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.

这项研究将提供有关DNA核苷酸序列微小差异可以影响柔性DNA结合蛋白的形状的重要见解。重要的是，蛋白质形状的这种变化导致生物学结果的显着差异，这对于正确利用遗传信息至关重要。该项目将为至少三名研究生，六名本科生，一名高中生和一名博士后研究员提供生化培训。这些学员将通过从事生物技术，学术界或相关领域的职业来为科学企业的未来做出贡献。由于这些受训人员中的大多数人都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。 DNA中存在的遗传信息必须以非常定义和特定的方式在活细胞中读取。遗传信息表达的第一步涉及在称为转录的过程中从DNA模板中合成RNA。在较高的生物体中，转录是由称为聚合酶I，II和III的三种不同的RNA聚合酶进行的。这些聚合酶中的每一个都参与了来自不同基因的RNA合成，并且必须通过不同的序列特异性DNA结合蛋白募集到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基因代码用于允许的小型RNA分子，涉及Messenger RNA的剪接。 U1和U6基因的启动子彼此非常相似，但在功能上具有显着差异。蛋白质复合物（SnRNA基因转录是独特所需的），由于与微小不同的DNA序列结合，对果蝇U1和U6基因启动子采用了不同的构象。假设SNAPC的这些独特的构象与RNA聚合酶II的募集有关以转录U1基因，但RNA聚合酶III却转录U6基因。该项目将研究SNAPC的不同构象如何导致差异RNA聚合酶募集。蛋白-DNA相互作用将由位点特异性蛋白-DNA光链链接与位点特异性蛋白质裂解相结合。蛋白质 - 蛋白质相互作用将通过交联的质谱法映射。整个项目将采用基本的生化技术。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聚合酶转录前发射复合物如何在SNRNA启动子上组装。