Biochemistry of Eukaryotic Messenger RNA Synthesis

描述(由申请人提供)： 我们的长期目标是帮助理解调控真核信使RNA合成的生化机制。这项研究的主要目标是在分子水平上确定RNA聚合酶II(Pol II)的启动子特异性转录是如何受一系列转录因子的作用控制的，这些转录因子可以指导体外转录产物的准确启动和有效延伸。我们的总体方法基本上与前面的建议中描述的相同：(I)用纯化的蛋白质在体外重组真核蛋白编码基因的忠实和高效转录，以及(Ii)利用这个重组系统来阐明哺乳动物Pol II转录启动和延伸的生化机制。本研究将沿着以下思路组织：(1)进一步分析Pol II延伸因子Elongin A的作用机制，以及它在紫外线诱导Pol II泛素化中的作用。(2)进一步研究Pol II延伸因子ELL的作用机制。(3)详细分析了两个新的人类依赖于三磷酸腺苷的染色质重构体，称为人INO80复合体和SNF2家族成员和癌基因ALC1(在肝癌中激活)的作用机制。这些研究应该提供关于控制真核信使RNA合成的基本机制的信息，它们也有可能对由于ELL及其相关蛋白和ALC1的异常表达而导致的癌症的分子基础产生新的见解。 公共卫生相关性： 众所周知，真核信使RNA合成是基因表达调控的主要部位，而且它的错误调控经常导致人类疾病，包括许多形式的癌症。真核生物信使RNA合成是一个复杂的生化过程，由RNA聚合酶II在多种转录因子的辅助下催化。尽管在过去的20年里取得了相当大的进展，但人类疾病中真核信使RNA合成的错误调控机制的关键方面还没有完全了解。因此，这项建议中描述的研究的目标是帮助理解哺乳动物细胞中信使RNA合成调节的生化机制。特别是，我们计划对实验室中发现的几种转录因子的作用机制进行研究。除了揭示哺乳动物信使RNA合成的基本机制外，这些研究还有可能揭示癌症的各个方面，因为我们计划研究的两种转录因子ELL和ALC1已被提议分别参与急性髓系白血病和肝细胞癌的病因学。

DESCRIPTION (provided by applicant): Our long range goal is to contribute to an understanding of the biochemical mechanisms underlying the regulation of eukaryotic messenger RNA synthesis. The major goal of the research described in this proposal is to determine, at the molecular level, how promoter-specific transcription by RNA polymerase II (Pol II) is controlled by the action of a set of transcription factors that direct accurate initiation and efficient elongation of transcripts in vitro. Our overall approach is essentially the same as that described in the previous proposals: (i) to reconstitute in vitro, with purified proteins, faithful and efficient transcription of eukaryotic protein-coding genes and (ii) to use this reconstituted system to elucidate the biochemical mechanisms governing transcription initiation and elongation by mammalian Pol II. This research will be organized along the following lines: (1) Further analysis of the mechanism of action of Pol II elongation factor Elongin A and of its newly described role in UV-induced ubiquitination of Pol II. (2) Further investigation of the mechanism of action of Pol II elongation factor ELL. (3) Detailed analysis of the mechanisms of action of two novel human ATP-dependent chromatin remodelers referred to as the human INO80 complex and the SNF2 family member and oncogene ALC1 (Activated in Liver Cancer 1). These studies should provide information on the basic mechanisms governing eukaryotic messenger RNA synthesis, and they also have the potential to yield new insights into the molecular bases of cancers resulting from abherrant expression of ELL and its associated proteins and of ALC1. PUBLIC HEALTH RELEVANCE: It is well established that eukaryotic messenger RNA synthesis is a major site for the regulation of gene expression and, further, that its misregulation often results in human diseases, including many forms of cancer. Eukaryotic messenger RNA synthesis is a complex biochemical process catalyzed by the enzyme RNA polymerase II with the assistance of a diverse collection of transcription factors. Despite considerable progress over the past 20 years, critical aspects of the mechanisms underlying misregulation of eukaryotic messenger RNA synthesis in human disease are not completely understood. As a consequence, the goal of the research described in this proposal is to contribute to an understanding of the biochemical mechanisms underlying the regulation of messenger RNA synthesis in mammalian cells. In particular, we plan to pursue investigations of the mechanisms of action of several transcription factors discovered in our lab. In addition to shedding light on the basic mechanisms governing mammalian messenger RNA synthesis, these studies have the potential to shed light on aspects of cancer, since two transcription factors, ELL and ALC1, which we plan to investigate have been proposed to participate in the etiology of acute myeloid leukemia and hepatocellular carcinoma, respectively.