Genetic information is stored in a manner that facilitates retrieval and promotes regulation of cellular processes. In eukaryotic genomes the largest collection of co-regulated genes is the transfer RNA (tRNA) gene family, transcribed by RNA polymerase III (Pol III). The budding yeast, Saccharomyces cerevisiae, has 274 tRNA genes widely dispersed throughout the 16 nuclear chromosomes, yet in three dimensions these genes cluster together at the nucleolus. This work investigates the mechanism and consequences of this spatial organization of tRNA genes.
Clustering of tRNA genes had initially been observed by fluorescence microscopy, but limits on resolution prevented seeing associations for individual tRNA genes. Here, in vivo chemical crosslinking identified physical interactions between genomic loci that are closely associated in three dimensions. This confirmed nucleolar clustering of tRNA genes and further demonstrated that specific association of tRNA genes along the nucleolar ribosomal RNA (rRNA) gene repeats is dependent upon tRNA gene identity. Although tRNA gene clustering is not necessarily the primary driving force of genome organization, the results suggest they are local organizers.
The mechanism of tRNA gene clustering was examined. Previous work showed the conserved condensin complex is required for clustering and is directly bound to tRNA gene transcription complexes in vivo. This work shows that binding of the Pol III transcription factor TFIIIC to the tRNA gene is necessary and sufficient for condensin to specifically recognize the tRNA gene.
Clustering of tRNA genes contributes to “silencing” of nearby transcription by RNA polymerase II (Pol II), but the molecular mechanisms are unknown. Work in both bacterial and mammalian systems has shown that other tRNA-related RNAs bind Pol II and inhibit transcription. However, this work shows not specific RNAs but a broad spectrum of RNAs directly binds to purified yeast Pol II, preventing it from subsequently binding DNA template. Globally, this result necessitates immediate ribonucleoprotein assembly and transport of nascent transcripts to sequester inhibitory RNAs away from the polymerase.
Overall, the findings from this dissertation further our understanding of how families of genes are spatially organized and reveal important consequences of nuclear organization on cellular processes.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91532/1/dapai_1.pd
