Mechanism and Regulation of Early Trasncription by RNA polymerase II

RNA聚合酶II早期转录的机制和调控

• 批准号: 0517296
• 负责人: Jennifer Kugel
• 金额: $ 42万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517296&HistoricalAwards=false
• 关键词: Mechanism; Regulation; Early; Trasncription; RNA

The expression of genes that are encoded by DNA is essential for sustained life. Transcription is the first step in gene expression. During transcription the enzyme RNA polymerase II, using DNA as a template, synthesizes mRNA molecules. The broad goal of this research is to understand in detail the steps that occur during early RNA polymerase II transcription and how they can be controlled. Such an understanding is important because transcription is a principal control point for regulating gene expression during growth, development, and normal cellular metabolism. Specifically, this research will identify which step in RNA synthesis is the slowest and therefore limits the rate at which mRNA is made. In addition, a factor in human nuclei that accelerates the rate of the slow step will be purified, identified, and characterized. This research will also investigate how large complexes containing proteins, DNA, and RNA, which are required for transcription to occur, both assemble and disassemble at genes that are being transcribed. The methods used in these studies center around a well established human transcription system that is assembled in vitro from highly purified proteins. The experiments utilize detailed quantitative assays specifically developed to study the steps of early RNA synthesis and their regulation. This will allow the identification of individual steps that dictate both the level and rate of RNA synthesis. In addition to the above research, an experimental module for an undergraduate biochemistry laboratory will be developed. This module will provide students with experience in designing and implementing a hypothesis driven experiment investigating protein-DNA interactions. Moreover, students will be exposed to state-of-the-art experimental techniques, thereby enhancing the scientific infrastructure of a teaching laboratory course. This research will impact the understanding of the critical first stages of mRNA transcription by defining where specific steps in the reaction occur and how they are interrelated, the role of the RNA transcript itself in early transcription, and how protein-DNA-RNA complexes assemble and disassemble. Moreover, the experiments will examine unique mechanisms by which transcription can be regulated. The knowledge derived from this research has the potential to stimulate new studies of transcriptional regulation in complex biological systems. In implementing this research, a significant amount of time will be spent training graduate students in experimental design, interpretation of data, creative thought, preparation and writing of manuscripts, and giving formal and informal oral presentations. In addition, developing an experimental module for an undergraduate laboratory underscores the importance of integrating research and education in order to promote teaching, training, and learning.

由DNA编码的基因的表达对于持续生命至关重要。 转录是基因表达的第一步。 在转录过程中，使用DNA作为模板，酶RNA聚合酶II合成mRNA分子。 这项研究的广泛目标是详细了解RNA聚合酶II早期转录期间发生的步骤以及如何控制它们。 这种理解很重要，因为转录是调节在生长，发育和正常细胞代谢过程中基因表达的主要控制点。 具体而言，这项研究将确定RNA合成中哪个步骤最慢，因此限制了制造mRNA的速率。 另外，将纯化，鉴定和表征的人核的一个因素将加速慢速步骤的速率。 这项研究还将研究转录发生的大型复合物，含有蛋白质，DNA和RNA，在要转录的基因上都组装和拆卸。 这些研究中使用的方法围绕着建立的人类转录系统，该系统由高度纯化的蛋白质体外组装而成。 这些实验利用专门开发的详细定量测定方法来研究早期RNA合成及其调节的步骤。 这将允许识别单个步骤，以决定RNA合成的水平和速率。 除上述研究外，还将开发针对本科生物化学实验室的实验模块。 该模块将为学生提供设计和实施假设驱动的实验的经验，研究蛋白-DNA相互作用。 此外，学生将接触到最先进的实验技术，从而增强教学实验室课程的科学基础设施。这项研究将通过定义反应中的特定步骤以及如何相互关联，RNA转录本身在早期转录中的作用以及蛋白-DNA-RNA复合物如何组装和拆卸来影响对mRNA转录的关键第一阶段的理解。 此外，实验将检查可以调节转录的独特机制。 从这项研究中得出的知识有可能刺激复杂生物系统中转录调控的新研究。 在实施这项研究时，将花费大量时间来培训研究生在实验设计，数据解释，创造性思想，手稿的准备和写作以及进行正式和非正式的口头演示中。 此外，为本科实验室开发一个实验模块强调了整合研究和教育以促进教学，培训和学习的重要性。