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

使用单分子荧光研究 RNA 聚合酶 II 转录和调节的机制

基本信息

批准号：
1817442
负责人：
Jennifer Kugel
金额：
$ 91.67万
依托单位：
University of Colorado at Boulder
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817442&HistoricalAwards=false
关键词：
Investigating Mechanisms RNA Polymerase II

项目摘要

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.

由DNA编码的基因的表达对于持续生命至关重要。该项目将研究基因表达的基本步骤。基因表达的第一步是转录：复制基因中一条DNA以形成RNA分子的过程。这要求许多不同的蛋白质和DNA组装成一个复合物，以便在正确的时间可以准确地发生基因表达，并在正确的细胞类型中进行。这项研究将研究含有转录至关重要的蛋白质和DNA的复合物的组装和拆卸。该研究将使用允许观察到单分子的实验。一次研究分子将揭示对蛋白质的多种行为和DNA对控制基因表达很重要的新见解。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开发，并鼓励人数不足的少数民族和女性科学家参与。该项目的具体目标是研究早期mRNA转录中的步骤机理，并了解转录激活蛋白p53与DNA结合的途径。实验利用了单分子荧光技术，这些技术已成为揭示生物复合物的动态行为和异质性的重要因素，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从早期转录人类复合物体外释放的机制。重要的是，将在主动复合物和非活性复合物之间进行区分，从而将TFIIB释放与活动直接相关。研究还将集中于了解p53如何结合其DNA响应元件，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很重要。解决蛋白质/DNA复合物中多样性的能力将揭示p53在DNA上形成不同的寡聚复合物的允许途径，以及这些途径中关联和解离事件的动力学。如何确定p53响应元件以及p53中的特定区域和氨基酸，将确定DNA结合的控制模式。这项研究所获得的知识将刺激新模型，这些模型将在体外和生物系统中广泛地为未来的转录调节研究提供信息。该奖项反映了NSF的法定任务，并被认为是通过基金会的智力优点和更广泛的影响来审查标准的评估值得支持的。