THE BYP1-3 ALLELE OF THE SACCHAROMYCES-CEREVISIAE GGS1 TPS1 GENE AND ITS MULTI-COPY SUPPRESSOR TRNA(GLN) (CAG) - GGS1/TPS1 PROTEIN-LEVELS RESTRAINING/

Byp1-3 is an amber nonsense allele of the Saccharomyces cerevisiae GGS 1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain- dependent manner. Overexpression of the isolated byp1-3 allele suppres sed the growth defect of a ggs1/tps1 Delta mutant. Expression of an in -vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 Delta mutan t. We have isolated, as an allele-specific multi-copy suppressor of th e growth defect of the byp1-3 mutant on fructose, the gene for tRNA(GL N) (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low l evel of read through of the internal nonsense codon by tRNA(GLN) (CAG) . Using overexpression of the isolated byp1-3 allele, as well as of th e tRNA(GLN) (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of G gs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cl oned tRNA(GLN) (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported seq uence of URA10, in particular in the non-coding region.