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.

摘要 智力优点：被称为 U1、U2、U4、U5 和 U6 的小核 RNA (snRNA) 包含一类高度丰富的代谢稳定的非聚腺苷酸化 RNA 分子，这些分子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 除外，U6 由 RNA 聚合酶 III 合成。尽管RNA聚合酶特异性存在差异，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节信号和重叠的转录因子组来表达。该项目的主要目标是了解聚合酶特异性转录复合物组装所涉及的结构功能关系，重点是无 TATA 的 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的非常微妙的变化如何导致显着不同的生物学结果。两类 snRNA 基因的转录都需要一种独特的多亚基转录因子，通常称为 snRNA 激活蛋白复合物 (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。在果蝇果蝇中，SNAPc 由三个亚基组成，它们共同对约 21 个碱基对 PSE（在果蝇中更具体地称为 PSEA）进行序列特异性识别。尽管 U1 PSEA 和 U6 PSEA 仅在 21 个核苷酸位置中的 5 个位置不同，但这种序列差异在确定昆虫 snRNA 基因的 RNA 聚合酶特异性方面起着重要作用。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招募正确 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，将采用一种将位点特异性蛋白质-DNA 光交联与蛋白质的特定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) 由 RNA 聚合酶 II 用于 U1 转录，但 TBP 相关因子 TRF1 由 RNA 聚合酶 III 用于 U6 转录。将确定参与与 TBP 或 TRF1 相互作用的各个 SNAPc 亚基的域。果蝇 U1 基因将作为研究转录复合物在无 TATA RNA 聚合酶 II 启动子上组装的优秀模型。为此，我们将研究 SNAPc 将 TBP 招募到无 TATA 的 U1 启动子的机制。 更广泛的影响：该研究将由正在攻读学士、硕士和博士学位的学生进行。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、升读研究生和专业学校或进入学术界提供培训。圣地亚哥州立大学由于其边境位置以及对本科和研究生教学的重视，为大量来自代表性不足的族裔群体的本科生提供服务。首席研究员活跃于本科生课堂教学，并在让代表性不足的学生参与 NSF 资助的研究方面拥有良好的记录和历史。该项目的结果将在同行评审的科学期刊上传播，并将为转录调控和基因表达领域做出广泛贡献。