DEVELOPMENT AND APPLICATION OF AN IN-VIVO SYSTEM TO STUDY YEAST RIBOSOMAL-RNA BIOGENESIS AND FUNCTION

We have developed a system for mutational analysis of Saccharomyces ce revisiae ribosomal RNA in vivo in which yeast cells can be made comple tely dependent on mutant rRNA and ribosomes by a simple switch in carb on source. The system is based on a yeast strain defective in RNA poly merase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. S ci. USA 88, 3962-3966]. This normally inviable strain was rescued by i ntegration of multiple copies of the complete 37S pre-rRNA operon unde r control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only gr ow on medium containing galactose as the carbon source. A second, epis omal vector was constructed in which the rDNA unit was placed under co ntrol of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that t he Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II sys tem' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable r egion V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysom al distribution of these subunits [Musters et al. (1989), Mol. Cell. B iol. 9, 551-559], is in fact a lethal mutation.