Initiation and Elongation in T7 RNA Polymerase

T7 RNA 聚合酶的起始和延伸

• 批准号: 7316488
• 负责人: Craig T Martin
• 金额: $ 26.16万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7316488
• 关键词: Bacteriophage T7; Bacteriophages; Biological Models; Cell physiology; Complex; DNA; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Disease; Enzymatic Biochemistry; Enzymes; Equilibrium; Foundations; Genetic; Genetic Transcription; Genomics; Learning; Left; Modeling; Molecular; Molecular Machines; Mutagenesis; Polymerase; Proteins; RNA; Regulation; Resolution; Site; Structure; T7 RNA polymerase; Testing; Work; base; biophysical chemistry; human disease; promoter; tool

DESCRIPTION (provided by applicant): Understanding genetic regulation is key to understanding human disease and to exploiting the wealth of information arising in the post-genomic era. Transcription, the controlled copying of RNA from DNA, is perhaps the premier step at which this regulation (or misregulation, in the case of many diseases) occurs. This key cellular process is carried out by a molecular machine with complex function and underlying requirements. While the molecular basis of transcription has been the focus of extensive study for 50 years, it has been only fairly recently that we have seen the determination of a variety of high resolution crystal structures for the multisubunit bacterial and eukaryotic RNA polymerases, and exciting new structures for the single subunit phage polymerase from bacteriophage T7. The latter presents an ideal model system for the study of fundamental issues in transcription. Although structurally distinct from the multi-subunit RNA polymerases, it shares many common functional and mechanistic attributes. Key questions in this work will focus on the balance of energetics in this complex molecular machine. We will test and refine specific models to explain a large rearrangement within the protein known to be essential as the enzyme leaves the promoter recognition site and transitions to an elongation complex capable of stably transcribing thousands of bases. Classic enzymology will be combined with protein mutagenesis and the tools of biophysical chemistry to test and further refine detailed models for structure and function. These studies will provide a foundation from which to understand energetics and mechanism in the key transition from initiation to elongation. Functional homologies suggest that the underlying lessons learned will be applicable to all RNA polymerases.

描述（由申请人提供）：了解基因调控是了解人类疾病和利用后基因组时代产生的大量信息的关键。转录，即从 DNA 中控制 RNA 的复制，可能是这种调节（或在许多疾病中的错误调节）发生的首要步骤。这一关键的细胞过程是由具有复杂功能和潜在要求的分子机器执行的。虽然转录的分子基础 50 年来一直是广泛研究的焦点，但直到最近我们才看到多亚基细菌和真核 RNA 聚合酶的各种高分辨率晶体结构的确定，以及令人兴奋的新结构用于来自噬菌体 T7 的单亚基噬菌体聚合酶。后者为研究转录中的基本问题提供了一个理想的模型系统。尽管在结构上与多亚基 RNA 聚合酶不同，但它具有许多共同的功能和机制属性。这项工作的关键问题将集中在这个复杂分子机器中的能量平衡。我们将测试和完善特定模型，以解释已知的蛋白质内的大重排，因为酶离开启动子识别位点并转变为能够稳定转录数千个碱基的延伸复合物。经典酶学将与蛋白质诱变和生物物理化学工具相结合，以测试和进一步完善结构和功能的详细模型。这些研究将为理解从起始到伸长的关键转变的能量学和机制提供基础。功能同源性表明，所学到的基本经验教训将适用于所有 RNA 聚合酶。