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.

抽象的智力优点：称为U1，U2，U4，U5和U6的小型核RNA（SNRNA）组成了一类高度丰富的代谢稳定的，非溶腺苷的RNA分子，这些分子在真核生物中需要预留前剂RNA所需的RNA所需的。除U6以外，剪接体SNRNA由RNA聚合酶II合成，u6是由RNA聚合酶III合成的。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基因利用类似的顺式作用调节信号和转录因子的重叠集来表达其表达。该项目的主要目的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，重点是无TATA-U1启动子，更一般而言，该项目将有助于我们了解大分子分子相互作用和组装的微妙变化如何导致显着不同的不同生物学成果。两类SNRNA基因的转录需要一个独特的多亚基转录因子，通常称为SNRNA激活蛋白复合物或SNAPC。该因素识别一个必不可少的启动子元素，该元素称为位于转录起始位点上游区域40-75个基本对中的PSE。在果蝇果蝇中，SNAPC由三个亚基组成，它们共同对〜21碱基对PSE进行序列特异性识别（更专门称为水果蝇中的psea）。即使在21个核苷酸位置中只有5个U1 psea和U6 psea不同，但该序列差异在确定昆虫SNRNA基因的RNA聚合酶特异性方面起主要作用。此外，与U1 psea相比，DMSNAPC的三个亚基采用不同的构象。这种构象差异被认为是募集正确的RNA聚合酶的原因。 为了更好地了解与PSEA结合的SNAPC亚基的结构排列，该技术将采用位点特异性蛋白DNA光合链接与特定的化学蛋白水解的新技术。这些实验将确定DNA上每个SNAPC亚基的方向或极性。在苍蝇中，TATA结合蛋白（TBP）用于RNA聚合酶II的U1转录，但与TBP相关的因子TRF1用于RNA聚合酶III的U6转录。将确定与TBP或TRF1相互作用的单个SNAPC亚基的域。 Fly U1基因将作为研究无TATA RNA聚合酶II启动子上转录复合物组件的绝佳模型。为此，将研究SNAPC将TBP招募到TATA-U1启动子的机制。 更广泛的影响：这项研究将由从事学士学位，M.S.和Ph.D.的学生进行。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，毕业和专业学校或学术界的职业提供培训。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。首席研究人员活跃于本科课堂教学，并具有良好的往绩和历史，使代表性不足的学生参与NSF资助的研究。该项目的结果将在经过同行评审的科学期刊中传播，并将为转录调节和基因表达的领域广泛贡献。