RNA Polymerase-Specific Transcription Complex Assembly on snRNA Genes: Structural and Functional Relationships

snRNA 基因上 RNA 聚合酶特异性转录复合物的组装：结构和功能关系

• 批准号: 0842770
• 负责人: William Stumph
• 金额: $ 48万
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0842770&HistoricalAwards=false
• 关键词: RNA; Polymerase; Specific; Transcription; Complex

ABSTRACT Intellectual Merit: 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. 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 structure-function relationships that are involved in polymerase-specific transcription complex assembly, with emphasis on the TATA-less U1 promoter, More generally, this project will help us understand how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes. 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 located within the region 40-75 base pairs upstream of the transcription start site. In the fruit fly Drosophila melanogaster, SNAPc is composed of three subunits that together carry out sequence-specific recognition of the ~21 base-pair PSE (known more specifically as the PSEA in fruit flies). 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 insect snRNA genes. Furthermore, the three subunits of DmSNAPc adopt a different conformation when bound to a U1 vs. U6 PSEA. This conformational difference is believed to be responsible for the recruitment of the correct RNA polymerase. To better understand the structural arrangement of the SNAPc subunits bound to a PSEA, a novel technique that combines site-specific protein-DNA photo-cross-linking with specific chemical proteolysis of the protein will be employed. These experiments will determine the orientation or polarity of each of the SNAPc subunits on the DNA. In flies, the TATA-binding protein (TBP) is used for U1 transcription by RNA polymerase II, but the TBP-related factor TRF1 is utilized for U6 transcription by RNA polymerase III. The domains of the individual SNAPc subunits involved in interactions with TBP or TRF1 will be identified. The fly U1 gene will serve as an excellent model for investigating transcription complex assembly on TATA-less RNA polymerase II promoters. Toward that end, the mechanism by which SNAPc recruits TBP to the TATA-less U1 promoter will be investigated. Broader Impacts: The research will be performed by students working on 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. 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 principal investigator is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented students in NSF-funded research. The results of the project will be disseminated in peer-reviewed scientific journals and will contribute widely to the areas of transcription regulation and gene expression.

知识价值：被称为U1、U2、U4、U 5和U6的小核RNA（snRNA）包含一类高度丰富的代谢稳定的非多聚腺苷酸化RNA分子，其是真核生物中前信使RNA剪接所需的。剪接体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，U6基因和RNA聚合酶II转录的snRNA基因利用相似的顺式作用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聚合酶特异性转录复合物组装中涉及的结构-功能关系，重点是TATA-less U1启动子，更一般地说，该项目将帮助我们了解大分子相互作用和组装中非常微妙的变化如何导致显着不同的生物学结果。这两类snRNA基因的转录需要一个独特的多亚基转录因子，通常被称为snRNA激活蛋白复合物，或SNAPc。该因子识别位于转录起始位点上游40-75个碱基对区域内的称为PSE的必需启动子元件。在果蝇Drosophila melanogaster中，SNAPc由三个亚基组成，它们共同进行~21个碱基对PSE（在果蝇中更具体地称为PSEA）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昆虫snRNA基因的RNA聚合酶特异性中起主要作用。此外，DmSNAPc的三个亚基在与U1和U6 PSEA结合时采用不同的构象。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PSEA结合的SNAPc亚基的结构排列，将采用将位点特异性蛋白质-DNA光交联与蛋白质的特异性化学蛋白水解相结合的新技术。这些实验将确定DNA上每个SNAPc亚基的方向或极性。在果蝇中，TATA结合蛋白（TBP）用于RNA聚合酶II的U1转录，但TBP相关因子TRF 1用于RNA聚合酶III的U6转录。将鉴定参与与TBP或TRF 1相互作用的单个SNAPc亚基的结构域。苍蝇U1基因将作为一个很好的模型，研究转录复合物组装TATA-少RNA聚合酶II启动子。为此，SNAPc招募TBP的TATA-少U1启动子的机制将进行研究。 更广泛的影响：这项研究将由攻读学士学位的学生进行，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。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。主要研究者活跃于本科课堂教学，并有很好的记录和历史，涉及代表性不足的学生在NSF资助的研究。该项目的成果将在同行评审的科学期刊上传播，并将对转录调节和基因表达领域作出广泛贡献。