The RNA polymerase II transcription complex

描述（由申请人提供）：该项目的目标是定义RNA聚合酶II、基础转录因子和染色质模板之间的相互作用，这些相互作用导致准确的转录起始和生产延伸。以酵母酿酒酵母为模型，研究基因表达的几个基本方面。本项目将重点研究组蛋白H3K4和H3K36共转录组蛋白甲基化的机制。很明显，Bur1激酶通过染色质促进转录，因为对Bur1的需求可以通过突变H3K36，或通过删除H3K36甲基转移酶Set2或其他几个染色质相关因子来绕过。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。虽然这些变化通常被认为是单独的事件，但初步数据表明它们并不是独立的。目前尚不清楚这些修饰如何影响转录，因此它们在影响转录伸长中的作用将通过一系列遗传和分子实验来探索。H3K4三甲基化定位在启动子附近，并且已经提出Set1/COMPASS复合体被招募到RNA聚合酶II c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之间有直接的相互作用。第三个具体目标将是测试COMPASS是否可以直接结合特定的CTD磷酸化形式，如果可以，是哪个亚基负责。第四个特定目标将跟进初步数据，表明RNA聚合酶II的Rpb4亚基参与介导转录复合物和延伸因子Spt6之间的相互作用。蛋白相互作用实验将检测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。亲和色谱法和质谱法将用于鉴定其他Rpb4/7结合蛋白，以确定其他染色质相关复合物是否通过该亚复合物与RNA聚合酶II相互作用。在最后一个具体目的中，将在固定模板上纯化在不同条件下形成的起始和延伸配合物，然后用质谱分析。虽然可能会发现一些与转录复合物相关的新因子，但更令人感兴趣的是在转录的各个阶段可能发生的因子交换。这五个特定目标的实验将显著增加我们对RNA聚合酶II转录反应及其与染色质模板相互作用的理解。这一基础知识对于理解转录因子和组蛋白修饰酶的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.