INITIATION OF TRANSCRIPTION BY T7 RNA POLYMERASE

T7 RNA 聚合酶启动转录

• 批准号: 6088373
• 负责人: Craig T Martin
• 金额: $ 0.54万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6088373
• 关键词: DNA directed RNA polymerase; DNA footprinting; RNA biosynthesis; active sites; bacteriophage T7; chemical kinetics; deoxyribonucleoside triphosphate; enzyme activity; enzyme mechanism; enzyme structure; genetic promoter element; genetic regulation; nucleoproteins; phosphoric ester; protein structure function; site directed mutagenesis; stop flow technique; temperature; thermodynamics; transcription factor

DESCRIPTION: The precise regulation of transcription is central to cellular gene expression and genomic regulation. The long term goal of this research is to understand the enzymology of transcription, including the chemical basis of promoter recognition and the initiation of transcription. The family of small RNA polymerases from the T7 related bacteriophages presents an ideal model system in which to study, not only the fundamental aspects of sequence-specific recognition of DNA in transcription, but also the basic mechanisms of polymerases in general. Toward the latter end, this family of simple polymerases shows sequence homology to a large family of DNA and RNA dependent polymerases, including replicases from polio, influenza, and Sendai viruses and reverse transcriptases from hepatitis B and human immunodeficiency viruses. The simplicity of the T7 model system lends itself well to studies directed at understanding how individual protein-DNA functional group interactions lead to specific recognition of DNA and to the initiation of catalysis at a well-defined site in the DNA. The overall aim of this proposal is to further refine an emerging structural model for promoter recognition and to extend that model to include mechanistic detail regarding the early stages of transcription. The combination of powerful kinetic and thermodynamic assays with the direct incorporation of functional group substitutions into oligonucleotide based DNA templates allows detailed probing of critical protein-DNA interactions. A spectroscopic probe will be employed to monitor the thermodynamics and kinetics of local helix melting by the RNA polymerase, while quench-flow studies will be used to follow limited-turnover synthesis of RNA. Following the development of a mechanistic proposal for initiation, specific perturbations will be introduced into the promoter to provide a link between structure and function. The nature of the rate limiting step(s) in initiation will also be perturbed by alteration of the reaction conditions, to allow probes of various mechanistic steps. The precise role of the template strand in positioning substrate at the active site will be explored via structural modifications of the promoter DNA. Finally, modifications in the promoter will be selected for their ability to increase the processivity of transcription during incorporation of the first few ribonucleotides, in order to understand relationships between promoter release and abortive cycling.

描述：转录的精确调控是细胞的中心。 基因表达和基因组调控。这项研究的长期目标是 是了解转录的酶学，包括化学物质 启动子识别和转录启动的基础。这个 来自T7相关噬菌体的小RNA聚合酶家族 一个理想的模型系统，在其中进行研究，不仅是 DNA在转录中的序列特异性识别，也是基本的 聚合酶的一般作用机制。到了最后，这个家族 简单聚合酶显示出与DNA和RNA大家族的序列同源性 依赖聚合酶，包括脊髓灰质炎、流感和 仙台病毒与乙肝病毒和人的逆转录酶 免疫缺陷病毒。 T7模型系统的简单性很适合进行定向研究 了解单个蛋白质-DNA功能基团如何相互作用 导致DNA的特异性识别和催化作用的启动 DNA中有明确的位置。这项建议的总体目标是 进一步完善新出现的启动子识别结构模型，并 扩展该模型以包括有关早期阶段的机械性细节 抄写。强大的动力学和热力学的结合 将官能团取代直接结合到 基于寡核苷酸的DNA模板允许详细探测关键的 蛋白质与DNA的相互作用。 将使用光谱探测器来监测热力学和 淬火流动时RNA聚合酶局部螺旋熔解的动力学 这些研究将用于跟踪有限周转率的RNA合成。跟随 制定一项机械性的启动、具体 扰动将被引入到启动子中，以在 结构和功能。限速步骤的性质(S) 引发也会被反应条件的改变所干扰， 以允许对各种机械步骤进行探测。的确切作用 将探索在活性部位定位底物的模板链 通过对启动子DNA进行结构修饰。最后，中的修改 选择发起人是因为他们有能力提高加工能力。 在掺入最初的几个核苷酸的过程中转录的 为了了解启动子释放与流产之间的关系 骑自行车。