Molecular Analysis of Eukaryotic Transcription

描述（由申请人提供）：真核RNA聚合酶（Pos）的转录是许多信号转导和发育途径的最终靶点，理解转录机制是理解基因调控机制的关键。从以前的研究中，很明显，基因特异性转录因子，染色质修饰因子或一般转录因子的突变破坏正常基因调控可导致癌症和其他疾病。该提案的广泛的长期目标是确定转录机制用于促进Pol II和Pol III的转录。这项工作将为在分子水平上理解正常和疾病状态下的基因调控提供基础。 这项工作的具体目标将利用生物化学，分子和遗传学方法来研究的结构和机制的转录机制指导RNA聚合酶II和III在S。啤酒。使用一种新开发的绘制大复合物结构排列的方法，将确定Pol II预引发复合物和开放复合物的结构。这些复合物的结构将在不同的启动子类型上进行比较。基于这些结果，模型的一般因素在转录起始的机制将被推导出来，这些模型将使用生化和遗传分析进行测试。将利用类似的方法来确定Pol III通用因子Brf 1在Pol III转录机制内的排列，从而允许比较不同核Pol所利用的保守和非保守机制。最后，两个细胞周期蛋白依赖性激酶，在延长过程中调节转录的机制将进行探讨。这些激酶的类似物敏感型将用于鉴定相关激酶靶标。将使用体外和体内测定来确定激酶活性调节转录的机制。

DESCRIPTION (provided by applicant): Transcription by eukaryotic RNA Polymerases (Pols) is the ultimate target for many signal transduction and developmental pathways, and understanding the mechanism of transcription is a key to understanding the mechanism of gene regulation. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription factors, chromatin modifying factors, or general transcription factors can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms utilized by the transcription machinery to promote transcription by Pol II and Pol III. The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, molecular, and genetic methods to examine the structures and mechanisms of the transcription machinery directing RNA Pols II and III in S. cerevisiae. Using a newly developed method for mapping the structural arrangement of large complexes, the structure of the Pol II pre-initiation Complex and Open Complex will be determined. The structures of these complexes will be compared on different promoter types. Based on these results, models for the mechanism of the general factors in transcription initiation will be derived and these models will be tested using biochemical and genetic assays. Similar methods will be utilized to determine the arrangement of the Pol III general factor Brf1 within the Pol III transcription machinery, permitting comparison of conserved and non conserved mechanisms utilized by different nuclear Pols. Finally, the mechanism of two cyclin-dependent kinases that regulate transcription during elongation will be explored. Analog-sensitive versions of these kinases will be utilized to identify relevant kinase targets. In vitro and in vivo assays will be used to determine the mechanisms whereby kinase activity regulates transcription.