Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5′-CAG-3′ (leucine) anticodon.

Abstract

From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5′‐CAG‐3′ anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl‐tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl‐tRNAs from the same species. In vitro tRNA‐charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR‐based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1–2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino‐terminal region of two RNA templates employed, namely the rabbit alpha‐ and beta‐globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an ‘omnipotent’ suppressor tRNA but rather may mediate a novel non‐standard translational event in vitro during the translation of the CUG codon. The possible nature of this non‐standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.

Functional Characterization of the MKC1 Gene of Candida albicans, Which Encodes a Mitogen-Activated Protein Kinase Homolog Related to Cell Integrity

“…Assuming that a similar signalling cascade might exist in C. albicans and that the corresponding homologous genes could be expressed in S. cerevisiae despite the deviation from universal translation rules for some Candida species (32,60,69), we were able to isolate a complementing C. albicans homolog of the S. cerevisiae SLT2 gene, suggesting that a signalling cascade homologous to the Pkc1-controlled cascade of S. cerevisiae is functional in this opportunistic fungus. The isolated gene, named MKC1, replaced the function of SLT2 in S. cerevisiae null mutants by restoring growth and preventing autolysis at 37ЊC, thus showing that the Candida gene is expressed in Saccharomyces cells.…”

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

“…Evidently, the fungal gene promoter is active in yeast (Figure 3), and fungal topoisomerase II can substitute for its yeast counterpart in discharging essential functions in chromosome condensation and segregation. Complementation occurs despite the fact that the C. albicans TOP2 gene carries six CUG codons (Figure 1) which are read as Ser in the non-universal code used in C. albicans [44][45][46] but are decoded as Leu in S. cere isiae. The full-length fungal protein made in yeast therefore carries six leucine substitutions (five when expressed from YEpWCa10).…”

“…It is known that, whereas human cells and S. cere isiae utilize the universal genetic code, in C. albicans, the CUG codon is decoded as serine and not leucine as would normally be expected [44][45][46]. The C. albicans TOP2 gene has six CUG codons (Figure 1), which presumably result in Ser-to-Leu protein substitutions when expressed in S. cere isiae.…”

Stop codon decoding in Candida albicans: from non‐standard back to standard

“…In vitro translation studies carried out with both yeast and rabbit reticulocyte translation systems programmed with reporter mRNAs suggest that this unique ser-tRNA CAG has the ability to translate the UAG and UGA termination codons (Tuite et al, 1986;Santos et al, 1993). This conclusion was reached because, for each reporter mRNA translated in presence of the C. albicans ser-tRNA CAG abnormally elongated polypeptides were synthesized which could be separated from the wild-type polypeptides on SDS-PAGE.…”

“…The dimorphic fungus Candida albicans encodes a novel transfer RNA, which decodes the standard leucine CUG codon as serine (Santos et al, 1993). In vitro translation studies carried out with both yeast and rabbit reticulocyte translation systems programmed with reporter mRNAs suggest that this unique ser-tRNA CAG has the ability to translate the UAG and UGA termination codons (Tuite et al, 1986;Santos et al, 1993).…”