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.

智力优点：称为U1，U2，U4，U5和U6的小核RNA（SNRNA）组成了高度较高的RNA剪接所需的高度丰富的RNA分子。这些SNRNA由RNA聚合酶II合成，除U6外，由RNA聚合酶III合成。尽管RNA聚合酶特异性差异存在差异，但U6基因和RNA聚合酶II转录的SNRNA基因利用了相似的调节信号和转录因子的重叠集以表达其表达。该项目的主要目的是了解募集正确的RNA聚合酶启动正确SNRNA的结构 - 功能关系。两类SNRNA基因的转录需要一个独特的蛋白质因子，称为SNRNA激活蛋白复合物（SNAPC）。该因素识别出一个必需的启动子元素，该元素称为RNA合成开始位点上游的DNA 40-75碱基对（BP）中的PSE。在果蝇D. melanogaster（该项目中使用的模型生物）中，DMSNAPC由三个亚基组成，这些亚基共同对〜21 bp long Psea（果蝇PSE）进行了序列特异性识别。即使在21个核苷酸位置中只有5个U1 psea和U6 psea不同，但该序列差在确定FLY SNRNA基因的RNA聚合酶特异性方面起主要作用。此外，当与U6与U1 psea结合时，这三个亚基假设了不同的构象。这种构象差异被认为是募集正确的RNA聚合酶的原因。为了更好地理解与U1和U6 PSEAS结合的DMSNAPC的构象差异，将采用PI实验室中开发的一种新技术，该技术将采用使用位点特异性蛋白DNA交叉链接，并在定义的位点结合了蛋白质的化学裂解。将最大的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，将研究在U1和U6启动子上的生存络合物（PIC）组件，主要强调RNA聚合酶II募集到无TATA-U1启动子上，但是将进行类似的实验，以研究U6启动子上的POL III PIC组件。最后，将通过冷冻电子显微镜研究与U1和U6 PSEA的DMSNAPC的总体轮廓形状，并将使用X射线晶体学研究DMSNAPC及其在原子水平的IT亚基的结构。该研究的结果将在RNA合成水平上对科学界对基因表达的理解做出广泛的贡献。果蝇U1基因是一种特别易于处理的范式，用于研究无TATA的启动子上的RNA聚合酶II转录复合物组件，这一过程目前非常了解这一过程。更一般地，该系统是理解大分子相互作用和组装的非常微妙的变化的绝佳模型，这会导致显着不同的生物学结果。BRODER的影响：该研究将由学生满意其B.S.，M.S.和Ph.D.进行。生物化学/分子生物学学位。该项目将为他们在生物技术行业的未来职业，毕业和专业学校的进步或学术界的职业以及在社区学院一级的教学提供培训。圣地亚哥州立大学由于其边境地理位置并强调本科生和研究生教学，为来自代表性不足的族裔群体的大量本科生提供服务。 PI活跃于本科课堂教学，并具有良好的往绩和历史，这些历史是从高中到研究生层面的代表性不足的学生参与研究。