Exploring how protein conformation influences assembly of transcriptional complexes on snRNA gene promoters

探索蛋白质构象如何影响 snRNA 基因启动子上转录复合物的组装

• 批准号: 1616487
• 负责人: William Stumph
• 金额: $ 90万
• 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: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616487&HistoricalAwards=false
• 关键词: Exploring; how; protein; conformation; influences

This research will provide key insights into the mechanism by which small differences in DNA nucleotide sequence can affect the shape of a flexible DNA-binding protein. Importantly, this change in protein shape leads to a significant difference in biological outcome that is crucial to the correct utilization of genetic information. The project will provide biochemical training for at least three graduate students, six undergraduate students, a high school student, and a postdoctoral fellow. These trainees will contribute to the future of the scientific enterprise by pursuing careers in biotechnology, academia, or related fields. As the majority of these trainees are expected to be from under-represented groups, the project will contribute to the diversity of the scientific workforce. Genetic information present in the DNA must be read out within living cells in a very defined and specific manner. The first step in the expression of genetic information involves the synthesis of RNA from the DNA template in a process termed transcription. In higher organisms, transcription is carried out by three different RNA polymerases termed polymerases I, II, and III. Each of these polymerases is involved in RNA synthesis from a distinct set of genes and must be recruited to the DNA promoter usually by distinct sets of sequence-specific DNA-binding proteins. These promoter binding proteins bind to DNA sequences near the transcription start site. The U1 and U6 snRNA genes code for essential small RNA molecules involved in the splicing of messenger RNAs. The promoters of the U1 and U6 genes are very similar to each other, yet contain functionally significant differences. A protein complex, SNAPc (which is uniquely required for snRNA gene transcription), adopts different conformations on fruit fly U1 and U6 gene promoters due to binding to minimally-different DNA sequences. It is hypothesized that these distinct conformations of SNAPc are involved in the recruitment of RNA polymerase II to transcribe the U1 gene but RNA polymerase III to transcribe the U6 gene. The project will investigate how the different conformations of SNAPc lead to differential RNA polymerase recruitment. Protein-DNA interactions will be mapped by site-specific protein-DNA photo-cross-linking combined with site-specific protein cleavage. Protein-protein interactions will be mapped by cross-linking mass spectrometry. Basic biochemical techniques will be employed throughout the project. The knowledge obtained from these experiments will be used to construct molecular models to aid in understanding how RNA polymerase transcription pre-initiation complexes assemble on snRNA promoters.

这项研究将为DNA核苷酸序列的微小差异能够影响柔性DNA结合蛋白形状的机制提供关键见解。重要的是，这种蛋白质形状的变化导致了生物学结果的显著差异，这对于正确利用遗传信息至关重要。该项目将为至少3名研究生、6名本科生、1名高中生和1名博士后提供生化培训。这些学员将通过从事生物技术、学术界或相关领域的职业，为科学事业的未来做出贡献。由于这些受训者中的大多数预计将来自代表性不足的群体，因此该项目将有助于科学劳动力的多样性。存在于DNA中的遗传信息必须在活细胞内以一种非常明确和特定的方式读出。遗传信息表达的第一步包括从DNA模板合成RNA，这一过程被称为转录。在高等生物体中，转录是由三种不同的RNA聚合酶进行的，称为聚合酶I、II和III。这些聚合酶中的每一种都参与从一组不同的基因合成RNA，并且通常必须通过不同的序列特异性DNA结合蛋白集将其招募到DNA启动子中。这些启动子结合蛋白与转录起始位点附近的DNA序列结合。U1和U6 snRNA基因编码参与信使RNA剪接的必需小RNA分子。U1和U6基因的启动子非常相似，但在功能上存在显著差异。一种蛋白质复合物SNAPc （snRNA基因转录所必需的）由于结合的DNA序列差异极小，在果蝇U1和U6基因启动子上呈现不同的构象。据推测，这些不同的SNAPc构象参与募集RNA聚合酶II转录U1基因和RNA聚合酶III转录U6基因。该项目将研究SNAPc的不同构象如何导致不同的RNA聚合酶募集。蛋白质- dna相互作用将通过位点特异性蛋白质- dna光交联结合位点特异性蛋白质切割来绘制。蛋白质之间的相互作用将通过交联质谱法绘制。基本的生化技术将贯穿整个项目。从这些实验中获得的知识将用于构建分子模型，以帮助理解RNA聚合酶转录起始前复合物如何在snRNA启动子上组装。