Mechanism and Regulation of Early Trasncription by RNA polymerase II

由DNA编码的基因的表达对维持生命至关重要。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。本研究的总体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这些步骤。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合成中哪一步是最慢的，从而限制mRNA的合成速度。此外，人类细胞核中加速缓慢步骤速率的因素将被纯化、鉴定和表征。这项研究还将研究转录发生所必需的包含蛋白质、DNA和RNA的大型复合体如何在正在转录的基因上组装和拆卸。这些研究中使用的方法围绕着一个建立良好的人类转录系统，该系统是在体外由高度纯化的蛋白质组装而成的。实验利用详细的定量分析，专门研究早期RNA合成的步骤及其调控。这将允许识别决定RNA合成水平和速率的单个步骤。除了上述研究外，还将开发一个本科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，从而加强教学实验课程的科学基础设施。这项研究将通过定义反应中发生的特定步骤以及它们如何相互关联，RNA转录本身在早期转录中的作用以及蛋白质- dna -RNA复合物如何组装和拆卸来影响对mRNA转录的关键第一阶段的理解。此外，这些实验将检验调节转录的独特机制。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。在实施这项研究的过程中，将花费大量的时间来训练研究生在实验设计、数据解释、创造性思维、手稿准备和写作以及正式和非正式的口头报告方面。此外，为本科实验室开发实验模块强调了将研究与教育相结合以促进教学、培训和学习的重要性。

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.