Recent studies of transfer RNA gene expression in the yeast Saccharomyces cerevisiae have led to major revisions in the picture of how these RNA polymerase III transcription units function. Characterization of the nucleoprotein structure of wild type and mutant genes in vivo at high resolution, combined with detailed biochemical reconstitution has shown that assembly of active tRNA genes in native chromatin or recombinant templates requires ordered binding of separate assembly and transcription factors. In the course of the in vivo studies in this laboratory it was found that the tRNA gene promoters not only controlled their own expression, but were capable of serving as silencer elements for nearby RNA polymerase II promoters. No direct steric interference between the promoters is involved and the relative orientation of the promoters is not critical, as long as the tRNA gene is actively transcribed. The experiments described in this proposal provide both physical and genetic approaches to investigating the mechanism of this type of negative transcriptional regulation in yeast. The results of these studies could have extensive implications for understanding the organization of transcriptionally active chromatin, since in larger eukaryotic genomes many highly repetitive, interspersed DNA elements (e.g. Alu elements) have tRNA-like promoters.
