Mechanism and Regulation of Early Trasncription by RNA polymerase II

由DNA编码的基因的表达对于维持生命是必不可少的。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，合成信使核糖核酸分子。这项研究的广泛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。这样的理解很重要，因为转录是生长、发育和正常细胞代谢过程中调节基因表达的主要控制点。具体地说，这项研究将确定RNA合成的哪一步是最慢的，因此限制了mRNA的合成速度。此外，还将提纯、鉴定和鉴定人类细胞核中加速慢步速度的因子。这项研究还将调查包含蛋白质、DNA和RNA的大型复合体是如何在正在转录的基因上组装和拆解的，这些都是转录发生所必需的。这些研究中使用的方法围绕着一个成熟的人类转录系统，该系统是由高度纯化的蛋白质在体外组装而成的。这些实验利用了专门为研究早期RNA合成的步骤及其调节而开发的详细的定量分析。这将允许识别决定RNA合成水平和速率的各个步骤。除上述研究外，还将为本科生生物化学实验室开发一个实验模块。本单元将为学生提供设计和实施假设驱动的实验的经验，该实验研究蛋白质与DNA的相互作用。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。这项研究将通过确定反应中特定步骤发生的位置以及它们之间的相互关系，RNA转录物本身在早期转录中的作用，以及蛋白质-DNA-RNA复合体的组装和拆解，影响对mRNA转录的关键第一阶段的理解。此外，这些实验还将检验转录调控的独特机制。从这项研究中获得的知识有可能激发对复杂生物系统转录调控的新研究。在实施这项研究时，将花费大量时间培训研究生在实验设计、数据解释、创造性思维、手稿的准备和写作以及进行正式和非正式口头陈述方面的培训。此外，为本科生实验室开发实验模块强调了整合研究和教育的重要性，以促进教学、培训和学习。

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.