Molecular Dissection of Transcription Termination Mechanisms in Eukaryotes

智力优势。 真核mRNA的转录是从基因组到细胞所需蛋白质表达的信息流中的一个必要步骤。转录的最后一步称为终止，涉及RNA聚合酶II（Pol II）和RNA从DNA模板中释放。 终止缺陷可由于DNA复制、染色体分离或转录起始所需的下游DNA元件上的通读转录的干扰而损害细胞功能。不良终止也可导致RNA的加工减少和降解增加以及基因启动子处的起始减少。尽管近年来取得了重大进展，终止仍然是最不了解的转录步骤之一。mRNA poly（A）位点下游的Pol II终止需要Rat 1核酸外切酶和在poly（A）位点识别并切割RNA的蛋白质的协同努力。Pol II通过基因体的转录既快速又进行性，但如果停滞，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，并诱导导致暂停和释放的变化。这项研究的目标是破译导致Rat 1介导的Pol II终止的分子机制，并寻找Rat 1如何促进转录核糖体RNA的Pol I释放的相似之处。通过酵母中的遗传筛选，使用定向和随机诱变，该项目应确定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以其他方式改变酶的终止行为。以前，缺乏一个明确的，易于操作的体外系统，在其中研究终止已经很难确定各个因素的贡献。这个问题已经通过开发一种新的体外测定来解决，在该测定中，突变体和野生型聚合酶可以被停止，然后用纯化的因子单独和组合来挑战。 该试验将纳入检查体内和细胞提取物中转录的综合策略中。更广泛的影响。从科学的角度来看，这个项目的成功完成应该会对转录终止的机制产生重要的新见解，并导致对转录周期中这一步骤可能在所有真核生物中受到调节的点的新的基本理解。此外，它还将为学生提供生物化学、分子生物学和遗传学等学科的严格培训，并为受训人员提供多种机会，使他们能够在书面和口头介绍其研究和指导年轻学生方面获得经验。此外，PI一直在积极开发新的方法来培养下一代科学家和增加生物研究的多样性，PI实验室的成员作为导师和学员参加这些计划。这些项目包括为代表性不足的学生提供的暑期研究和博士后项目，以及一个博士后培训项目，该项目为研究员成功的学术生涯做好准备，包括生物科学的研究，指导和教学本科生。

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.