Ribosomal RNA synthesis in Saccharomyces cerevisiae.

Ribosomal RNA biosynthesis in metabolically active spheroplasts of the yeast Saccharomyces cerevisiae was selected as a model system for the study of nucleo-cytoplasmic interactions. Examination of the total cellular RNA synthesized during short exposures to labeled nucleosides demonstrated the existence of three large, short-lived RNA species of 35 s, 27 s and 20 s, in addition to the mature ribosomal RNA's, 25 s and 18 s. Little or no RNA larger than 35 s is found. The kinetics of synthesis of these RNA molecules was pursued by continuous labeling and pulse-chase experiments, using [methyl-3H]methionine to label only ribosomal species. These results demonstrated the following precursor-product relationship: In the presence of cycloheximide the terminal processing steps leading to the mature ribosomal RNA's are severely inhibited, indicating that the maturation of ribosomal precursor RNA requires continuous protein synthesis. RNA denaturation studies using dimethyl sulfoxide showed the precursors to be single polyribonucleotide chains larger than the mature rRNA's, rather than aggregates of smaller molecules, or conformational isomers of the ribosomal species. Molecular sizes of these RNA's were estimated by electrophoretic mobility in acrylamide gels. The 35 s precursor is 2.5 × 106 daltons. Its cleavage appears to be conservative, producing intermediate molecules of 1.6 × 106 and 0.8 × 106 daltons. The final maturation steps yield mature rRNA's of 1.3 × 106 and 0.7 × 106 daltons with the concomitant loss of about 20% of the original precursor molecule. In addition, it was concluded that the small RNA species, 5.8 s (6 × 104 daltons) found non-covalently bound to the 25 s RNA, is generated during the final cleavage of the 27 s precursor molecule. In contrast, 5 s RNA is synthesized independently. These results support the contention that all eukaryotes synthesize ribosomes similarly and that yeast may be a useful organism for the investigation of intracellular communication, particularly across the nuclear membrane.