Yeast Ribosomal Protein L24 Affects the Kinetics of Protein Synthesis and Ribosomal Protein L39 Improves Translational Accuracy, While Mutants Lacking Both Remain Viable

Abstract

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.

A Dispensable Yeast Ribosomal Protein Optimizes Peptidyltransferase Activity and Affects Translocation

“…As shown in Table II, the error frequency of the wild-type strain YKS99-2A was 26 errors every 10 4 codons. This error rate is almost identical to that reported recently for 2D-J809, another wild-type strain (21) and it is within the range reported in the literature (36,37).…”

“…Mutant ribosomes lacking L41 showed the same extent of polyphenylalanine synthesis activity as wild-type ribosomes (Table II). Moreover, ribosomes from the quintuple mutant YKS121, despite a 3-fold slower growth rate, displayed the same extent (Table II) but lower initial rates (not shown) of polyphenylalanine formation compared with the wild-type ribosomes, similar to the values reported for ribosomes from the triple mutant (21). Thus, the absence of L41 from the YKS99-2C or YKS121 strains does not affect polyphenylalanine synthesis.…”

“…Biochemical Procedures-Experiments to measure the sensitivity of cells to paromomycin, the preparation of a yeast cell-free system for translation in vitro, the translation of poly(U) templates in vitro, the Pand A-site binding studies as well as the time course measurements of polyphenylalanine synthesis, the sucrose gradient analysis, and the preparation of ribosomal proteins were all performed as described recently (21).…”

“…The first lacked the two genes encoding L41. The second strain lacked L41 in addition to the absence of L24 and L39, two eukaryote-specific proteins studied recently (21,24). The fact that the cells remained viable upon removal of the two genes encoding L41 in addition to the absence of the two genes encoding L24 and the single gene encoding L39, allowed for the first time the study of a quintuple mutant in yeast as well as the study of the role of L41 in combination with the effects of the other two proteins.…”

“…Also, they are crucial for the assembly of functional ribosomes (16). Several ribosomal proteins of the small subunit (17)(18)(19)(20) and at least one of the large subunit (21) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22)(23)(24).…”