Poising of RNA Polymerase II: Features and Functions

知识价值。RNA聚合酶II （RNAPII）在某些基因的转录激活之前的占据地位的最初发现发生在果蝇中超过25年前。由于全基因组分析的最新进展，RNAPII在非活性状态下的占用可以在整个进化谱和不同的基因组中检测到。本项目的目标是通过关注酵母中的平衡RNAPII和作为氧化应激反应的调控系统来研究基因募集后调控机制。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），适合不同水平的研究人员（研究生、本科生和小学生）。因此，对氧化应激的转录反应将重点研究结合前和平衡的RNAPII复合体，通过测试两个主要假设：1)平衡启动子具有与招募调节启动子不同的共同特征；2)平衡启动子在调控基因表达方面具有功能优势。由于招募后调控在人类和其他生物中普遍存在，这些发现将显著影响基因表达领域，并有可能改变我们的整体观点。因此，这些研究非常及时，意义重大。更广泛的影响。该项目至少在四个方面具有广泛的影响。首先，这些研究为本科生学员提供了合适的项目，他们可能有时间限制，而且实验室经验有限。其次，对技术和时间要求较高的研究为研究生在多个不同学科（遗传学、基因组学、细胞和分子生物学）提供了出色的交叉训练经验。这些技术是非常通用的。如果学员将来选择研究其他关键的遗传机制（DNA修复、复制、重组、RNA代谢、表观遗传学等），他们将利用这些技术。第三，为了传播外展的传统，本科生和研究生将参与将基因表达研究带入小学教室。作为先前资助的NSF项目的一部分，五年级学生和他们的老师帮助开发了一个名为“生物化学是基础”的新颖扩展项目。这个创新的课程有8个独立的1小时课程，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。由于廉价和生物安全的试剂以及在项目中创建的详细工作手册，如果许多科学家接受了该计划的培训，他们可以为当地社区提供类似的东西。因此，该计划将扩大到包括一个互动网站。最后，该项目的结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发表。总之，这个项目直接支持了NSF的使命，即推进发现和理解，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.