INITIATION OF TRANSCRIPTION BY T7 RNA POLYMERASE

T7 RNA 聚合酶启动转录

• 批准号: 2023602
• 负责人: Craig T Martin
• 金额: $ 12.24万
• 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/2023602
• 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：转录的精确调控对于细胞至关重要 基因表达和基因组调控。本研究的长期目标 是了解转录的酶学，包括化学 启动子识别和转录起始的基础。这 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，不仅要研究其基本方面 转录中 DNA 的序列特异性识别，也是基本的 聚合酶的一般机制。到最后，这个家庭 简单聚合酶与 DNA 和 RNA 大家族显示序列同源性 依赖性聚合酶，包括来自脊髓灰质炎、流感和 仙台病毒以及乙型肝炎和人类的逆转录酶 免疫缺陷病毒。 T7 模型系统的简单性非常适合直接研究 了解单个蛋白质-DNA 官能团如何相互作用 导致DNA的特异性识别并在一定条件下启动催化 DNA 中明确的位点。该提案的总体目标是 进一步完善启动子识别的新兴结构模型，并 扩展该模型以包括有关早期阶段的机制细节 的转录。强大的动力学和热力学的结合 将官能团取代直接掺入的测定 基于寡核苷酸的 DNA 模板可以详细探测关键的 蛋白质-DNA 相互作用。 将使用光谱探针来监测热力学和 RNA 聚合酶局部螺旋熔化的动力学，同时淬灭流 研究将用于跟踪 RNA 的有限周转合成。下列的 制定启动、具体的机制建议 扰动将被引入启动子中以提供之间的联系 结构和功能。速率限制步骤的性质 反应条件的改变也会干扰引发， 允许探测各种机械步骤。的精确作用 将探索将底物定位在活性位点的模板链 通过启动子 DNA 的结构修饰。最后，修改为 将根据其提高持续生产能力的能力来选择启动子 掺入前几个核糖核苷酸期间的转录， 为了了解启动子释放和流产之间的关系 骑自行车。