Biochemical and Genetic Analysis of the RNA Polymerase Specificity of Small Nuclear RNA Genes

小核 RNA 基因的 RNA 聚合酶特异性的生化和遗传分析

• 批准号: 0131151
• 负责人: William Stumph
• 金额: $ 37.5万
• 依托单位: San Diego State University Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0131151&HistoricalAwards=false
• 关键词: Biochemical; Genetic; Analysis; RNA; Polymerase

The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 comprise a highly abundant class of metabolically stable, non-polyadenylated RNA molecules that are required for pre-messenger RNA splicing in eukaryotic organisms. These snRNAs are all synthesized by RNA polymerase II, with the exception of U6, which is synthesized by RNA polymerase III. Despite this difference in RNA polymerase specificity, U6 genes and the RNA polymerase II-transcribed snRNA genes utilize similar cis-acting regulatory signals and overlapping sets of transcription factors for their expression. The main goal of this project is to gain an understanding of the molecular mechanisms that are responsible for the selection of the correct enzyme (either RNA polymerase II or RNA polymerase III) at individual snRNA gene promoters. In higher eukaryotes, transcription of both classes of snRNA genes requires an essential proximal sequence element (PSE) within the region 40-75 base pairs upstream of the transcription start site. In the fruit fly Drosophila melanogaster, the PSE is recognized by the PSE binding protein (termed DmPBP) that contains three distinct subunits that closely approach the DNA. Previous work in the principal investigator's lab has led to a working model in which the U1 and U6 PSEs act as differential allosteric effectors of DmPBP. Conformational differences in DmPBP, in turn, are believed to be responsible for the subsequent downstream recruitment of the correct RNA polymerase. Two distinct yet highly synergistic approaches will be undertaken to test the validity of this model. First, germline transformation and Drosophila genetics will be employed to select mutants that have an altered RNA polymerase specificity at snRNA gene promoters. The second approach involves targeted in vitro mutagenesis of the genes that code for the subunits of DmPBP. The biochemical mechanisms, including conformational differences in DmPBP, that lead to changes in RNA polymerase specificity will be examined. Particular interest will be focused upon identifying functional domains or amino acid residues required specifically for the recruitment of one RNA polymerase but not the other. For the genetic information that resides in DNA to be correctly read out, it is critical that the correct RNA polymerase must be recruited to any particular gene of interest. This project will shed light on how this is accomplished at the molecular level. The system under investigation in the principal investigator's laboratory also serves as a general model for macromolecular assembly and for understanding how subtle changes in macromolecular interactions can lead to significantly different biological outcomes. The project will help us to understand how decisions among alternative biological pathways are made within cells. Research training will be provided for students engaged in acquiring their B.S., M.S., and Ph.D. degrees in biochemistry and molecular biology. Students with disabilities and from underrepresented groups will be active participants.

称为U1，U2，U4，U5和U6的小核RNA（SNRNA）组成了一类高度丰富的代谢稳定的，非多烯基化的RNA分子，这些分子在真核生物中需要预几个预习体RNA所需的RNA所需。 这些SNRNA都是由RNA聚合酶II合成的，除U6外，由RNA聚合酶III合成。 尽管RNA聚合酶特异性差异存在差异，但U6基因和RNA聚合酶II转录的SNRNA基因利用类似的顺式作用调节信号和转录因子的重叠集来表达其表达。 该项目的主要目的是了解负责在单个SNRNA启动子上选择正确酶（RNA聚合酶II或RNA聚合酶III）的分子机制。 在较高的真核生物中，两类SNRNA基因的转录需要在转录起始位点上游的40-75个基本对区域内的基本近端序列元件（PSE）。 在果蝇果蝇中，PSE被PSE结合蛋白（称为DMPBP）识别，该蛋白包含三个与DNA紧密接近的不同亚基。 主要研究者实验室的先前工作已导致了一个工作模型，其中U1和U6 PSE充当DMPBP的差异效应子。 反过来，DMPBP的构象差异被认为是随后的下游募集正确的RNA聚合酶。 将采用两种不同但高度协同的方法来测试该模型的有效性。 首先，将使用种系转化和果蝇遗传学来选择在SnRNA基因启动子上具有改变RNA聚合酶特异性的突变体。 第二种方法涉及针对DMPBP亚基的基因的体外诱变。 将研究导致RNA聚合酶特异性变化的生化机制，包括DMPBP中的构象差异。 特别的兴趣将集中在识别专门募集一种RNA聚合酶而不是另一种的功能域或氨基酸残基所需的功能域或氨基酸残基。为了正确读取DNA中的遗传信息，必须将正确的RNA聚合酶募集到任何特定感兴趣的基因中至关重要。 该项目将阐明如何在分子水平上完成。 主要研究者实验室中正在研究的系统也是大分子组装的一般模型，并了解大分子相互作用的细微变化如何导致显着不同的生物学结果。 该项目将帮助我们了解细胞内替代生物学途径之间的决策。 将为从事获得学士学位，M.S.和博士学位的学生提供研究培训。生物化学和分子生物学学位。 残疾学生和人数不足的团体将是活跃的参与者。