RNA Polymerase-Specific Transcription Complex Assembly on snRNA Genes: Structural and Functional Relationships

San Diego State University Foundation, San Diego CA

Investigators

Abstract

ABSTRACT Intellectual Merit: The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 comprise a highly abundant class of metabolically stable, non-polyadenylated 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 structure-function relationships that are involved in polymerase-specific transcription complex assembly, with emphasis on the TATA-less U1 promoter, More generally, this project will help us understand how very subtle changes in macromolecular interactions and assembly can lead to significantly different biological outcomes. 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 located within the region 40-75 base pairs upstream of the transcription start site. In the fruit fly Drosophila melanogaster, SNAPc is composed of three subunits that together carry out sequence-specific recognition of the ~21 base-pair PSE (known more specifically as the PSEA in fruit flies). 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 insect snRNA genes. Furthermore, the three subunits of DmSNAPc adopt a different conformation when bound to a U1 vs. U6 PSEA. This conformational difference is believed to be responsible for the recruitment of the correct RNA polymerase. To better understand the structural arrangement of the SNAPc subunits bound to a PSEA, a novel technique that combines site-specific protein-DNA photo-cross-linking with specific chemical proteolysis of the protein will be employed. These experiments will determine the orientation or polarity of each of the SNAPc subunits on the DNA. In flies, the TATA-binding protein (TBP) is used for U1 transcription by RNA polymerase II, but the TBP-related factor TRF1 is utilized for U6 transcription by RNA polymerase III. The domains of the individual SNAPc subunits involved in interactions with TBP or TRF1 will be identified. The fly U1 gene will serve as an excellent model for investigating transcription complex assembly on TATA-less RNA polymerase II promoters. Toward that end, the mechanism by which SNAPc recruits TBP to the TATA-less U1 promoter will be investigated. Broader Impacts: The research will be performed by students working on 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. 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 principal investigator is active in undergraduate classroom teaching and has a strong track record and history of involving underrepresented students in NSF-funded research. The results of the project will be disseminated in peer-reviewed scientific journals and will contribute widely to the areas of transcription regulation and gene expression.