tRNA Genes and Genetic Mobility in S. cerevisiae

University Of California-Irvine, Irvine CA

Investigators

Abstract

Transfer RNA genes (tDNA's) are important not only for the RNA's they encode, but also for their roles in genomic function and evolution. They have been identified as boundaries of heterochromatic DNA, repressors of pol II transcription, sites of genomic rearrangement and ectopic recombination, sites of pathogenic island insertion, and targets of long terminal repeat (LTR) retrotransposon integration. The Sandmeyer laboratory studies Ty3, a retroviruslike element in Saccharomyces cerevisiae that inserts at RNA polymerase III transcription initiation sites. It is known from an in vitro system that includes viruslike particles and a plasmid-borne target, that the pol III transcription factor TFIIIB bound to DNA is sufficient for integration in vitro. However, much remains to be learned about the relationship between this element, tDNA's, and their genomic contexts. This project has three goals: 1) Characterization of the Ty3 preintegration complex (PIC) and reconstitution of a defined in vitro Ty3 integration system. A central step in the Ty3 lifecycle is conversion of the VLP into the integration-competent cDNA-associated PIC. A PIC fraction will be isolated based on cDNA integrating activity. It will be characterized for Ty3 and host proteins and imaged using AFM. 2) Identification of specific interactions between the Ty3 PIC and its target. The minimal target requirement for in vitro integration of Ty3 is comprised of the TATA binding protein (TBP) and Brf1 bound to a RNA pol III promoter. The domains in the PIC and target which interact will be identified by testing for interactions between Ty3 proteins, particularly integrase, and Brf1. If those candidates do not interact, Ty3 will be mutagenized and a genetic screen will be used to identify mutants affected in position specificity. 3) Characterization of features of chromosomal targets of Ty3 integration. The relationship of in vivo chromosome-based transcriptional activity and Ty3 targeting will be addressed using a diverse subset of Pol III-transcribed genes. TFIIIB and TFIIIC and Pol III occupancy and transposition will be monitored using chromatin immunoprecipitation quantitative PCR, respectively. Retrotransposons and genes for tRNA's are major forces affecting mobilization in prokaryotic and eukaryotic genomes yet relatively little is actually understood about the ability of tRNA genes to act as delimiters of chromosomal regions or why retrotransposons prefer integration in some regions over others. This research will lead to a better understanding of these two interacting and dynamic genomic components. It will also be used by the PI to introduce summer high school students, undergraduate biology majors, and graduate students to the principles of yeast molecular biology and genomic research. UCI has an active Minority Scientist Program, and students from a wide range of educational institutions, including community colleges and medical schools, are recruited into this program and are eligible for this project. Data will be disseminated by publication and by posting a database summarizing the findings at a public website.