Inhibition of protein synthesis by an efficiently expressed mutation in the yeast 5.8S ribosomal RNA. Nucleic Acids Res

Recent studies on the inhibition of protein synthesis by specific antl 5.8S rRNA oligonucleotldes strongly suggested that this RNA plays an Important role in eukaryotic rlbosome function. To evaluate this possi-bility further, a ribosomal DNA transcription unit from Schlzosaccharomyces pombe was cloned Into yeast shuttle vectors with copy numbers ranging from 2 to approximately 90 per cell; to allow direct detection of expressed RNA and to disrupt the function of the 5.8S rRNA molecule, a five base Insertion was made in a universally conserved GAAC sequence. The altered mobility of the mutant RNA was readily detected by gel electrophoresls and analyses indicated that mutant RNA transcription reflected the ratio of plasmld to endogenous rDNA. The highest copy number plasmid resulted In about 40- 50 % mutant RNA. This mutant RNA was readily integrated into the rlbosome structure resulting In an In vivo rlbosome population which was also about 40- 50 % mutant; the rates of growth and protein synthesis were equally reduced by approxi-mately 40%. A comparable level of Inhibition in protein synthesis was demonstrated In vitro and polyribosomal profiles revealed a consistent increase In size. Subse-quent RNA analyses indicated a normal distribution of mutant RNA in both monoribosomes and polyribo-somes, but elevated tRNA levels in mutant poly-ribosomes. Additional mutations in alternate GAAC sequences revealed similar but cumulative effects on both protein synthesis and polyrlbosome profiles. Taken together, these results suggest little or no effect on Initiation but provide In vivo evidence of a functional role for the 5.8S rRNA in protein elongation