Molecular Dissection of Transcription Termination Mechanisms in Eukaryotes

智力上的功绩。真核细胞信使核糖核酸的转录是从基因组到细胞所需蛋白质表达的信息流中必不可少的一步。转录的最后一步称为终止，涉及从DNA模板释放RNA聚合酶II(POL II)和RNA。由于直读转录干扰了DNA复制、染色体分离或转录启动所需的下游DNA元件，终止缺陷会损害细胞的功能。不良的终止性也会导致加工减少和RNA降解增加，以及基因启动子的启动减少。尽管近年来取得了重大进展，但终止仍然是人们最不了解的转录步骤之一。PolII在mRNA聚(A)位点下游的终止需要Rat1核酸外切酶和蛋白质的协同作用，这些蛋白质识别并作用于聚(A)位点的RNA。POL II通过基因体内的转录是快速和渐进的，但如果停滞不前，POL II与DNA的结合是非常稳定的。然而，终止因子与聚合酶的相互作用克服了这些挑战，并引发了导致暂停和释放的变化。本研究的目的是破译导致Rat1介导的Pol II终止的分子机制，并寻找Rat1如何促进转录核糖体RNA的Pol I的释放。通过酵母中的遗传筛选，使用定向和随机突变，该项目应该确定RNA聚合酶的关键区域，这些区域可能直接与终止因子相互作用，或者以其他方式改变酶的终止行为。以前，由于缺乏一个明确的、易于操作的体外系统来研究终止，使得很难确定各种因素对个体的贡献。这个问题已经通过开发一种新的体外实验来解决，在这种实验中，突变和野生型聚合酶可以被停滞，然后用纯化因子单独和联合挑战。这项测试将被整合到一个综合策略中，以检查体内和细胞提取物中的转录。更广泛的影响。从科学上讲，这个项目的成功完成将为转录终止的机制提供重要的新见解，并导致对所有真核生物转录周期中这一步骤可能受到调控的点的新的基本理解。此外，它将为学生提供生物化学、分子生物学和遗传学学科的严格培训，并为受训人员提供多种机会，让他们在书面和口头陈述研究和指导年轻学生方面获得经验。此外，国际和平研究所一直积极开发新的方法来培训下一代科学家，并增加生物研究的多样性，国际和平研究所实验室的成员以导师和受训者的身份参加这些计划。这些项目包括针对代表性不足的学生的暑期研究和学士后项目，以及一个博士后培训项目，该项目旨在为成功的学术生涯做准备，涉及生物科学领域的本科生的研究、指导和教学。

Intellectual Merit. The transcription of eukaryotic mRNA is an obligate step in the flow of information from the genome to expression of proteins needed by the cell. The final step of transcription is called termination, and involves release of RNA polymerase II (Pol II) and the RNA from the DNA template. Defects in termination can impair cell function due to the interference of read-through transcription on downstream DNA elements needed for DNA replication, chromosomal segregation, or the initiation of transcription. Poor termination can also lead to decreased processing and increased degradation of the RNA as well as reduced initiation at the gene's promoter. Despite significant advances in recent years, termination remains one of the least understood steps of transcription. Pol II termination downstream of mRNA poly(A) sites requires the concerted efforts of the Rat1 exonuclease and proteins which recognize and act to cleave RNA at the poly(A) site. Transcription by Pol II through a gene's body is both rapid and processive, yet if stalled, the association of Pol II with DNA is remarkably stable. Nevertheless, interactions of termination factors with the polymerase overcome these challenges and induce changes that lead to pausing and release. The goal of this research is to decipher the molecular mechanisms that lead to Rat1-mediated termination of Pol II and to seek parallels in how Rat1 also facilitates release of Pol I, which transcribes ribosomal RNA. Through genetic screens in yeast, using both directed and random mutagenesis, this project should identify critical regions of RNA polymerase that might interact directly with termination factors or otherwise alter the termination behavior of the enzyme. Previously, the lack of a defined, easily manipulated in vitro system in which to study termination has made it difficult to determine individual contributions of the various factors. This problem has been addressed by developing a new in vitro assay in which mutant and wild-type polymerase can be stalled and then challenged with purified factors alone and in combination. This assay will be incorporated into an integrated strategy that examines transcription in vivo and in cell extract. Broader impacts. Scientifically, the successful completion of this project should give significant new insight into the mechanism of transcription termination and lead to a new fundamental understanding of points at which this step in the transcription cycle might be regulated in all eukaryotes. Furthermore, it will provide rigorous training for students in the disciplines of biochemistry, molecular biology and genetics as well as multiple opportunities for trainees to gain experience in the written and oral presentation of their research and in mentoring younger students. In addition, the PI has been active in developing new ways to train the next generation of scientists and to increase diversity in biological research, and the members of the PI's lab participate in these programs as mentors and trainees. These programs include summer research and postbaccalaureate programs for underrepresented students, and a postdoctoral training program that prepares fellows for successful academic careers that involve research, mentoring and teaching undergraduates in the biological sciences.