Single Molecule Studies of Transcription Complexes

真核生物中蛋白质编码基因的转录是一个高度调控的过程，它是由 当无数转录激活子识别特定的DNA元件近端和远端 核心推动者。这些DNA结合的激活剂然后指导RNA的动态组装 聚合酶II、通用转录因子和启动子DNA上的辅因子复合体启动 抄写。由于启动转录所需的确切事件序列很难理解， 对转录激活剂如何调控和指导转录的机械性理解 从它们的近端和远端识别位置形成的复杂队形对于理解 基因的表达是受调控的。 为了更好地理解RNAPII介导的转录机制，我们将开发 并应用单分子技术来(I)测量GTF的速率和序列 和RNAPII与启动子DNA结合，(Ii)确定 GTFS和RNAPII与启动子DNA结合受转录激活剂和辅助剂的调节. 激活剂，以及(Iii)决定远端增强剂如何进行激活。 在这些单分子研究中获得的关于转录启动动力学的信息将 补充通过建议的生化、EM和x射线获得的结构信息 该PPG的结晶学研究。对转录复合体形成机制的理解 将为抗癌治疗提供新的潜在蛋白质靶点。

Transcription of protein-encoding genes in eukaryotes is a highly regulated process that is initiated when a myriad of transcriptional activators recognize specific DNA elements proximal and distal to core promoters. These DNA bound activators then direct the dynamic assembly of RNA polymerase II, general transcription factors, and cofactor complexes on promoter DNA to initiate transcription. As the exact sequence of events needed to initiate transcription is poorly understood, a mechanistic understanding of how transcription activators regulate and direct transcription complex formation from their proximal and distant recognition sites is crucial to understanding how genes expression is regulated. Toward a better understanding of the mechanism of RNAPII mediated transcription we will develop and apply single molecule techniques to (i) measure the rates and the sequence by which the GTFs and RNAPII bind to promoter DNA, (ii) determine how the rates and the sequence by which the GTFs and RNAPII bind to promoter DNA are modulated by transcriptional activators and co- activators, and (iii) determine how activation occurs by distal enhancers. Information on dynamics of transcription initiation obtained in these single-molecule studies will complement the structural information obtained by the proposed biochemical, EM, and x-ray crystallographic studies of this PPG. Mechanistic understanding of transcription complex formation will provide new potential protein targets for anticancer therapies.