Electron Microscpy Studies of Gene Regulatory Complexes

人类基因表达的调节是精确控制的，以确保适当的基因表达。 对环境和发展线索的反应。事实上，异常的基因表达导致 无数的问题，最明显的是癌症。虽然这种调节大部分是由激活器施加的， 结合特定的DNA序列，它们通常需要额外的“共激活剂”复合物， 将它们的信号传递给转录机器。我们建议分析结构 CRSP（也称为介体）共激活剂的功能特性（使用cryo-EM） 复杂，以了解它如何调节转录。具体来说，我们将研究如何 基因表达的CRSP依赖性活化可被“ARC-L特异性”（cdk 8， 细胞周期蛋白C、Med 230和Med 240）多肽。我们还将描述 CRSP-RNA聚合酶II二元复合物。这一分析将确定CRSP的具体亚单位 以及对其功能至关重要的结构域，从而鉴定治疗的潜在靶点。 真核基因包装到染色质中提供了一个额外的，可微调的 转录调控机制。特别地，将DNA包裹到核小体中可以 通过使启动子或增强子序列 无法接近达到必要转录水平所需的蛋白质。atp依赖 染色质重塑复合物改变了核小体的结构， 核小体DNA，而不去除或修饰组蛋白。在这里，我们建议描述 四个相关的人类和果蝇重塑复合物的结构属于 SWI-SNF家族，通过Cryo-EM。我们的目标是确定定义酶核心的共同特征， 以及查明进化，功能，和特异性相关的差异之间的 配合物为了获得重塑机制的信息，我们将研究 这些复合物与重组核小体，并在不同的核苷酸的存在下。 最终的目标是能够产生一个机制模型，这些ATP驱动的分子 机器影响核小体结构。

Regulation of gene expression in humans is precisely controlled in order to ensure proper response to environmental and developmental cues. Indeed, aberrant gene expression leads to myriad problems, most notably cancer. While much of this regulation is imposed by activators binding specific DNA sequences, they typically require additional "co-activator" complexes to communicate their signals to the transcriptional machinery. We propose to analyze the structural (using cryo-EM) and functional characteristics of the CRSP (also called Mediator) co-activator complex in order to understand how it regulates transcription. Specifically, we will examine how CRSP-dependent activation of gene expression can be repressed by the "ARC-L specific" (cdk8, cyclin C, Med230, and Med240) polypeptides. We will also characterize the tight association of the CRSP-RNA polymerase II binary complex. This analysis will establish specific CRSP subunits and domains that are essential for its function and thus identify potential targets for therapeutics. The packaging of eukaryotic genes into chromatin provides an additional, finely-tunable mechanism of transcriptional regulation. In particular, wrapping of DNA into nucleosomes can result in repression of gene expression by making either promoter or enhancer sequences inaccessible to proteins required to achieve the necessary levels of transcription. ATP-dependent chromatin remodeling complexes alter nucleosomal structure and therefore the accessibility of nucleosomal DNA, without removal or modification of histones. Here we propose to characterize the architecture of four related human and Drosophila remodeling complexes belonging to the SWI-SNF family by Cryo-EM. Our aim is to identify common features defining an enzymatic core, as well as to pinpoint evolutionary, functional, and specificity-related differences between the complexes. To gain information on the mechanism of remodeling, we will study the interaction of these complexes with recombinant nucleosomes and in the presence of different nucleotides. The final goal is to be able to generate a mechanistic model of how these ATP-driven molecular machines affect nucleosome structure.