Initiation and Elongation in T7 RNA Polymerase

描述（由申请人提供）：理解遗传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，即从DNA复制RNA的受控过程，可能是这种调节（或在许多疾病中的误调节）发生的第一步。这个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进行的。虽然转录的分子基础已经是50年来广泛研究的焦点，但直到最近，我们才看到多亚基细菌和真核RNA聚合酶的各种高分辨率晶体结构的确定，以及来自噬菌体T7的单亚基噬菌体聚合酶的令人兴奋的新结构。后者提出了一个理想的模型系统的转录的基本问题的研究。虽然在结构上与多亚基RNA聚合酶不同，但它具有许多共同的功能和机制属性。这项工作的关键问题将集中在这个复杂的分子机器的能量平衡。我们将测试和完善特定的模型，以解释已知的蛋白质内的大重排是必不可少的酶离开启动子识别位点，并过渡到一个能够稳定转录数千个碱基的延伸复合物。经典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。这些研究将提供一个基础，从中了解能量和机制的关键转变，从启动到伸长。功能同源性表明，潜在的经验教训将适用于所有的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.