| The genome of Saccharomyces cerevisiae contains 274 tRNA genes that are linearly scattered throughout all sixteen chromosomes. In this work, we have found that despite their apparently random distribution, tRNA genes are clustered together at the nucleolus and that this nucleolar localization depends on the active transcription of a tRNA gene. We went on to characterize the effect of this surprising arrangement on transcription near tRNA gene loci.; The active transcription of a tRNA gene by RNA polymerase III can antagonize transcription from a linearly adjacent polymerase II promoter, an effect termed tRNA gene mediated (tgm) silencing. We have now identified several mutations in nucleolar proteins that cause tgm silencing to be released. These mutants also show tRNA genes and pre-tRNAs dispersed throughout the nucleoplasm instead of their normal nucleolar localization. Thus, the nucleolar clustering of tRNA genes appears to contribute to tgm silencing.; Extensive spatial organization of the S. cerevisiae genome must exist in order to gather these genes, and we investigated whether structural elements were involved in the tRNA gene localization mechanism. After treatment with the microtubule-depolymerizing drug nocodazole, tRNA genes were still clustered together, but the clusters were divorced from the nucleolus. This role for microtubules appears to be unrelated to cell cycle stage, because we found that unsynchronized cells throughout the cell cycle showed tRNA gene signal concentrated at the nucleolus. These observations suggest that microtubules are involved in the nucleolar localization of tRNA gene clusters, which are initially formed by a separate mechanism.; To investigate the initial clustering mechanism, we examined the condensin complex, which has been found to bind to tRNA genes along S. cerevisiae chromosomes. Condensin is involved in the compaction of mitotic chromosomes and is concentrated at the nucleolus during anaphase. We have now found that temperature sensitive mutants of condensin subunits are able to release tgm silencing and mislocalize tRNA genes.; This work has shown that clustering of the linearly dispersed tRNA genes is a major force in spatial organization of the yeast genome and has provided the first mechanistic insights as to how this clustering is accomplished. |
