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

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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.