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

U1和U6 snRNA基因启动子上RNA聚合酶特异性转录复合物组装的机制

• 批准号: 1157549
• 负责人: William Stumph
• 金额: $ 61.29万
• 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: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1157549&HistoricalAwards=false
• 关键词: Mechanisms; RNA; polymerase; specific; transcription

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.

智力优势：被称为U1、U2、U4、U 5和U6的小核RNA（snRNA）包含高度丰富的一类RNA分子，其是高等生物体中前信使RNA剪接所需的。这些snRNA由RNA聚合酶II合成，但U6由RNA聚合酶III合成。尽管RNA聚合酶特异性存在这种差异，但U6基因和RNA聚合酶II转录的snRNA基因利用相似的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结构-功能关系，这些关系涉及招募正确的RNA聚合酶来启动正确的snRNA的合成。两类snRNA基因的转录都需要一种独特的蛋白质因子，称为snRNA激活蛋白复合物（SNAPc）。该因子识别位于RNA合成起始位点上游40-75个碱基对（bp）的DNA中称为PSE的必需启动子元件。果蝇D.在黑腹果蝇（本项目中使用的模式生物）中，DmSNAPc由三个亚基组成，这三个亚基一起进行~21 bp长的PSEA（果蝇PSE）的序列特异性识别。尽管U1 PSEA和U6 PSEA仅在21个核苷酸位置中的5个不同，但这种序列差异在确定果蝇snRNA基因的RNA聚合酶特异性中起主要作用。此外，当结合至U6与U1 PSEA时，三个亚基呈现不同的构象。这种构象差异被认为是负责招募正确的RNA聚合酶。为了更好地理解与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，该技术使用位点特异性蛋白质-DNA交联结合蛋白质在定义位点的化学裂解。将最大DmSNAPc亚基和U6启动子DNA之间的接触点与该亚基和U1启动子DNA之间的接触点进行比较。接下来，将研究U1和U6启动子上的前起始复合物（PIC）组装，主要强调RNA聚合酶II向无TATA的U1启动子的募集，但将进行类似的实验以研究U6启动子上的Pol III PIC组装。最后，结合到U1和U6 PSEAs的DmSNAPc的整体轮廓形状将通过冷冻电子显微镜进行研究，并且X射线晶体学将用于在原子水平上研究DmSNAPc及其亚基的结构。研究结果将广泛有助于科学界在RNA合成水平上理解基因表达。果蝇U1基因作为一个特别容易处理的范例，用于研究RNA聚合酶II转录复合物组装TATA-少启动子，一个过程，目前是非常了解。更一般地说，这个系统作为一个很好的模型，了解如何非常微妙的变化，大分子的相互作用和组装可以导致显着不同的生物结果。更广泛的影响：该研究将由学生进行满意他们的学士学位，医学硕士，和博士生物化学/分子生物学学位。该项目将为他们未来在生物技术行业的职业生涯提供培训，以促进研究生院和专业学校的发展，或在学术界的职业生涯以及在社区学院一级的教学。圣地亚哥州立大学，由于其边界位置和强调本科以及研究生教学，服务于来自代表性不足的民族群体的本科生的大机构。PI在本科课堂教学中很活跃，从高中到研究生阶段，在研究中涉及代表性不足的学生方面有着良好的记录和历史。