ARCHITECTURE OF THE EUKARYOTIC TRANSCRIPTION APPARATUS IN SOLUTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 转录是基因表达途径的第一步，也是关键的控制点。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，核糖核酸聚合酶II(PolI)负责信使RNA的转录，使Polo？S调控成为基因表达的中心。POL含有12个亚基，约0.5Mda，但尽管其大小和复杂性很高，POL不能识别和启动特定启动子的转录。为了从特定的启动子转录，POL至少需要5个通用转录因子(IID、IIB、IIE、IIF和IIH)。除了五种常见的转录因子外，Poll还需要一种名为Mediator的1.5MDA复合体的作用，以便对转录激活物和抑制物做出反应。本研究的目的是用溶液散射的方法研究参与激活转录的各种复合体，包括Pol60多肽复合体、5种常见的转录因子和介体。这些大型组件的结构确定带来了巨大的技术挑战。我们正试图通过结合X射线结晶学、电子显微镜和溶液X射线散射来应对这些挑战。这一实验方案构成了我们使用溶液散射的研究工作，我们认为，在亚兆道尔顿络合物的强度和在溶液中研究它们的能力方面，这填补了X射线结晶学和低温EM之间的技术空白。这项研究的意义可以概括为：它将提供充分理解转录基本机制所需的结构信息；它将为转录调控研究奠定结构基础；它可能表明未来如何通过组合结构方法解决其他异质多蛋白组件的结构问题。

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.