Characterization of serine and leucine tRNAs in an asporogenic yeastCandida cylindraceaand evolutionary implications of genes for tRNA^SerCAG responsible for translation of a non-universal genetic code

Abstract

Five serine and three leucine isoaceptor tRNAs were purified from the asporogenic yeast Candida cylindracea, in which codon CUG is translated as serine instead of leucine, and their primary structures were determined. From the wobble hypothesis, it was assumed that one of the tRNA(Leu) species (Leu1), with the anticodon CmAA, corresponded to the UUG leucine codon, and that the remaining two leucine tRNAs (Leu2 and Leu3), with the same IAG anticodon sequence would decode the CUU, CUC and CUA codons as leucine, but not the CUG codon; this was clarified by an in vitro translation experiment with C.cylindracea using synthetic mRNAs containing the CUA or CUG codons. One of the serine tRNAs (Ser1) has already been demonstrated to have the anticodon CAG and to be responsible for translation of the codon CUG in C.cylindracea. Three of the other species of tRNA(Ser) (Ser2,3 and 4), with the anticodon sequences cm5UGA, IGA and CGA, can translate all four codons in the UCN codon box, while the remaining species (Ser5), with the anticodon GCU, corresponds to AGU and AGC serine codons. The gene sequences for these five serine and three leucine tRNAs were also determined, with the finding that only tRNA(Ser)CAG (Ser1) has an intron. At least five different types of tRNA(Ser)CAG genes exist in the genome of C.cylindracea. The nucleotide sequences of the flanking regions of these tRNA(Ser)CAG genes indicated that the tRNA(Ser)CAG gene has duplicated at least three times on the genome. The existence of multiple genes for tRNA(Ser)CAG on the genome may account for the observation that codon CUG is used very frequently in C.cylindracea. All of these tRNASerCAG genes contain the CCA sequence in their 3' termini, suggesting the possibility that during their multiplication process in the evolution of the C.cylindracea genome, the tRNA(Ser)CAG molecule was integrated into DNA via reverse transcription.

Genomic Exploration of the Hemiascomycetous Yeasts: 15. Pichia sorbitophila

“…Similar observations were previously made in several Candida species [13,14] and Debaryomyces hansenii [15]. Unfortunately, we have not been able to identify the corresponding tRNA gene suggesting that it is present at a low copy number.…”

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

“…Purified tRNA Tyr accepted at least 1.4 nmol of tyrosine/A 260 unit, determined in an aminoacylation assay described elsewhere (11). To isolate tRNA Ser acceptors, the tRNA was serylated using pure seryl-tRNA synthetase, derivatized by naphthoxyacetylation (36), and purified by chromatography on a benzoylated DEAE-cellulose column (37). The aliquots from the chromatography fractions were subjected to electrophoresis on a 8% polyacrylamide gel containing 8 M urea.…”

A new method for identifying the amino acid attached to a particular RNA in the cell

“…In order to demonstrate the validity of this method, we selected a serine tRNA having the anticodon CAG (tRNASerCAG), corresponding to the universal leucine codon CUG, from Candida cylindracea [15,16]. We have already reported that the universal leucine codon CUG is read as serine in several Candida species by a specific serine tRNA corresponding to the codon CUG (tRNASerCAG) [17,18], following an experiment showing that the codon CUG can in fact be translated as serine in an in vitro translation with this serine tRNA using synthetic mRNA containing the in-frame CUG codons [15,16]. Here, we attempted to analyze the aminoacylated form of this serine tRNA in the cell to ensure that the tRNA is also charged with serine even in vivo.…”