We have developed a method for the direct measurement, in eukaryotic cells, of the synthesis of ribosomal proteins, irrespective of the synthesis of ribosomes. In this way the synthesis of ribosomal proteins has been examined in mutant strains of Saccharomyces cerevisiae, which are unable to synthesize ribosomes under nonpermissive conditions. The results suggest that the synthesis of more than 40 ribosomal proteins is under coordinate control. Under nonpermissive conditions, the synthesis of each protein declines exponentially to a basal level which is 10-20% of normal. The kinetics of that decline suggest that an early, if not primary, result of the nonpermissive conditions is the cessation of production of new mRNA for-each of the ribosomal proteins. The coordinate regulation appears not to be influenced directly by the rate of transcription of ribosomal precursor RNA. The eukaryotic ribosome is assembled from several species of RNA, synthesized within the nucleus, and more than 50 proteins, synthesized in the cytoplasm (1). The genetically tractable eukaryote, Saccharomyces cerevisiae, is useful for studying the coordination between such nuclear and cytoplasmic processes. Hartwell and coworkers have identified 10 unlinked genes, rna 2-rna 11, in which temperature sensitive mutants are unable to synthesize ribosomes at the restrictive temperature (2), although protein synthesis continues normally. While such cells continue to transcribe ribosomal precursor RNA at up to 80% of the control rate (ref. 3; R. Shulman, unpublished), the RNA is not properly processed, and is eventually degraded.Indirect experiments suggested that the synthesis of ribosomal proteins is also inhibited at the restrictive temperature (3). In the present communication we describe a method, adapted from that of Dennis (4), to measure the synthesis of ribosomal proteins directly. We show that mutations in gene ma 2 or gene rna 9 cause the coordinate inhibition of the synthesis of at least 40 ribosomal proteins. The kinetics of the inhibition suggest that an early effect of each mutation is the suppression of the synthesis of mRNA for the ribosomal proteins.
METHODSCells and Media. The parent strain of S. cerevisiae, A364A, (ATCC no. 22244) was obtained from L. Hartwell. Temperature sensitive mutants derived from this strain are: ts 136 (rna 1-), in which no RNA passes from the nucleus to the cytoplasm at the restrictive temperature, 36°(5); ts 368 (rna 2-), and ts 257 (ma 9-), in which no ribosomes are made at temperatures of 330 or above (2). Media, growth conditions, and procedures for preparing spheroplasts have been described (6, 7).Analysis of Data. Analysis of Ribosomal Proteins. To study the synthesis of ribosomal proteins directly, we extracted the total protein complement of the cell and analyzed it by two-dimensional polyacrylamide gel electrophoresis. Because most of the ribosomal proteins are small and basic, they can be clearly separated from nonribosomal proteins (Fig. 1). The ribosomal proteins in Fig. lb
