Investigating mechanisms of RNA polymerase II transcription and regulation using single molecule fluorescence

在DNA中编码的基因的表达始于转录--复制一条DNA链来制造RNA的过程。转录需要许多不同的蛋白质和DNA组装成大的复合体，以便在适当的细胞环境中准确和及时地表达基因。这个项目将使用显微镜技术来研究转录早期阶段涉及的关键复合体的组装和拆卸，这种技术允许观察单分子。监测单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。该项目还将通过培训研究生和本科生，开发新的实验室课程，增加代表不足的少数民族和女性科学家对STEM研究的参与，产生广泛的教育影响。该项目的具体目标是研究支配结构蛋白和转录因子与核小体结合的大分子相互作用，以及RNA聚合酶II(POL II)转录复合体中发生的分子转化。这些实验采用了体外单分子荧光技术，非常适合于研究被研究系统中的快速开/关相互作用、分子扫描和异质性，从而提供了对功能的深入了解。一个目的是研究人类建筑蛋白HMGB1如何能够快速穿越核小体，并帮助转录因子P53结合其识别位点。第二个目标是解决一般转录因子TFIIE与Pol II复合体的结合/解离如何控制转录活性。这项研究的独特之处在于，将区分活性和非活性复合体，从而直接将TFIIE行为与转录活性联系起来。从这项研究中获得的知识将刺激新的模型，这些模型将增加对转录调控的理解。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。

Expression of genes encoded in DNA starts with transcription – the process whereby one strand of DNA is copied to make RNA. Transcription requires that many different proteins and the DNA assemble into large complexes to enable accurate and timely gene expression in appropriate cellular contexts. This project will study the assembly and disassembly of key complexes involved in the early stages of transcription using microscopy techniques that allow observation of single molecules. Monitoring individual molecules will reveal new insights into diverse behaviors of proteins and DNA that are important for controlling gene expression. This project will also have broad educational impacts through training of graduate and undergraduate students, development of a new laboratory curriculum, and increased participation of underrepresented minority and women scientists in STEM research.The specific objectives of the project are to investigate the macromolecular interactions that govern binding of architectural proteins and transcription factors to nucleosomes, and the molecular transformations that occur in RNA polymerase II (Pol II) transcription complexes. The experiments employ in vitro single molecule fluorescence techniques, which are ideally suited to study rapid on/off interactions, molecular scanning, and heterogeneity in the systems under investigation, thus providing deep insights into function. One objective is to investigate how the human architectural protein HMGB1 is able to rapidly traverse nucleosomes and aid transcription factor p53 in binding its recognition site. The second objective is to resolve how the binding/dissociation of general transcription factor TFIIE to Pol II complexes controls transcriptional activity. Unique to this study, active and inactive complexes will be distinguished, thereby directly relating TFIIE behavior to transcriptional activity. The knowledge gained from this research will stimulate new models that will increase understanding of transcriptional regulation.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.