Biology and Mechanism of the Single Subunit RNA Polymerases

描述(申请人提供)：自从近40年前首次发现T7噬菌体的单多肽RNA聚合酶以来，它一直是研究基本转录机制的模型系统。过去15年的研究表明，这种RNA聚合酶的功能超出了噬菌体基因的转录范围，T7 RNA聚合酶的同源物广泛存在于真核细胞的线粒体中，也存在于哺乳动物的细胞核中，在那里它们形成了核RNAPs的第四大类(SpRNAPIV)。然而，与我们对T7RNAP结构和转录机制的详细了解相比，我们对该酶的转录外功能或其线粒体和核同源物的机制和生物学的了解是有限的。为了解决这些理解上的差距，我们将：(1)定义T7RNAP及其调控因子T7溶菌酶在T7 DNA包装机械的招募和组装中的作用，并利用整体和单分子实验描述T7 DNA包装反应的分子细节；(2)确定酵母线粒体RNAP延伸和起始复合体的晶体结构，并表征该RNA聚合酶识别启动子及其由线粒体转录因子激活的机制；(3)鉴定受核spRNAPIV调控的基因以及呼吸激活或分解代谢抑制对spRNAPIV活性的影响。这些研究将促进我们对真核细胞线粒体和核内大分子复合体组装和转录过程的基本机制的理解。与公共健康相关的RNA聚合酶是表达遗传信息的核心角色。RNA聚合酶活性异常可导致人类疾病。我们的工作将增加我们对RNA聚合酶如何控制癌细胞中启动的基因以及参与细胞碳水化合物和能量代谢的基因的理解，这些基因是衰老和糖尿病的重要过程。

DESCRIPTION (provided by applicant): The single-polypeptide RNA polymerase of the T7 bacteriophage has been a model system for studying fundamental mechanisms of transcription ever since it was first identified almost 4 decades ago. Work over the past 15 years has revealed that the functions of this RNA polymerase extend beyond transcription of the phage genes, and that homologues of T7 RNA polymerase are widespread, occurring not only in eukaryotic mitochondria but also in mammalian nuclei, where they form the 4th major class of nuclear RNAPs (spRNAPIV). However, when compared to our detailed understanding of the structure and transcriptional mechanisms of T7RNAP, our understanding of the extra-transcriptional functions of this enzyme or of the mechanisms and biology of its mitochondrial and nuclear homologues is limited. To address these gaps in understanding we will: (1) Define the role of T7RNAP and its regulator T7 lysozyme in recruitment and assembly of the T7 DNA packaging machinery and, using both ensemble and single molecule experiments, describe the molecular details of the T7 DNA packaging reaction , (2) Determine crystal structures of yeast mitochondrial RNAP elongation and initiation complexes, and characterize the mechanism of promoter recognition by this RNA polymerase and of its activation by the mitochondrial transcription factor, (3) Identify the genes regulated by nuclear spRNAPIV and the effects of activation of respiration or of catabolite repression on spRNAPIV activity. These studies will advance our understanding of fundamental mechanisms of macromolecular complex assembly and of transcription processes in the mitochondrial and nuclear compartments of eukaryotic cells. PUBLIC HEALTH RELEVANCE RNA polymerases are the central players in the expression of genetic information. Aberrant activity of RNA polymerases leads to human disease. Our work will increase our understanding of how RNA polymerases control genes that are turned on in cancer cells and genes that are involved in cellular carbohydrate and energy metabolism, processes important in aging and diabetes.