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灵活的DNA结合蛋白的形状的机制提供关键的见解。重要的是，这种蛋白质形状的变化导致生物结果的显著差异，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化培训。这些受训人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。由于预计这些受训人员中的大多数将来自代表性不足的群体，该项目将有助于增加科学工作者的多样性。DNA中存在的遗传信息必须以非常明确和具体的方式在活细胞内读出。表达遗传信息的第一步是从DNA模板合成RNA，这一过程称为转录。在高等生物中，转录是由三种不同的RNA聚合酶进行的，分别称为聚合酶I、聚合酶II和聚合酶III。这些聚合酶中的每一个都参与了一组不同的基因的RNA合成，并且通常必须通过不同的序列特异性DNA结合蛋白集被招募到DNA启动子。这些启动子结合蛋白结合在转录起始点附近的DNA序列上。U1和U6短链RNA基因编码参与信使RNA剪接的基本小RNA分子。U1和U6基因的启动子彼此非常相似，但在功能上存在显著差异。SNAPc是一种蛋白质复合体，在果蝇U1和U6基因启动子上由于与差异最小的DNA序列结合而采用不同的构象。推测SNAPc的这些不同构象参与了RNA聚合酶II转录U1基因而RNA聚合酶III转录U6基因的过程。该项目将研究SNAPc的不同构象如何导致不同的RNA聚合酶招募。蛋白质-DNA的相互作用将通过特定部位的蛋白质-DNA光交联和特定部位的蛋白质切割相结合来绘制。蛋白质之间的相互作用将通过交联质谱图进行绘制。基本的生化技术将在整个项目中使用。从这些实验中获得的知识将被用来构建分子模型，以帮助理解RNA聚合酶转录预启动复合体是如何组装在SnRNA启动子上的。

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.