Biology and Mechanism of the Single Subunit RNA Polymerases

T7噬菌体的单多肽RNA聚合酶已成为研究基础生物学的模型系统。 自从它在40年前被首次发现以来，它就一直在研究转录机制。 过去15年的工作 揭示了这种RNA聚合酶的功能超出了噬菌体基因的转录， T7 RNA聚合酶的同源物广泛存在，不仅存在于真核线粒体中，而且存在于 哺乳动物细胞核，在那里它们形成核RNAP的第四大类（spRNAPIV）。 不过比起 我们对T7 RNAP的结构和转录机制的详细了解，我们对T7 RNAP的理解， 这种酶的转录外功能或其线粒体和核的机制和生物学 同源物有限。 为了解决这些理解上的差距，我们将：（1）定义T7 RNAP的作用及其 调节剂T7溶菌酶在T7 DNA包装机器的募集和组装中的作用，并使用整体和 单分子实验，描述T7 DNA包装反应的分子细节;（2）晶体测定 酵母线粒体RNAP延伸和起始复合物的结构，并表征其机制， 该RNA聚合酶的启动子识别及其线粒体转录因子的激活，（3） 鉴定受核spRNAPIV调控的基因以及呼吸或分解代谢物激活的影响 抑制spRNAPIV活性。 这些研究将促进我们对基本机制的理解， 大分子复合物组装和转录过程中的线粒体和核隔室的 真核细胞

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.