Initiation and Elongation in T7 RNA Polymerase

描述(由申请人提供)：了解基因调控是理解人类疾病和利用后基因组时代产生的丰富信息的关键。转录，即DNA中RNA的受控复制，可能是这种调控(或在许多疾病中的错误调控)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。尽管转录的分子基础50年来一直是广泛研究的焦点，但直到最近，我们才看到多亚单位细菌和真核RNA聚合酶的各种高分辨晶体结构的确定，以及来自噬菌体T7的单亚单位噬菌体聚合酶的令人兴奋的新结构。后者为研究抄写中的基本问题提供了一个理想的模式系统。尽管它在结构上与多亚基RNA聚合酶不同，但它有许多共同的功能和机制属性。这项工作中的关键问题将集中在这台复杂的分子机器中的能量平衡上。我们将测试和完善特定的模型，以解释在酶离开启动子识别位点并过渡到能够稳定转录数千个碱基的延伸复合体时，蛋白质中已知的大规模重排是必不可少的。经典的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延伸的关键转变中的能量学和机制提供基础。功能同源性表明，所学的基本经验教训将适用于所有RNA聚合酶。

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.