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的模式系统 自近40年前首次被发现以来，转录机制一直是人类研究的热点。在过去15年的工作中， 揭示了这种RNA聚合酶的功能超越了噬菌体基因的转录，并且 T7RNA聚合酶的同源物广泛存在，不仅存在于真核细胞的线粒体中，也存在于 哺乳动物的细胞核，在那里它们形成了核RNAPs的第四大类(SpRNAPIV)。然而，当与 我们对T7RNAP结构和转录机制的详细理解，我们对T7RNAP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 同源基因是有限的。为了解决这些理解上的差距，我们将：(1)定义T7RNAP及其 调节剂T7溶菌酶在T7 DNA包装机械的招募和组装中，并使用全套和 单分子实验，描述T7DNA包装反应的分子细节，(2)测定晶体 酵母线粒体RNAP延伸和起始复合体的结构，并对其作用机制进行了表征 这种RNA聚合酶对启动子的识别和线粒体转录因子对其激活的识别，(3) 确定受核spRNAPIV调控的基因以及呼吸或分解代谢激活的影响 对spRNAPIV活性的抑制。这些研究将促进我们对心力衰竭的基本机制的理解。 大分子复合体的组装及其在线粒体和核间的转录过程 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.