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

被称为U1、U2、U4、U 5和U6的小核RNA（snRNA）包含一类高度丰富的代谢稳定的非多聚腺苷酸化RNA分子，其是真核生物中前信使RNA剪接所需的。 这些snRNA都是由RNA聚合酶II合成的，除了U6是由RNA聚合酶III合成的。 尽管RNA聚合酶特异性存在这种差异，U6基因和RNA聚合酶II转录的snRNA基因利用相似的顺式作用调控信号和重叠的转录因子集进行表达。 该项目的主要目标是了解负责在单个snRNA基因启动子处选择正确酶（RNA聚合酶II或RNA聚合酶III）的分子机制。 在高等真核生物中，两类snRNA基因的转录都需要在转录起始位点上游40-75个碱基对区域内的必需近端序列元件（PSE）。 在果蝇Drosophila melanogaster中，PSE被PSE结合蛋白（称为DmPBP）识别，该蛋白含有三个不同的亚基，这些亚基与DNA密切相关。 主要研究者实验室的先前工作已经产生了一个工作模型，其中U1和U6 PSE作为DmPBP的差异变构效应物。 DmPBP的构象差异，反过来，被认为是负责正确的RNA聚合酶的后续下游招聘。 将采取两种不同但高度协同的方法来测试这一模式的有效性。 首先，将采用种系转化和果蝇遗传学来选择在snRNA基因启动子处具有改变的RNA聚合酶特异性的突变体。 第二种方法涉及编码DmPBP亚基的基因的靶向体外诱变。 生化机制，包括DmPBP的构象差异，导致RNA聚合酶特异性的变化将进行检查。 特别感兴趣的将集中在识别功能结构域或氨基酸残基，特别是一个RNA聚合酶的招聘，但不是其他所需的。为了正确读出DNA中的遗传信息，必须将正确的RNA聚合酶募集到任何特定的感兴趣基因中，这一点至关重要。 这个项目将阐明这是如何在分子水平上完成的。 主要研究者实验室中正在研究的系统也可作为大分子组装的一般模型，并用于理解大分子相互作用中的细微变化如何导致显著不同的生物学结果。 该项目将帮助我们了解细胞内替代生物途径的决定是如何做出的。 研究培训将提供给从事获得学士学位的学生，医学硕士，和博士生物化学和分子生物学学位。 残疾学生和来自代表性不足群体的学生将积极参与。

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.