RUI: Abortive Initiation and Promoter Escape by E. coli RNA Polymerase

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AbstractMCB-0077941Lilian HsuHsu Abortive initiation and promoter escape are reaction processes that accompany the transitional steps from initiation to elongation. For some promoters, this last stage of transcription initiation contains the rate-limiting step governing the extent of productive gene expression. Previously, it was shown that extensive replacement of the initial transcribed sequence could result in a promoter whose expression is severely limited at the promoter escape step. Such a promoter produces an unusually high level of abortive RNA. Thus, the initial transcribed sequence is an important factor influencing abortive initiation and promoter escape. The overall goal of this research is to decipher the mechanism(s) by which the initial transcribed sequence exerts a rate block at productive transcription, and seek conditions/factors that can alleviate the rate limitation. The first objective is to define the characteristics, if any, of initial transcribed sequences that impede or facilitate promoter escape. This will employ the quantitative analysis of random initial transcribed sequence mutants. Recent evidence suggests that highly abortive promoters do so due to the formation of a high fraction of unproductive initial transcribing complexes that only carry out abortive RNA synthesis. Thus, for promoters that are severely limited at promoter escape, a second objective will be to examine the formation of the productive versus unproductive complexes and identify conditions/factors that modulate the partitioning of the enzyme. Finally, it is observed that supercoiling greatly increases the efficiency of promoter escape. To pinpoint the exact step of activation, reaction rates after open complex formation will be measured from linear versus supercoiled templates. In addition, the extent of productive and unproductive complex formation will be compared between the linear and supercoiled templates. The use of supercoiled template is the first step toward mimicking the in vivo transcription condition and will shed light on the in vivo relevance of abortive initiation and promoter escape. Taken together, the combination of questions and approaches will clarify the role of initial transcribed sequence in promoter activity and offer many insights toward a better understanding of the mechanism of abortive initiation and promoter escape. Given the universal nature of these reactions, the work in the E. coli system, supported by further progress on structural analysis of the transcription complexes, will have greater impact on achieving a general knowledge of transcription initiation.