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; 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）：理解遗传调节是理解人类疾病并利用在后代后时代产生的信息丰富的关键。转录是从DNA中对RNA的受控复制，也许是这种调节（或许多疾病的情况）发生的首要步骤。该关键的细胞过程是由具有复杂功能和潜在要求的分子机进行的。虽然50年来，转录的分子基础一直是广泛研究的重点，但直到最近，我们才确定了多种亚基细菌和真核生物RNA聚合酶的多种高分辨率晶体结构，以及来自细菌噬菌体T7的单个亚基噬菌体聚合酶的令人兴奋的新结构。后者为研究转录基本问题提供了理想的模型系统。尽管结构上与多亚基RNA聚合酶不同，但它具有许多常见的功能和机械属性。这项工作中的关键问题将集中在这款复杂的分子机器中的能量平衡上。我们将测试和完善特定模型，以解释该蛋白质内的大重排，因为酶使启动子识别位点并过渡到延伸复合物，能够稳定转录数千个碱基。经典的酶学将与蛋白质诱变和生物物理化学的工具结合使用，以测试并进一步完善结构和功能的详细模型。这些研究将为从启动到伸长的主要过渡中理解能量和机制的基础。功能同源性表明，所学习的潜在教训将适用于所有RNA聚合酶。