Near-cognate peptidyl-tRNAs promote +1 programmed translational frameshifting in yeast.

Translational frameshifting is a ubiquitous, if rare, form of alternative decoding in which ribosomes spontaneously shift reading frames during translation elongation. In studying +1 frameshifting in Ty retrotransposons of the yeast S. cerevisiae, we previously showed that unusual P site tRNAs induce frameshifting. The frameshift-inducing tRNAs we show here are near-cognates for the P site codon. Their abnormal decoding induces frameshifting in either of two ways: weak codon-anticodon pairing allows the tRNA to disengage from the mRNA and slip +1, or an unusual codon-anticodon structure interferes with cognate in-frame decoding allowing out-of-frame decoding in the A site. We draw parallels between this mechanism and a proposed mechanism of frameshift suppression by mutant tRNAs.

Allele shuffling: conjugational transfer, plasmid shuffling and suppressor analysis in Saccharomyces cerevisiae.

Trans-acting suppressor analysis represents a powerful genetic technique capable of revealing interactions among biochemical pathways in vivo. Suppressor characterization in Saccharomyces cerevisiae has traditionally utilized meiotic segregation for the requisite manipulation of strain genotypes. Meiotic segregation is not compatible with all yeast genotypes and can be prohibitively labor intensive when examining large collections of suppressors. To facilitate rapid phenotypic analysis of suppressor mutations, we have devised a novel genetic strategy called 'allele shuffling'. This plasmid-based method should in principle identify allele-specific, allele-dependent and bypass suppressors. A centromere vector (YCp) was developed that can be directly transferred from Escherichia coli to yeast via 'trans-kingdom' conjugation. Suppressors of a thermolabile cdc23 allele, cdc23-39, were isolated in the background of a yeast host strain harboring the mutant cdc23-39 gene positioned on a counterselectable plasmid. CDC23 or cdc23-39 genes cloned into a mobilizable YCp vector were then transferred directly from E. coli cultures to each suppressed yeast strain on the surfaces of agar plates. Plasmid shuffling of the cdc23-39 allele transconjugants segregated away the original cdc23-39 gene present during mutagenesis, allowing the intra- or extragenic nature of suppression to be determined. Phenotypes (if any) produced by suppressor mutations were revealed in those transconjugants receiving the wild-type CDC23-containing episome. The allele shuffling method should be generally applicable to the analysis of suppressors of any essential yeast gene.

Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis.

Cdc16p, Cdc23p and Cdc27p are all essential proteins required for cell cycle progression through mitosis in Saccharomyces cerevisiae. All three proteins contain multiple tandemly repeated 34 amino acid tetratricopeptide repeats (TPRs). Using two independent assays, two-hybrid analysis in vivo and co-immunoprecipitation in vitro, we demonstrate that Cdc16p, Cdc23p and Cdc27p self associate and interact with one another to form a macromolecular complex. A temperature sensitive mutation in the most highly conserved TPR domain of Cdc27p results in a greatly reduced ability to interact with Cdc23p, but has no effect on interactions with wild-type Cdc27p or Cdc16p. The specificity of this effect indicates that TPRs can mediate protein-protein interactions and that this mutation may define an essential interaction for cell cycle progression in yeast. The conservation of at least two of the three proteins from yeast to man suggests that this protein complex is essential for mitosis in a wide range of eukaryotes.

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.