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持续的生命至关重要。 转录是基因表达的第一步。 在转录过程中，RNA聚合酶II以DNA为模板合成mRNA分子。 这项研究的主要目标是详细了解早期RNA聚合酶II转录过程中发生的步骤以及如何控制它们。 这样的理解是重要的，因为转录是在生长、发育和正常细胞代谢过程中调节基因表达的主要控制点。 具体来说，这项研究将确定RNA合成中哪一步最慢，从而限制mRNA的合成速度。 此外，人类细胞核中加速慢步骤速率的因子将被纯化、鉴定和表征。 这项研究还将研究如何包含蛋白质，DNA和RNA的大复合物，这些复合物是转录发生所必需的，在被转录的基因上组装和分解。 这些研究中使用的方法围绕着一个完善的人类转录系统，该系统是在体外由高度纯化的蛋白质组装而成的。 这些实验利用专门开发的详细定量分析来研究早期RNA合成及其调控的步骤。 这将允许确定决定RNA合成水平和速率的各个步骤。 除了上述研究外，还将开发一个本科生生物化学实验室的实验模块。 本模块将为学生提供设计和实施假设驱动实验研究蛋白质-DNA相互作用的经验。 此外，学生将接触到最先进的实验技术，从而提高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发生的位置以及它们如何相互关联，RNA转录本本身在早期转录中的作用以及蛋白质-DNA-RNA复合物如何组装和分解来影响对mRNA转录关键第一阶段的理解。 此外，实验将研究转录可以调节的独特机制。 从这项研究中获得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，大量的时间将花费在培训研究生的实验设计，数据的解释，创造性思维，准备和写作的手稿，并给予正式和非正式的口头报告。 此外，为本科实验室开发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。