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

这项研究将为DNA核苷酸序列的微小差异可以影响柔性DNA结合蛋白的形状的机制提供关键见解。重要的是，蛋白质形状的这种变化导致生物学结果的显著差异，这对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提供生物化学培训。这些学员将通过在生物技术，学术界或相关领域从事职业生涯，为科学企业的未来做出贡献。由于预计这些受训人员大多数来自代表性不足的群体，该项目将有助于科学工作队伍的多样性。存在于DNA中的遗传信息必须在活细胞内以非常明确和特定的方式读出。遗传信息表达的第一步涉及在称为转录的过程中从DNA模板合成RNA。在高等生物中，转录由三种不同的RNA聚合酶进行，称为聚合酶I，II和III。这些聚合酶中的每一种都参与来自不同基因组的RNA合成，并且通常必须通过不同的序列特异性DNA结合蛋白组被募集到DNA启动子。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基因的启动子彼此非常相似，但包含功能上的显著差异。一种蛋白质复合物SNAPc（snRNA基因转录所需的唯一蛋白质），由于与差异极小的DNA序列结合，在果蝇U1和U6基因启动子上采用不同的构象。据推测，SNAPc的这些不同的构象参与招募RNA聚合酶II转录U1基因，但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差异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-蛋白质相互作用将通过交联质谱法绘制。整个项目将采用基本的生物化学技术。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聚合酶转录前起始复合物如何在snRNA启动子上组装。

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.