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

由DNA编码的基因的表达对于维持生命是必不可少的。这个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：复制基因中的一条DNA链来制造RNA分子的过程。这需要许多不同的蛋白质和DNA组装成一个复合体，这样基因表达才能准确地在适当的时间在正确的细胞类型中发生。这项研究将研究含有蛋白质和DNA的复合体的组装和拆卸，这是转录发生所必需的。这项研究将使用能够观察到单分子的实验。每次研究一个分子将揭示蛋白质和DNA的不同行为，这些行为对控制基因表达非常重要。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，并鼓励代表不足的少数群体和女科学家参与。该项目的具体目标是研究早期mRNA转录的步骤机制，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，这些技术已经成为揭示生物络合物的动态行为和异质性的关键因素，从而为了解它们的功能提供了独特的见解。实验将研究通用转录因子TFIIB在体外从早期转录的人类复合体中释放的机制。重要的是，活性和非活性复合体之间将有区别，从而将TFIIB的释放与活性直接联系起来。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非常重要。解决蛋白质/DNA复合体多样性的能力将揭示P53在DNA上形成不同寡聚复合体的可能途径，以及这些途径中结合和解离事件的动力学。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结合的模式将被确定。从这项研究中获得的知识将激发新的模型，为未来转录调控的研究提供广泛的信息，无论是在体外还是在生物系统中。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.