Decreased peptidyltransferase activity correlates with increased programmed −1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae

Abstract

Increased efficiencies of programmed −1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed −1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed −1 ribosomal frameshift signals. Keywords: Frameshifting; ribosome; virusProgrammed ribosomal frameshift (PRF) events most commonly induce translating ribosomes to slip by a single base in either the 5Ј (−1) or 3Ј (+1) direction, though examples of ribosomal "hops," "shunts," and "bypasses" have also been documented (for review, see Jacks 1990;Farabaugh 1996;Gesteland and Atkins 1996). Such translational recoding signals have been valuable in addressing questions relating to ribosome structure and function. For viruses that utilize PRF, the efficiencies of frameshift events are critical: They determine the stoichiometry of viral structural to enzymatic proteins available for virus particle assembly, and altering PRF frequencies have dire consequences for virus propagation (for review, see Dinman et al. 1998). Thus, it is important to understand how frameshifting efficiencies are controlled. The most widespread mechanisms involve inducing ribosomes to stall with their associated tRNAs positioned over specific mRNA sequences called "slippery sites" such that, in the event of slippage, the tRNAs are able to base pair with the out-of-frame codon or codons. Although the cis-acting signals are relatively well characterized, the trans-acting factors and the biophysical parameters that contribute to determine PRF efficiencies are less well understood. Genetic, biochemical, molecular, and pharmacological methods have been employed toward this end. In general, parameters that can affect PRF efficiencies include: (1) changes in the residence time of ribosomes at a particular PRF signal and the precise steps of the elongation cycle that such kinetic changes might occur; (2) changes in the stabilities of ribosome-bound tRNAs and/or ribosome catalytic function due to alterations in intrinsic ribosomal components such as ribosomal proteins, rRNAs, and codon:antidcodon interactions; and (3) defects in the abilities of the translational apparatus to recognize and correct errors (for review, see Harger et al. 2002).The genetic manipulability of the yeast Saccharomyces

Ribosomal Protein L3: Influence on Ribosome Structure and Function

“…These biochemical alterations have important implications for the development of antiviral agents: the resulting increased efficiencies in programmed -1 ribosomal frameshifting leads to an imbalance in the ratio of viral Gag to Gag-pol proteins, which negatively impact on viral particle assembly. The findings presented here also support our previously described model in which W255 plays an important role in L3 function, the nearby proline forms an important turn in the protein so as to properly position the tryptophan, and the isoleucine is only important insofar as it is part of the platform upon which the "finger" of L3 is positioned (13). Figure 5 depicts the large subunit rRNA nucleotides and L3 amino acid residues identified in this study within the context of the 2.5Å H. marismortui ribosome structure.…”

“…Small local changes would impinge upon and distort the immediate neighboring space, in turn promoting the outward radiation of spatial displacement. Given the high degree of limitation on the primary amino acid structure of L3, it is not surprising that so few mutants were discovered in the original screen (13). We suggest that the reason why so few lethal mutants were identified was because most of them were probably dominant, and thus either killed or severely inhibited cell growth even in the presence of the wild-type gene.…”

“…Construction of an RPL3::HIS3 gene deletion strain and of TRP1-CEN6 based rpl3 alleles were previously described (13). Methods to monitor cell growth, rates of protein synthesis, and drug sensitivity phenotypes were previously described (13 ;33 ).…”

“…A mutant allele of RPL3 called tcm1-1 promoted simultaneous resistance to the peptidyltransferase inhibitors trichodermin and anisomycin (5 ;8-10 ), and the mak8-1 allele of RPL3 prevented cells from propagating the yeast 'killer' virus (11). In a previous publication, we demonstrated that a defect in programmed -1 ribosomal frameshifting (PRF) was responsible for the inability of mak8-1 cells to maintain the killer virus (12), and more recently we demonstrated that peptidyltransferase defects were the underlying cause of the altered frameshifting and virus maintenance phenotypes (13). In that study, a saturation mutagenesis approach identified four rpl3 alleles having these properties.…”

Identification ofSaccharomyces cerevisiaeRibosomal Protein L3 as a Target of Curvularol, a G1-Specific Inhibitor of Mammalian Cells

“…The conservation was especially high in the sequences surrounding the residue 255Trp. 6) To confirm that the CUR resistance caused by the YCR1 mutations was due to the Trp255Cys and Trp255Leu mutations, each mutation was generated using appropriate mutagenic PCR primers as shown in Table 1. An rpl3Á strain harboring centromeric plasmid carrying a mutant allele was constructed by tetrad analysis of the spores derived from a RPL3/rpl3Á heterodiploid strain (YFK31) transformed with the mutant plasmid.…”