Investigating Mechanisms of RNA Polymerase II Transcription and Regulation Using Single Molecule Fluorescence

由DNA编码的基因的表达对持续的生命至关重要。该项目将研究基因表达的基本步骤是如何发生的。基因表达的第一步是转录：基因中的一条DNA链被复制成RNA分子。这需要许多不同的蛋白质和DNA组装成一个复合物，这样基因表达才能在正确的时间在正确的细胞类型中准确地发生。 这项研究将研究含有蛋白质和DNA的复合物的组装和拆卸，这些蛋白质和DNA对转录的发生至关重要。这项研究将使用允许观察单个分子的实验。一次研究一个分子将揭示对控制基因表达重要的蛋白质和DNA的不同行为的新见解。重要的是，该项目将促进对研究生和本科生的培训，使新的教育课程得以制定，并鼓励代表性不足的少数民族和女科学家的参与。该项目的具体目标是研究早期mRNA转录步骤的机制，并了解转录激活蛋白p53与DNA结合的途径。这些实验利用了单分子荧光技术，该技术已成为揭示生物复合物动态行为和异质性的重要贡献者，从而为其功能提供了独特的见解。实验将研究一般转录因子TFIIB在体外从早期转录的人类复合物中释放的机制。重要的是，将在活性和非活性复合物之间进行区分，从而将TFIIB释放与活性直接相关。研究还将侧重于了解p53如何结合其DNA反应元件，这对于控制对调节细胞周期和程序性细胞死亡至关重要的基因的转录至关重要。分辨蛋白质/DNA复合物多样性的能力将揭示p53在DNA上形成不同寡聚复合物的允许途径，以及这些途径中缔合和解离事件的动力学。将确定不同的p53反应元件以及p53中的特定区域和氨基酸如何控制DNA结合模式。从这项研究中获得的知识将激发新的模型，将广泛地为未来的体外和生物系统中的转录调控研究提供信息。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

The expression of genes that are encoded by DNA is essential for sustained life. This project will investigate how basic steps in gene expression occur. The first step in gene expression is transcription: the process in which one strand of DNA in a gene is copied to make an RNA molecule. This requires that many different proteins and the DNA assemble into a complex, such that gene expression can occur accurately, and in the correct cell type at the proper time. This research will study the assembly and disassembly of complexes containing proteins and DNA that are essential for transcription to occur. The research will use experiments that allow single molecules to be observed. Studying molecules one at a time will reveal new insight into the diverse behaviors of proteins and DNA important for controlling gene expression. Importantly, this project will promote the training of graduate and undergraduate students, enable new educational curriculum to be developed, and encourage participation of underrepresented minority and women scientists. The specific objectives of the project are to investigate mechanisms of steps in early mRNA transcription and to understand the pathways by which the transcriptional activator protein p53 binds to DNA. The experiments take advantage of single molecule fluorescence techniques, which have emerged as essential contributors to revealing the dynamic behavior and heterogeneity of biological complexes, thus providing unique insight into their function. Experiments will investigate the mechanism by which the general transcription factor TFIIB releases from early transcribing human complexes in vitro. Importantly, distinctions will be made between active and inactive complexes, thereby directly relating TFIIB release to activity. Studies will also focus on understanding how p53 binds its DNA response elements, which is important for controlling transcription of genes critical to regulating the cell cycle and programmed cell death. The ability to resolve diversity in protein/DNA complexes will reveal the allowable pathways by which p53 forms different oligomeric complexes on DNA, as well as the kinetics of association and dissociation events in these pathways. How different p53 response elements, as well as specific regions and amino acids in p53, control modes of DNA binding will be determined. The knowledge gained from this research will stimulate new models that will broadly inform future studies of transcriptional regulation, both in vitro and in biological systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.