In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.nucleolus ͉ RNA polymerase III I t has previously been shown that tRNA-class RNA polymerase III (pol III) promoters can exert negative transcriptional regulation on neighboring DNA in the budding yeast, Saccharomyces cerevisiae (1). The degree to which this tRNA genemediated silencing (tgm silencing) affects nearby RNA polymerase II-transcribed genes varies among different pol II promoters. Although complete inhibition of some nearby pol II promoters has been achieved in selected artificial juxtapositions, promoters that are found bordering the 274 tRNA genes in their normal chromosomal locations must somehow be exempt, or at least conditionally resistant to this form of negative regulation.There is a particularly close association of tRNA genes with naturally occurring copies of most classes of the Ty retrotransposons (2). One possible selective pressure for maintaining an association between tRNA genes and neighboring pol II promoters is that the proximity serves some regulatory function that is beneficial for maintenance of the retrotransposon at that position. Although the presence of a Ty3 sigma element does not strongly affect expression of a neighboring tRNA gene (3), temperature-dependent silencing of the chromosomal Ty3 and sigma elements was shown to be dependent on RNA polymerase III, suggesting possible involvement of the neighboring tRNA transcription units (1). It is interesting to note that the one class of Ty retrotransposon that is not found adjacent to tRNA genes, the Ty5 class, is found instead at other silenced locations, namely telomeres and the silent mating type loci (4, 5).At this time, it is not clear what relationship the mechanism of tgm silencing might have to silencing at silent mating type loci, telomeres, ribosomal RNA genes, or other forms of negative regulation, but several types of interference between the transcription units appear to be ruled out. It seems unlikely that either readthrough by pol III or positive supercoils propaga...