Study of Ris1

Name of Research Sponsor: Dr. David Engelke

Dates of Involvement: Fall 2009 - Fall 2010

Abstract

The 274 tRNA genes in Saccharomyces cerevisiae are scattered throughout the linear genome, but are clustered together in the nucleolus in 3-demensional space. This spatial arrangement of tRNA genes contributes to tRNA gene-mediated (tgm) silencing of RNA polymerase II transcription adjacent to tRNA genes. Deletions of the MOD5, MAF1, and RIS1 genes have been shown to alleviate tgm silencing. Previous work has been done on MOD5 and MAF1, but the details of the effect of RIS1 are unknown. To determine whether the Ris1 protein affects silencing by direct binding to the tRNA gene, I will perform chromatin immunoprecipitation (ChIP). The presence of Ris1 at tRNA genes will be assessed by forming protein-DNA cross-links and then using an antibody to the Ris1 protein which will pull out the Ris1 protein and any cross-linked DNA. This work will further the common understanding of how eukaryotic genomes are organized.

My Contributions

I worked on this project for about a year. That being said, little progress had been made. I worked to tag the C-terminus for the majority of that time. Although the sequencing data showed that Ris1 was tagged, I could not produce a Western blot with that data. I also worked to tag the N-terminus. A fellow lab member is currently working on this.