THE RET1 GENE OF YEAST ENCODES THE 2ND-LARGEST SUBUNIT OF RNA POLYMERASE-III - STRUCTURAL-ANALYSIS OF THE WILD-TYPE AND RET1-1 MUTANT ALLELES

We have previously reported on the isolation of the ret1-1 mutation in yeast, which reduces the efficiency of transcription termination by RNA polymerase III. We have cloned the RET1 gene by complementation of an ochre suppression phenotype in ret1-1 cells. The RET1 gene was mapped to near the HIS3 gene on the right arm of chromosome 15 by using hybridization to OFAGE gels. Sequencing of the RET1 gene has identified its product as the second-largest subunit of RNA polymerase III. We have carried out an extensive sequence alignment with other RNA polymerase second-largest subunits and discuss the conservation of several functional domains. The RET1 gene was used to recover the ret1-1 mutant allele from genomic DNA using an integration/excision technique. Plasmid-based fine mapping and fragment swapping were used to localize the ret1-1 mutation for sequencing. We discuss the ret1-1 sequence lesion with regard to possible roles in transcription. In particular, the recessive nature of the ret1-1 mutation may have major implications for our understanding of the transcription process.