Fidelity of Transcription by RNA Polymerase II

该项目旨在了解细胞维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，这种酶在模式生物酿酒酵母（面包酵母）中含有12种不同的蛋白质成分。保持转录保真度是很重要的，因为即使是一小部分改变的RNA转录物也会对细胞产生非常负面的影响，比如产生显性负性蛋白，可能会破坏其他细胞功能。初步结果表明，Rpb9是pol II的一个小的非必需亚基，是精确转录所必需的。该研究旨在回答以下问题：Rpb9如何促进转录保真度？容易出错的转录是缺乏rpb9的pol II的固有特性吗？还有哪些蛋白质影响转录保真度？这些问题将用三种方法来解决。第一种方法将检测rpb9缺陷pol II的转录特性。转录活性将在体外进行检测，以直接评估NTP错误整合和错误整合后的延伸率，以及错误整合的核苷酸的核溶解校对率。Rpb9对Km结合正确和错误核苷酸的潜在影响也将被探讨。初步研究已经证实，当pol II中Rpb9缺失时，模板CTP的UTP错接率会增加，此外，新生RNA 3'端错接的U不太可能通过校对被切除。第二种实验方法将确定对维持保真度很重要的Rpb9的氨基酸或结构域。突变将被设计在破坏功能或改变与其他蛋白质相互作用的位点上，并且一种替代的，无偏的策略将被利用来产生RPB9的随机突变。这些突变导致的功能改变将在体内和体外进行评估。第三种方法将根据在RPB9缺失的背景下被删除时的合成致死率或通过其抑制RPB9突变的能力来确定对转录保真度重要的其他基因。至少有一个编码染色质重塑共激活因子SAGA亚基的基因，已经在初步工作中显示对体内转录保真度有影响。更广泛的影响：从更广泛的角度来看，拟议的研究不仅会产生新的知识，而且还将有助于培养研究生和本科生，包括nsf赞助的REU项目的参与者。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著和未充分利用的资产，而PI在他的实验室培养本科生和高中生有着悠久的历史。

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.