The RNA polymerase II transcription complex

描述(申请人提供)：本项目的目标是确定RNA聚合酶II、基本转录因子和染色质模板之间的相互作用，从而导致准确的转录启动和有效的延伸。以酿酒酵母为模型，我们将研究基因表达的几个基本方面。本项目期间将重点研究针对组蛋白H3K4和H3K36共转录的组蛋白甲基化的机制。很明显，Bur1激酶通过染色质促进转录，因为突变H3K36或删除H3K36甲基转移酶Set2或其他几个染色质相关因子可以绕过对Bur1的要求。第一个具体目标是进一步探索Bur1的底物和功能。第二个目标的实验将探索H3K4和H3K36甲基化之间的相互作用。尽管这些修改通常被认为是独立的事件，但初步数据表明，它们并不是独立的。目前还不清楚这些修饰是如何影响转录的，因此它们在影响转录延长方面的作用将通过一系列遗传和分子实验来探索。H3K4的三甲基化定位于启动子附近，有观点认为Set1/Compass复合体被招募到RNA聚合酶II C末端结构域(CTD)的丝氨酸5磷酸化形式。然而，没有实验证据表明COMPASS和CTD之间存在直接的相互作用。第三个具体目标将是测试COMPASS是否可以直接结合特定的磷酸化形式的CTD，如果是，应该由哪些亚基负责。第四个具体目标将根据初步数据跟进，这些数据表明RNA聚合酶II的Rpb4亚单位参与了转录复合体和延伸因子Spt6之间的相互作用。蛋白质相互作用实验将测试Spt6与Rpb4/7异源二聚体之间的直接相互作用，而染色质免疫沉淀实验将探索Rpb4缺失对组蛋白沉积和修饰的影响。亲和层析和质谱学将被用来鉴定其他Rpb4/7结合蛋白，看看其他与染色质相关的复合体是否通过这个亚复合体与RNA聚合酶II相互作用。在最后一个具体目标中，在不同条件下形成的起始和延伸复合体将在固定化模板上进行纯化，然后用质谱仪进行分析。虽然可能会鉴定出一些与转录复合体相关的新因子，但更令人感兴趣的是可能在转录的不同阶段发生的因子的交换。在这五个特定目标上的实验将大大增加我们对RNA聚合酶II转录反应及其与染色质模板相互作用的了解。这一基础知识对于理解转录因子和组蛋白修饰酶突变如何导致癌症和发育缺陷等疾病至关重要。

DESCRIPTION (provided by applicant): The goal of this project is to define the interactions between RNA polymerase II, the basal transcription factors, and the chromatin template that lead to accurate transcription initiation and productive elongation. Using the yeast Saccharomyces cerevisiae as a model, several fundamental aspects of gene expression will be studied. This project period will focus on the mechanisms for targeting co-transcriptional histone methylation of histone H3K4 and H3K36. It is clear that the Bur1 kinase promotes transcription through chromatin, as the requirement for Bur1 can be bypassed by mutating H3K36, or by deleting the H3K36 methyltransferase Set2 or several other chromatin-related factors. The first specific aim is to further probe the substrates and functions of Bur1. Experiments in the second aim will explore the interactions between the methylations at H3K4 and H3K36. Although these modifications have typically been considered as separate events, preliminary data indicates they are not independent. It is still not clear exactly how these modifications affect transcription, so their role in affecting transcription elongation will be probed by a series of genetic and molecular experiments. H3K4 tri-methylation is localized near promoters and it has been proposed that the Set1/COMPASS complex is recruited to the Serine 5 phosphorylated form of the RNA polymerase II C-terminal domain (CTD). However, there is no experimental evidence for a direct interaction between COMPASS and the CTD. The third specific aim will be to test whether COMPASS can directly bind specific phosphorylated forms of the CTD, and if so, which subunits are responsible. The fourth specific aim will follow up on preliminary data suggesting that the Rpb4 subunit of RNA polymerase II is involved in mediating interactions between the transcription complex and the elongation factor Spt6. Protein interaction experiments will test for direct interactions between Spt6 and the Rpb4/7 heterodimer, while chromatin immunoprecipitation experiments will explore the effect of Rpb4 deletion on histone deposition and modification. Affinity chromatography and mass spectrometry will be used to identify other Rpb4/7 binding proteins to see if other chromatin-related complexes interact with RNA polymerase II through this subcomplex. In the last specific aim, initiation and elongation complexes formed under various conditions will be purified on immobilized templates and then analyzed by mass spectometry. Although it is possible some new factors associated with transcription complexes will be identified, what is of greater interest is the exchange of factors that are likely to occur at various stages of transcription. The experiments in these five specific aims will significantly increase our understanding of the RNA polymerase II transcription reaction and its interactions with the chromatin template. This fundamental knowledge is essential for understanding how mutations in transcription factors and histone modifying enzymes lead to diseases such as cancer and developmental defects.