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

摘要智力优势：被称为U1、U2、U4、U5和U6的小核RNA(SnRNAs)包括一类高度丰富的代谢稳定的非多腺化RNA分子，这些RNA分子是真核生物中前信使RNA剪接所必需的。剪接体SnRNA由RNA聚合酶II合成，U6除外，U6是由RNA聚合酶III合成的。尽管U6基因和RNA聚合酶II转录的SnRNA基因在RNA聚合酶专一性上存在差异，但它们利用相似的顺式作用调控信号和重叠的转录因子来表达。该项目的主要目标是了解聚合酶特异性转录复合体组装中涉及的结构-功能关系，重点是无TATA的U1启动子，更广泛地说，该项目将帮助我们了解大分子相互作用和组装中非常细微的变化如何导致显著不同的生物学结果。这两类SnRNA基因的转录都需要一个独特的多亚基转录因子，通常被称为SnRNA激活蛋白复合体，或SNAPc。该因子识别一个重要的启动子元件，称为PSE，位于转录起始点上游40-75个碱基对的区域内。在果蝇中，SNAPc由三个亚基组成，它们共同对~21碱基对PSE(在果蝇中更确切地称为PSEA)进行序列特异性识别。尽管U1 PSEA和U6 PSEA在21个核苷酸位置中只有5个不同，但这种序列差异在决定昆虫SnRNA基因的RNA聚合酶专一性方面发挥了重要作用。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导致正确的RNA聚合酶招募的原因。为了更好地了解与PSEA结合的SNAPc亚基的结构安排，将采用一种新的技术，将特定位点的蛋白质-DNA光交联与蛋白质的特定化学蛋白分解相结合。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)被RNA聚合酶II用于U1转录，但TBP相关因子TRF1被RNA聚合酶III用于U6转录。与TBP或TRF1相互作用的单个SNAPc亚基的结构域将被确定。Fly U1基因将成为研究TATA缺失的RNA聚合酶II启动子上转录复合体组装的极佳模型。为此，将研究SNAPc将TBP招募到TATA缺失U1启动子的机制。更广泛的影响：这项研究将由攻读生物化学/分子生物学学士、硕士和博士学位的学生进行。该项目将为他们未来在生物技术行业的职业生涯、升入研究生院和专业学校或在学术界的职业生涯提供培训。圣地亚哥州立大学由于地处边境，重视本科生和研究生教学，为大量来自代表人数较少的民族的本科生提供服务。这位首席调查员活跃在本科课堂教学中，在国家科学基金会资助的研究中有很强的记录和让未被充分代表的学生参与的历史。该项目的成果将在同行评议的科学期刊上传播，并将对转录调控和基因表达领域作出广泛贡献。

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.