Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.     

Translation elongation factor-3 (EF-3): An evolving eukaryotic ribosomal protein?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, andPneumocystis carinii). Computer-assisted analysis of the predictedS. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an aminoterminal region showing significant similarity to theE. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal accuracy center, which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of theE. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes. Correspondence to: M.F. Tuite     

Expression of Ty-lacZ fusions in Saccharomyces cerevisiae

We have determined the nucleotide sequence of about 520 bp spanning the 5' delta regions (Figure 1) of two Tyl elements. There is an open reading frame running out of the deltas for at least 180 nucleotides into the internal region of each element. The functional significance of these open reading frames has been tested by fusing them to a defective E.coli lacZ gene. Expression of B-galactosidase in yeast transformants containing these fusions shows that Tyl elements contain functional translation signals.     

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.     

Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.