Intellectual Merit: The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 comprise a highly abundant class of RNA molecules that are required for pre-messenger RNA splicing in higher organisms. These snRNAs are synthesized by RNA polymerase II with the exception of U6, which is synthesized by RNA polymerase III. Despite this difference in RNA polymerase specificity, U6 genes and the RNA polymerase II-transcribed snRNA genes utilize similar regulatory signals and overlapping sets of transcription factors for their expression. The main goal of the project is to gain an understanding of the structure-function relationships that are involved in recruiting the correct RNA polymerase to initiate synthesis of the correct snRNAs. Transcription of both classes of snRNA genes requires a unique protein factor referred to as the snRNA-activating protein complex (SNAPc). This factor recognizes an essential promoter element termed the PSE located in the DNA 40-75 base pairs (bp) upstream of the start site of RNA synthesis. In the fruit fly D. melanogaster (the model organism used in this project), DmSNAPc is composed of three subunits that together carry out sequence-specific recognition of the ~21 bp long PSEA (the fruit fly PSE). Even though a U1 PSEA and a U6 PSEA differ at only 5 of 21 nucleotide positions, this sequence difference plays a major role in determining the RNA polymerase specificity of fly snRNA genes. Furthermore, the three subunits of assume a different conformation when bound to a U6 versus a U1 PSEA. This conformational difference is believed to be responsible for the recruitment of the correct RNA polymerase. To better understand the conformational differences of DmSNAPc bound to U1 and U6 PSEAs, a novel technique developed in the PI's lab will be employed that uses site-specific protein-DNA cross-linking combined with chemical cleavage of the protein at defined sites. Contact points made between the largest DmSNAPc subunit and U6 promoter DNA will be compared with the contact points made between this subunit and U1 promoter DNA. Next, pre-initiation complex (PIC) assembly on U1 and U6 promoters will be investigated, with primary emphasis on RNA polymerase II recruitment to the TATA-less U1 promoter, but analogous experiments will be performed to investigate Pol III PIC assembly on the U6 promoter. Finally, the overall contour shape of DmSNAPc bound to the U1 and U6 PSEAs will be studied by cryo-electron microscopy, and X-ray crystallography will be employed to investigate the structure of DmSNAPc and it subunits at the atomic level. The results of the research will contribute widely to the scientific community's understanding of gene expression at the level of RNA synthesis. Fruit fly U1 genes serve as a particularly tractable paradigm for investigating RNA polymerase II transcription complex assembly on TATA-less promoters, a process that is currently very poorly understood. More generally, this system serves as an excellent model for understanding how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes.
Broader Impacts: The research will be performed by students in satisfaction of their B.S., M.S., and Ph.D. degrees in biochemistry/molecular biology. The project will provide training for their future careers in the biotechnology industry, for advancement to graduate and professional schools, or to careers in academia as well as teaching at the community college level. San Diego State University, due to its border location and emphasis on undergraduate as well as graduate instruction, serves a large body of undergraduate students from underrepresented ethnic groups. The PI is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented students in research, from high school through the graduate level.
