ARCHITECTURE OF THE EUKARYOTIC TRANSCRIPTION APPARATUS IN SOLUTION

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 转录是基因表达途径的第一步，也是基因表达的关键控制点。 转录调控是发育、肿瘤发生和其他基本过程的基础。 在真核生物中，RNA聚合酶II（pol）负责信使RNA的转录，使pol？的调控中心的基因表达。 Pol含有12个亚基，分子量约为0.5 Mda，但尽管其大小和复杂性，Pol不能识别并启动特定启动子的转录。 为了从特异性启动子转录，pol需要至少五种通用转录因子（IID、IIB、IIE、IIF和IIH）。 除了五种一般的转录因子外，pol还需要一种1.5 MDa的复合物（称为介体）的作用，以响应转录的激活因子和抑制因子。本研究的目的是利用溶液散射研究参与激活转录的各种复合物，包括pol的60多肽复合物、5种通用转录因子和介体。这些大型组件的结构确定提出了艰巨的技术挑战。我们正试图通过结合X射线晶体学，电子显微镜和溶液X射线散射来应对这些挑战。这个实验性的建议构成了我们的研究工作，使用溶液散射，我们相信填补了X射线晶体学和cryoEM之间的技术差距，其强度在亚兆道尔顿复合物和它的能力，在解决方案中研究它们。该研究的意义可以概括为：它将提供所需的结构信息，以充分了解转录的基本机制;它将建立一个结构基础的转录调控的研究;它可能会表明如何在未来的异质性多蛋白组装体的结构可以解决的组合结构的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transcription is the first step and the key control point in the pathway of gene expression. Transcriptional regulation underlies development, oncogenesis, and other fundamental processes. In eukaryotes the enzyme RNA Polymerase II (pol) is responsible for transcription of messenger RNA making pol?s regulation central to gene expression. Pol contains 12 subunits massing about 0.5 Mda, but despite its size and complexity pol cannot recognize and initiate transcription from a specific promoter. In order to transcribe from a specific promoter pol requires at least five general transcriptions factors (IID, IIB, IIE, IIF and IIH). In addition to the five general transcription factors, pol requires the action of a 1.5 MDa complex called mediator in order to respond to activators and repressors of transcription. The purpose of this proposal is to use solution scattering to study the various complexes involved in activated transcription up to and including the 60 polypeptide complex of pol, the 5 general transcription factors and mediator. Structure determination of these large assemblies presents formidable technical challenges. We are attempting to meet these challenges through a combination of x-ray crystallography, electron microscopy, and solution x-ray scattering. This experimental proposal constitutes our research effort using solution scattering, which we believe fills the technological gap between x-ray crystallography and cryoEM in terms of its strength on submega Dalton complexes and its ability to study them in solution. The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription; it will establish a structural basis for studies of transcriptional regulation; and it may indicate how structures of other heterogeneous multiprotein assemblies can be solved in the future by the combined structural approach.