Fidelity of Transcription by RNA Polymerase II

该项目旨在了解细胞保持转录保真度的方式，这是利用遗传信息的第一步。蛋白质编码基因的转录由RNA聚合酶II（pol II）催化，RNA聚合酶II是一种在模式生物酿酒酵母（面包酵母）中含有12种不同蛋白质组分的酶。维持转录保真度是重要的，因为即使是一小群改变的RNA转录物也可能对细胞产生非常负面的影响，例如产生可能破坏其他细胞功能的显性负性蛋白。初步结果表明，Rpb9，一个小的，非必需的亚基pol II，是需要准确的转录。该研究旨在回答以下问题：Rpb9如何有助于转录保真度？易错转录是Rpb9缺陷型pol II的固有特性吗？还有哪些蛋白质影响转录保真度？这些问题将通过三种方法来解决。第一种方法将检查Rpb9缺陷型pol II的转录特性。将在体外检查转录活性，以直接评估NTP错误掺入和错误掺入后延伸的速率，以及错误掺入核苷酸的溶核校正速率。还将探索Rpb 9对掺入正确和不正确核苷酸的Km的潜在影响。初步研究已经确定，当Rpb9从pol II中缺失时，UTP错误掺入模板CTP的速率增加，此外，新生RNA 3 '端的错误掺入U不太可能通过校对被切除。第二种实验方法将确定Rpb9的氨基酸或结构域，这些氨基酸或结构域对保持保真度很重要。突变将在旨在破坏功能或改变与其他蛋白质相互作用的位点进行工程化，并将利用替代的无偏见策略在RPB 9中产生随机突变。将在体内和体外评估由这些突变引起的功能改变。第三种方法将基于当它们在RPB 9中缺失的情况下缺失时的合成致死性或通过它们抑制RPB 9中突变的能力来鉴定对转录保真度重要的其他基因。 至少有一个基因，其编码的染色质重塑共激活子佐贺的亚基，已显示在初步工作中，在体内转录保真度的影响。更广泛的影响：从更广泛的角度来看，拟议的研究不仅将产生新的知识，但它也将有助于培养研究生和本科生，包括参与NSF赞助的REU计划。大型公立大学的研究活动是他们为学生提供学习经验的最显着和未充分利用的资产，PI在他的实验室中培养本科生和高中生的历史悠久。

This project seeks to understand the ways that cells maintain the fidelity of transcription, the first step in the utilization of genetic information. Transcription of protein-coding genes is catalyzed by RNA polymerase II (pol II), an enzyme that contains 12 different protein components in the model organism Saccharomyces cerevisiae (baker's yeast). Maintaining transcriptional fidelity is important, as even a small population of altered RNA transcripts could have very negative consequences for the cell, such as production of dominant negative proteins that could disrupt other cellular functions. Preliminary results have demonstrated that Rpb9, a small, non-essential subunit of pol II, is required for accurate transcription. The research is designed to answer the following questions: How does Rpb9 contribute to transcriptional fidelity? Is error-prone transcription an inherent property of Rpb9-deficient pol II? What other proteins affect transcriptional fidelity? These questions will be addressed with three approaches. The first approach will examine the transcriptional properties of Rpb9-deficient pol II. Transcriptional activity will be examined in vitro to directly assess the rates of NTP misincorporation and extension after misincorporation, as well as the rate of nucleolytic proofreading of a misincorporated nucleotide. Potential effects of Rpb9 on the Km for incorporation of correct and incorrect nucleotides will also be explored. Preliminary studies have established that when Rpb9 is missing from pol II, the rate of misincorporation of UTP for a templated CTP is increased, and, furthermore, that the misincorporated U at the 3'-end of the nascent RNA is less likely to be excised by proofreading. A second experimental approach will identify amino acids or domains of Rpb9 that are important for maintaining fidelity. Mutations will be engineered at sites designed to disrupt function or alter interactions with other proteins, and an alternative, unbiased strategy will be exploited to generate random mutations in RPB9. Altered functions resulting from these mutations will be assessed both in vivo and in vitro. The third approach will identify other genes important for transcriptional fidelity based on synthetic lethality when they are deleted in the context of a deletion in RPB9 or by their ability to suppress mutations in RPB9. At least one gene, which encodes a subunit of the chromatin remodeling coactivator SAGA, has been shown in preliminary work to have an effect on transcriptional fidelity in vivo. Broader Impacts: From a broader perspective, the proposed research will not only generate new knowledge, but it will also serve to train graduate and undergraduate students, including participants in an NSF-sponsored REU program. The research activities at large public universities are their most notable and under-utilized asset for providing learning experiences for students, and the PI has a long history of training undergraduate and high school students in his lab.