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

RUI：大肠杆菌 RNA 聚合酶的失败启动和启动子逃逸

• 批准号: 0077941
• 负责人: Lilian Hsu
• 金额: $ 10万
• 依托单位: Mount Holyoke College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2002-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0077941&HistoricalAwards=false
• 关键词: RUI; Abortive; Initiation; Promoter; Escape

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.

败育起始和启动子逃逸是伴随着起始到延伸的过渡步骤的反应过程。 对于一些启动子，转录起始的最后阶段包含控制生产性基因表达程度的限速步骤。 以前，它表明，广泛取代的初始转录序列可能会导致启动子的表达是严重限制在启动子逃逸步骤。 这样的启动子产生异常高水平的败育RNA。 因此，起始转录序列是影响启动子失败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性转录中施加速率阻滞的机制，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止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体的定量分析。 最近的证据表明，高度失败的启动子这样做是由于形成了高比例的非生产性的初始转录复合物，只进行失败的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性复合物与非生产性复合物的形成，并鉴定调节酶分配的条件/因素。 最后，观察到超螺旋大大增加了启动子逃逸的效率。 为了确定活化的确切步骤，将从线性模板与超螺旋模板测量开放复合物形成后的反应速率。 此外，将比较线性和超螺旋模板之间的生产性和非生产性复合物形成的程度。超螺旋模板的使用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。 两者合计，问题和方法的结合将澄清启动子活性的初始转录序列的作用，并提供了许多见解，更好地了解流产启动和启动子逃逸的机制。 考虑到这些反应的普遍性，E.大肠杆菌系统，进一步的支持下，转录复合物的结构分析的进展，将有更大的影响，实现转录起始的一般知识。