Mechanisms of RNA Polymerase Specificity at Small Nuclear RNA Gene Promoters

被称为U1、U2、U4、U5和U6的小核RNA(SnRNAs)是真核生物中前信使RNA剪接所必需的RNA分子。剪接体SnRNA由RNA聚合酶II合成，U6除外，U6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，但它们利用相似的顺式作用调控信号和重叠的转录因子来表达。该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)的分子机制和结构差异。在高等真核生物中，这两类SnRNA基因的转录都需要一个独特的多亚基转录因子，通常被称为SnRNA激活蛋白复合体，或SNAPc。该因子识别一种称为PSE的基本启动子元件。PI的实验室已经证明，PSE的准确序列是决定果蝇U1和U6基因RNA聚合酶专一性的主要决定因素。DmSNAPc含有三个多肽亚基(DmSNAP190、DmSNAP50和DmSNAP43)，蛋白质-DNA光交联分析表明，当DmSNAPc与U1和U6 PSE结合时，DmSNAPc的构象不同，尽管这些PSE在21个核苷酸位置中只有5个不同。这导致了一个工作模型，在该模型中，当DmSNAPc与U1和U6 PSES结合时，构象的差异被认为是导致随后正确的RNA聚合酶的下游招募的原因。将进行更深入的实验，以探讨决定RNA聚合酶在SnRNA基因启动子上的专一性的分子机制。具有突变亚基的DmSNAPc将在同源表达系统中产生，并将确定DmSNAPc组装、PSE结合和转录激活所需的蛋白质结构域。先前的工作表明，切换U1和U6PSES可以防止体内形成生产性转录复合体。将进行实验，以确定在哪个阶段，预引发复合体组装在含有错误的PSE的启动子上被阻止。这项研究的结果将有助于理解储存在DNA中的遗传信息表达的分子机制。总的来说，正在研究的问题是一个很好的模型，可以理解大分子相互作用和组装中非常细微的变化如何导致显著不同的生物结果。这项研究将由攻读生物化学和分子生物学学士、硕士和博士学位的学生进行。该项目将为他们未来在生物技术行业、研究生院和专业学校以及学术界的职业生涯提供培训。国际学生联合会积极参与本科生课堂教学，在让少数族裔参与研究方面有着良好的记录和历史。

The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 are essential RNA molecules that are required for pre-messenger RNA splicing in eukaryotic organisms. The spliceosomal snRNAs are 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 the project is to gain an understanding of the molecular mechanisms and structural differences responsible for the selection of the correct enzyme (RNA polymerase II or RNA polymerase III) at individual snRNA gene promoters. In higher eukaryotes, transcription of both classes of snRNA genes requires a unique multi-subunit transcription factor most commonly referred to as the snRNA-activating protein complex, or SNAPc. This factor recognizes an essential promoter element termed the PSE. The PI's laboratory has shown that the exact sequence of the PSE is a major determinant of the RNA polymerase specificity of Drosophila melanogaster U1 and U6 genes. DmSNAPc contains three polypeptide subunits (DmSNAP190, DmSNAP50, and DmSNAP43), and protein-DNA photocrosslinking assays have shown that the conformation of DmSNAPc is different when bound to U1 and U6 PSEs, even though these PSEs differ at only 5 of 21 nucleotide positions. This has led to a working model in which a difference in the conformation of DmSNAPc, when bound to U1 and U6 PSEs, is believed to be responsible for the subsequent downstream recruitment of the correct RNA polymerase. Experiments will be carried out to probe more deeply into the molecular mechanisms that determine RNA polymerase specificity at snRNA gene promoters. Versions of DmSNAPc with mutated subunits will be generated in a homologous expression system, and protein domains required for DmSNAPc assembly, PSE binding, and transcriptional activation will be identified. Previous work revealed that switching the U1 and U6 PSEs prevents the formation of productive transcription complexes in vivo. Experiments will be performed to determine at which stage preinitiation complex assembly is blocked on promoters that contain the "wrong" PSE. The results of the research will contribute widely toward understanding the molecular mechanisms involved in the expression of genetic information stored in the DNA. In general, the problem under investigation serves as an excellent model for understanding how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes. The research will be performed by students working on their B.S., M.S., and Ph.D. degrees in biochemistry and molecular biology. The project will provide training for their future careers in the biotechnology industry, graduate and professional schools, and academia. The PI is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented minorities in research.