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

被称为U1、U2、U4、U5和U6的小核RNA(SnRNAs)组成了一类高度丰富的代谢稳定的非多腺化RNA分子，是真核生物中前信使RNA剪接所必需的。这些SnRNA都是由RNA聚合酶II合成的，除了U6，它是由RNA聚合酶III合成的。尽管U6基因和RNA聚合酶II转录的SnRNA基因在RNA聚合酶专一性上存在差异，但U6基因和RNA聚合酶II转录的SnRNA基因利用相似的顺式调控信号和重叠的转录因子来表达。这个项目的主要目标是了解在单个SnRNA基因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。在高等真核生物中，这两类SnRNA基因的转录都需要在转录起始点上游40-75个碱基对的区域内有一个基本的近端序列元件(PSE)。在果蝇中，PSE是由PSE结合蛋白(称为DmPBP)识别的，PSE结合蛋白包含三个与DNA密切相关的不同亚基。首席研究员实验室之前的工作已经导致了一个工作模型，在该模型中，U1和U6 PSE充当DmPBP的差异变构效应器。DmPBP的构象差异被认为是随后正确的RNA聚合酶下游招募的原因。将采取两种截然不同但高度协同的方法来测试这一模型的有效性。首先，将利用生殖系转化和果蝇遗传学来选择在SnRNA基因启动子上具有改变的RNA聚合酶专一性的突变体。第二种方法涉及对编码DmPBP亚单位的基因进行有针对性的体外突变。生化机制，包括DmPBP的构象差异，导致RNA聚合酶专一性的变化将被检验。特别感兴趣的将是确定特定于一个RNA聚合酶而不是另一个RNA聚合酶招募所需的功能结构域或氨基酸残基。为了正确读出DNA中的遗传信息，必须将正确的RNA聚合酶招募到任何特定的感兴趣基因上，这一点至关重要。这个项目将阐明这一点是如何在分子水平上实现的。首席研究员实验室正在研究的系统也是大分子组装的通用模型，并用于理解大分子相互作用的细微变化如何导致显著不同的生物结果。该项目将帮助我们了解细胞内如何在不同的生物途径之间做出决定。将为致力于获得生物化学和分子生物学学士、硕士和博士学位的学生提供研究培训。残疾学生和代表人数不足的群体将是积极的参与者。

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.