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

起始流产和启动子逃逸是伴随起始到延伸过渡的反应过程。对于某些启动子，转录起始的最后阶段包含控制高产基因表达程度的限速步骤。先前的研究表明，初始转录序列的大量替换可能导致启动子的表达在启动子逃逸步骤受到严重限制。这样的启动子产生异常高水平的流产RNA。因此，起始转录序列是影响起始流产和启动子逃逸的重要因素。本研究的总体目标是破译初始转录序列在生产转录中施加速率障碍的机制，并寻找可以减轻速率限制的条件/因素。第一个目标是定义阻碍或促进启动子逃逸的初始转录序列的特征（如果有的话）。这将采用随机初始转录序列突变的定量分析。最近的证据表明，高度流产的启动子之所以这样做，是因为形成了很大一部分非生产性初始转录复合物，这些复合物只进行流产的RNA合成。因此，对于在启动子逃逸方面受到严重限制的启动子，第二个目标将是检查生产与非生产复合物的形成，并确定调节酶分配的条件/因素。最后，观察到超卷绕大大提高了启动子逸出的效率。为了确定激活的确切步骤，将从线性模板和超螺旋模板中测量开放式配合物形成后的反应速率。此外，将比较线性和超卷曲模板之间的生产性和非生产性复杂地层的程度。使用超螺旋模板是模拟体内转录条件的第一步，并将阐明流产起始和启动子逃逸的体内相关性。综上所述，这些问题和方法的结合将阐明初始转录序列在启动子活性中的作用，并为更好地理解启动子流产和启动子逃逸的机制提供许多见解。鉴于这些反应的普遍性，在大肠杆菌系统中的工作，以及转录复合物结构分析的进一步进展，将对实现转录起始的一般知识产生更大的影响。

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.