Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

Abstract

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The In eucaryotes, ribosomes initiate translation of mRNA by binding at or near the 5'-terminal cap structure. Bound ribosomes then scan the mRNA in the 5' to 3' direction to find the first AUG codon in optimal sequence context (for reviews, see references 11, 14, and 19). Presumably, this cap-dependent pathway represents the major translation initiation pathway in eucaryotes. More recently, however, alternative initiation pathways have been described. (i) Ribosomes can reinitiate translation after termination of translation of an open reading frame (reinitiation) (15,17,20), and (ii) ribosomes can initiate translation internally, e.g., initiate at a downstream open reading frame without translating the upstream open reading frame (internal initiation) (21). Whereas the initiation factor requirements for cap-dependent translation are known (for reviews, see references 11 and 19), the mechanisms and factor requirements for reinitiation and internal initiation have yet to be determined. Since initiation factor functions appear to be conserved between higher and lower eucaryotes, as indicated by amino acid sequence similarities between mammalian and yeast (Saccharomyces cerevisiae) initiation factors (1, 2, 7, 16) and substitution of a mouse initiation factor for the homologous yeast factor in vivo (M. Altmann, P. P. Muller, J. Pelletier, N. Sonenberg, and H. Trachsel, J. Biol. Chem., in press), we use S. cerevisiae as a model system to study factor requirements for translation of different mRNAs. Here, we describe the construction of an initiation factor 4E (eIF-4E)-dependent system and provide evidence for eIF-4E-independent mRNA translation. (ii) In vitro mutagenesis. pMDA-101 (about 50 ,ug) was treated with 1 M hydroxylamine (neutralized by adding NaOH to a final concentration of 0.5 M)-10 mM EDTA at 65°C for 3 h. The transformation efficiency (LiOAc method) (3) of the mutagenized plasmid pool was determined to be about 15% of that of nonmutagenized plasmid. Mutagenized plasmid was subjected to extensive phenol extraction (at least five times) and two ethanol precipitations. MATERIALS AND METHODSIsolation of temperature-sensitive yeast strains. The mutagenized plasmid pool was transformed into T93C (a eIF-4E::LEU2 ura3 trpl leu2[pGAL1-eIF-4E URA3]), a haploid S. cerevisiae strain having its chromosomal copy of the eIF-4E gene removed and replaced by the LEU2 gene (3). In these cells, the eIF-4E function is provided by the plasmid pGAL1-eIF-4E (Altmann et al., in press

Poliovirus 2Apro expression inhibits growth of yeast cells

“…It is unclear if p220 cleavage is responsible for the poliovirusinduced host-cell shut-off of protein synthesis or if another event is responsible for this phenomenon [6,7,26,27]. Thus, it was of interest to analyze the effects of 2A pr° expression on S. cerevisiae macromolecular synthesis, since the protein synthesis machinery of this organism is similar to that of mammalian cells [28] and some of the factors involved in translation in these systems are interchangeable [29].…”

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…The yeast strain T93C (Altmann et al, 1989), containing a chromosomal deletion of eIF4E and a plasmid with eIF4E under control of a Gal promoter (eIF4E::LEU2 ura3 trp1 leu2 [pGal1-eIF4E URA3]), was transformed with pG-1 vectors (with an added NcoI site N terminus to BamHI in the cloning region) containing Arabidopsis eIF4E constructs. pG-1 provides constitutive gene expression and a TRP1 marker (Schena et al, 1991).…”

“…The ability of Arabidopsis eIF4E proteins to complement deletion of the eIF4E gene in yeast. Complementation was tested by introducing pG-1 plasmids for constitutive expression of Arabidopsis eIF4E genes into a yeast strain (T93C; Altmann et al, 1989) with eIF4E under control of a GAL promoter. Serial dilutions of midlog phase yeast were plated in 10-fold serial dilutions on SCM-Trp plates containing 2% Gal (A) or 2% Glc (B) and incubated at 30˚C for 48 h. The experiment was performed in three biological replicates; representative results are shown.…”

The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae

“…Temperature-sensitive alleles of the CDC33 gene lead to a defect in translation initiation in vivo and an arrest in the G 1 phase of the cell cycle (8)(9)(10)(11). Recently, the analysis of deletion variants of eIF4E with reduced affinity for the 5Ј cap indicated that eIF4E mediates the selectivity of yeast ribosomes for capped mRNAs (12).…”