reiterative transcription cycle. We also examine the effects of the length of homopolymeric tracts on reiterative transcription. Finally we used high throughput sequencing of mRNA 5' ends to determine the number of promoters in vivo which have homopolymeric tracts at their 5' ends as well as determine the prevalence of productive reiterative transcription in vivo. We demonstrate that sequence of the initially transcribed region is the primary factor which determines if reiterative transcription will occur and if the transcript will be elongated or released. We also find that promoters with homopolymeric tracts are common in vivo and that productive reiterative transcription occurs at a high frequency in vivo.