Mechanisms of RNA Polymerase Specificity at Small Nuclear RNA Gene Promoters

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The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 are essential RNA molecules that are required for pre-messenger RNA splicing in eukaryotic organisms. The spliceosomal 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 cis-acting regulatory signals and overlapping sets of transcription factors for their expression. The main goal of the project is to gain an understanding of the molecular mechanisms and structural differences responsible for the selection of the correct enzyme (RNA polymerase II or RNA polymerase III) at individual snRNA gene promoters. In higher eukaryotes, transcription of both classes of snRNA genes requires a unique multi-subunit transcription factor most commonly referred to as the snRNA-activating protein complex, or SNAPc. This factor recognizes an essential promoter element termed the PSE. The PI's laboratory has shown that the exact sequence of the PSE is a major determinant of the RNA polymerase specificity of Drosophila melanogaster U1 and U6 genes. DmSNAPc contains three polypeptide subunits (DmSNAP190, DmSNAP50, and DmSNAP43), and protein-DNA photocrosslinking assays have shown that the conformation of DmSNAPc is different when bound to U1 and U6 PSEs, even though these PSEs differ at only 5 of 21 nucleotide positions. This has led to a working model in which a difference in the conformation of DmSNAPc, when bound to U1 and U6 PSEs, is believed to be responsible for the subsequent downstream recruitment of the correct RNA polymerase. Experiments will be carried out to probe more deeply into the molecular mechanisms that determine RNA polymerase specificity at snRNA gene promoters. Versions of DmSNAPc with mutated subunits will be generated in a homologous expression system, and protein domains required for DmSNAPc assembly, PSE binding, and transcriptional activation will be identified. Previous work revealed that switching the U1 and U6 PSEs prevents the formation of productive transcription complexes in vivo. Experiments will be performed to determine at which stage preinitiation complex assembly is blocked on promoters that contain the "wrong" PSE. The results of the research will contribute widely toward understanding the molecular mechanisms involved in the expression of genetic information stored in the DNA. In general, the problem under investigation serves as an excellent model for understanding how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes. The research will be performed by students working on their B.S., M.S., and Ph.D. degrees in biochemistry and molecular biology. The project will provide training for their future careers in the biotechnology industry, graduate and professional schools, and academia. The PI is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented minorities in research.