Biochemical and Genetic Analysis of the RNA Polymerase Specificity of Small Nuclear RNA Genes

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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. These snRNAs are all 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 this project is to gain an understanding of the molecular mechanisms that are responsible for the selection of the correct enzyme (either RNA polymerase II or RNA polymerase III) at individual snRNA gene promoters. In higher eukaryotes, transcription of both classes of snRNA genes requires an essential proximal sequence element (PSE) within the region 40-75 base pairs upstream of the transcription start site. In the fruit fly Drosophila melanogaster, the PSE is recognized by the PSE binding protein (termed DmPBP) that contains three distinct subunits that closely approach the DNA. Previous work in the principal investigator's lab has led to a working model in which the U1 and U6 PSEs act as differential allosteric effectors of DmPBP. Conformational differences in DmPBP, in turn, are believed to be responsible for the subsequent downstream recruitment of the correct RNA polymerase. Two distinct yet highly synergistic approaches will be undertaken to test the validity of this model. First, germline transformation and Drosophila genetics will be employed to select mutants that have an altered RNA polymerase specificity at snRNA gene promoters. The second approach involves targeted in vitro mutagenesis of the genes that code for the subunits of DmPBP. The biochemical mechanisms, including conformational differences in DmPBP, that lead to changes in RNA polymerase specificity will be examined. Particular interest will be focused upon identifying functional domains or amino acid residues required specifically for the recruitment of one RNA polymerase but not the other. For the genetic information that resides in DNA to be correctly read out, it is critical that the correct RNA polymerase must be recruited to any particular gene of interest. This project will shed light on how this is accomplished at the molecular level. The system under investigation in the principal investigator's laboratory also serves as a general model for macromolecular assembly and for understanding how subtle changes in macromolecular interactions can lead to significantly different biological outcomes. The project will help us to understand how decisions among alternative biological pathways are made within cells. Research training will be provided for students engaged in acquiring their B.S., M.S., and Ph.D. degrees in biochemistry and molecular biology. Students with disabilities and from underrepresented groups will be active participants.