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.
SummaryThe ribosome is a macromolecular machine responsible for protein synthesis in all organisms. Despite the enormous progress in studies on the structure and function of prokaryotic ribosomes, the respective molecular details of the mechanism by which the eukaryotic ribosome and associated factors construct a polypeptide accurately and rapidly still remain largely unexplored. Eukaryotic ribosomes possess more RNA and a higher number of proteins than eubacterial ribosomes. As the tertiary structure and basic function of the ribosomes are conserved, what is the contribution of these additional elements? Elucidation of the role of these components should provide clues to the mechanisms of translation in eukaryotes and help unravel the molecular mechanisms underlying the differences between eukaryotic and eubacterial ribosomes. This article focuses on a class of eukaryotic ribosomal proteins that do not have a eubacterial homologue. These proteins play substantial roles in ribosomal structure and function, and in mRNA binding and nascent peptide folding. The role of these proteins in human diseases and viral expression, as well as their potential use as targets for antiviral agents is discussed.
A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6 ), and 912 (rdn4 ) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.
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