Mechanisms of RNA polymerase-specific transcription complex assembly on U1 and U6 snRNA gene promoters

智力优势：被称为U1、U2、U4、U5和U6的小核RNA(SnRNAs)包括一类高度丰富的RNA分子，这些RNA分子是高等生物体中前信使RNA剪接所必需的。除了由RNA聚合酶III合成的U6外，这些SnRNAs都是由RNA聚合酶II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招募正确的RNA聚合酶以启动正确的SnRNA合成所涉及的结构-功能关系。这两类SnRNA基因的转录都需要一种独特的蛋白质因子，称为SnRNA激活蛋白复合体(SNAPc)。该因子识别一种重要的启动子元件，称为PSE，位于RNA合成起始点上游的DNA 40-75碱基对(BP)上。在果蝇(本项目中使用的模式生物)中，DmSNAPc由三个亚基组成，它们共同对~21bp长的PSEA(果蝇PSE)进行序列特异性识别。尽管U1 PSEA和U6 PSEA在21个核苷酸位置中只有5个不同，但这种序列差异在决定苍蝇SnRNA基因的RNA聚合酶专一性方面发挥了重要作用。此外，当与U6与U1 PSEA结合时，的三个亚基呈现不同的构象。这种构象差异被认为是导致正确的RNA聚合酶招募的原因。为了更好地了解与U1和U6 PSEA结合的DmSNAPc的构象差异，将采用PI实验室开发的一种新技术，该技术使用定点特定的蛋白质-DNA交联，并在指定的位点对蛋白质进行化学切割。将最大的DmSNAPc亚基与U6启动子DNA之间的接触点与该亚基与U1启动子DNA之间的接触点进行比较。接下来，我们将研究U1和U6启动子上的预起始复合体(PIC)组装，主要重点是RNA聚合酶II在无TATA的U1启动子上的招募，但将进行类似的实验来研究Pol III PIC在U6启动子上的组装。最后，将用冷冻电子显微镜研究DmSNAPc与U1和U6 PSEA结合的整体轮廓形状，并用X射线结晶学在原子水平上研究DmSNAPc及其亚基的结构。这项研究的结果将有助于科学界在RNA合成水平上理解基因表达。果蝇U1基因是研究RNA聚合酶II转录复合体在无TATA启动子上组装的一个特别容易的范例，目前对这一过程了解很少。更广泛地说，这个系统是一个很好的模型，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。广泛的影响：这项研究将由获得生物化学/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的职业生涯、升入研究生院和专业学校、或在学术界的职业生涯以及在社区大学一级的教学提供培训。圣地亚哥州立大学由于地处边境，重视本科生和研究生教学，为大量来自代表人数较少的民族的本科生提供服务。PI活跃在本科课堂教学中，有着从高中到研究生阶段让代表性不足的学生参与研究的良好记录和历史。

Intellectual Merit: The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 comprise a highly abundant class of RNA molecules that are required for pre-messenger RNA splicing in higher organisms. These 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 regulatory signals and overlapping sets of transcription factors for their expression. The main goal of the project is to gain an understanding of the structure-function relationships that are involved in recruiting the correct RNA polymerase to initiate synthesis of the correct snRNAs. Transcription of both classes of snRNA genes requires a unique protein factor referred to as the snRNA-activating protein complex (SNAPc). This factor recognizes an essential promoter element termed the PSE located in the DNA 40-75 base pairs (bp) upstream of the start site of RNA synthesis. In the fruit fly D. melanogaster (the model organism used in this project), DmSNAPc is composed of three subunits that together carry out sequence-specific recognition of the ~21 bp long PSEA (the fruit fly PSE). Even though a U1 PSEA and a U6 PSEA differ at only 5 of 21 nucleotide positions, this sequence difference plays a major role in determining the RNA polymerase specificity of fly snRNA genes. Furthermore, the three subunits of assume a different conformation when bound to a U6 versus a U1 PSEA. This conformational difference is believed to be responsible for the recruitment of the correct RNA polymerase. To better understand the conformational differences of DmSNAPc bound to U1 and U6 PSEAs, a novel technique developed in the PI's lab will be employed that uses site-specific protein-DNA cross-linking combined with chemical cleavage of the protein at defined sites. Contact points made between the largest DmSNAPc subunit and U6 promoter DNA will be compared with the contact points made between this subunit and U1 promoter DNA. Next, pre-initiation complex (PIC) assembly on U1 and U6 promoters will be investigated, with primary emphasis on RNA polymerase II recruitment to the TATA-less U1 promoter, but analogous experiments will be performed to investigate Pol III PIC assembly on the U6 promoter. Finally, the overall contour shape of DmSNAPc bound to the U1 and U6 PSEAs will be studied by cryo-electron microscopy, and X-ray crystallography will be employed to investigate the structure of DmSNAPc and it subunits at the atomic level. The results of the research will contribute widely to the scientific community's understanding of gene expression at the level of RNA synthesis. Fruit fly U1 genes serve as a particularly tractable paradigm for investigating RNA polymerase II transcription complex assembly on TATA-less promoters, a process that is currently very poorly understood. More generally, this system serves as an excellent model for understanding how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes.Broader Impacts: The research will be performed by students in satisfaction of their B.S., M.S., and Ph.D. degrees in biochemistry/molecular biology. The project will provide training for their future careers in the biotechnology industry, for advancement to graduate and professional schools, or to careers in academia as well as teaching at the community college level. San Diego State University, due to its border location and emphasis on undergraduate as well as graduate instruction, serves a large body of undergraduate students from underrepresented ethnic groups. The PI is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented students in research, from high school through the graduate level.