Poising of RNA Polymerase II: Features and Functions

智力上的功绩。在果蝇中，最初发现RNA聚合酶II(RNAPII)在转录激活之前占据某些基因的位置是在四分之一个世纪前。由于全基因组分析的最新进展，RNAPII处于不活跃状态的情况可以在整个进化谱和不同的基因组中检测到。这个项目的目标是通过关注酵母中稳定的RNAPII和作为调控系统的氧化应激反应来研究招募后基因调控的机制。这些研究充分利用了现有的方法(遗传学、分子生物学、生物化学、细胞生物学、基因组学)，适合不同层次的研究人员(研究生、本科生和小学生)。因此，我们将通过检验两个主要假说来研究对氧化应激的转录反应，重点是预先结合和稳定的RNAPII复合体：1)稳定的启动子具有不同于招募调控启动子的共同特征；2)稳定的启动子为基因表达的调控提供功能优势(S)。由于招募后的调控在人类和其他生物中广泛存在，这些发现将显著影响基因表达领域，具有改变我们整体观点的特殊潜力。因此，这些研究是非常及时和非常有意义的。更广泛的影响。该项目至少在四个方面产生了广泛的影响。首先，这些研究为本科生提供了合适的项目，他们可能有时间限制以及有限的实验室经验。其次，对技术和时间要求更高的研究为研究生提供了许多不同学科(遗传学、基因组学、细胞和分子生物学)的出色交叉培训经验。这些技术是高度通用的。如果学员选择研究其他关键的遗传机制(DNA修复、复制、重组、RNA新陈代谢、表观遗传学等)，他们将在未来利用这些技术。第三，为了推广外展的传统，本科生和研究生将参与到小学课堂上进行基因表达研究。作为之前资助的NSF项目的一部分，五年级的学生和他们的老师帮助开发了一个新的外展项目，名为“生物化学是小学”。这个创新的课程有八个单独的一小时的课程，包括实践活动，旨在探索涉及生物化学和酵母遗传学的科学推理的基本概念。由于廉价且对生物安全的试剂，以及在项目期间创建的详细工作手册，如果许多科学家接受该项目的培训，他们可以提供与当地社区类似的东西。因此，这一计划将扩大到包括一个互动网站。最后，该项目的成果将发表在基础广泛的科学期刊上，并在当地和国际会议上展示。总而言之，该项目直接支持国家科学基金会的使命，即促进发现和理解，同时促进教学、培训和学习。

Intellectual Merit. The initial discovery of the occupancy of RNA polymerase II (RNAPII) at certain genes prior to their transcriptional activation occurred over a quarter century ago in Drosophila. Due to recent advances in whole genome analyses, occupancy of RNAPII in an inactive state can be detected in organisms across the evolutionary spectrum and at diverse sets of genes. The goal of this project is to investigate post-recruitment mechanisms of gene regulation by focusing on poised RNAPII in yeast and the response to oxidative stress as the regulatory system. These studies take full advantage of the available approaches (genetics, molecular biology, biochemistry, cell biology, genomics), and are amenable to researchers at a variety of levels (graduate, undergraduate and elementary school students). Thus, the transcriptional response to oxidative stress will be investigated with a focus on the pre-bound and poised RNAPII complex by testing two overarching hypotheses: 1) Poised promoters share common features that are distinct from recruitment-regulated promoters; and 2) Poised promoters offer functional advantage(s) for the regulation of gene expression. Since post-recruitment regulation is widespread in humans and other organisms, these findings will significantly impact the field of gene expression with exceptional potential to alter our overall views. Thus, these studies are extremely timely and highly significant. Broader Impacts. The project has broad impact in at least four ways. First, the studies offer suitable projects for undergraduate trainees, who may have time constraints as well as limited laboratory experience. Second, studies with higher technical and time demands provide outstanding cross-training experiences for graduate students in a number of different disciplines (genetics, genomics, cell and molecular biology). These techniques are highly versatile. Trainees will utilize these techniques in the future if they choose to investigate other critical genetic mechanisms (DNA repair, replication, recombination, RNA metabolism, epigenetics, etc.). Third, to propagate the tradition of outreach, undergraduate and graduate students will be involved in taking gene expression studies into elementary school classrooms. As part of a previously funded NSF project, fifth graders and their teachers helped to develop a novel outreach program called 'Biochemistry is Elementary'. This innovative program has eight separate 1 hour sessions involving hands-on activities designed to explore basic concepts of scientific reasoning involving biochemistry and yeast genetics. Due to the inexpensive and biologically safe reagents and the detailed workbooks created during the project, many scientists could offer something similar to their local community if they received training on the program. As such, this program will be expanded to include an interactive website. Finally, results from this project will be published in broad based scientific journals, and presented at local and international meetings. Taken together, this project directly supports the mission of the NSF to advance discovery and understanding, while promoting teaching, training and learning.