A yeast arginine specific tRNA is a remnant aspartate acceptor

Abstract

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.

Figure 1

Sequence comparison and tertiary structural elements of yeast aspartate and arginine isoacceptor tRNAs. (A) tRNA^Arg isoacceptors 1, 2 and 3 (the three major species decoding 96.1% of the arginine codons) show 53% identity. Isoacceptor 4 is distinguished from the other three in sharing only 33% of common nucleotides (this minor species decodes only 3.9% of the arginine codons). Percentage of identical residues was calculated considering all nucleotides (also those conserved in all canonical tRNAs) shared between the compared sequences. The secondary structural domains are indicated below the primary sequences. (B) Sequence comparison of tRNA₄^Arg and the single tRNA^Asp isoacceptor indicates 57% identity. These two sequences share a same length in their variable region and in the α and β domains of the D-loop.

Figure 2

Sequence distances in the 48 yeast tRNA genes highlight proximity between tRNA^Asp and tRNA₄^Arg. The dendrogram was established with the ClustalV program from MegAlign (Lasergene from DNAstar). Genes for tRNA^Arg and tRNA^Asp are highlighted as well as either short or long distance between them. A total of 48 tRNAs are identified by their anticodon triplet (the isoacceptor species with sequence variations outside the anticodon triplet are identified by single primes and double primes).

Figure 3

Cloverleaf sequences of the four yeast tRNA^Arg isoacceptors. Full red dots highlight the set of arginine identity elements experimentally determined for tRNA₃^Arg (19). Insertion of the aspartate identity set (17,18) indicated in green, generates an alternative arginine identity set (19) in tRNA₄^Arg (nucleotides in red circles).

Figure 4

Northern-blot analysis of tRNA samples extracted from yeast cells under acidic conditions. Experimental procedure is described in Materials and Methods. Levels of aminoacylation of tRNA₄^Arg and tRNA₄^Arg/C38/G73 were determined by measurement of the ratio of acylated (− lanes) and deacylated tRNA (+ lanes, obtained by alkaline treatment of the RNA extracts). The blot was probed with ³²P-labeled oligonucleotides complementary to 5S RNA (control of RNA content), to tRNA^Leu (control of the acidic extraction procedure) and to tRNA₄^Arg. Lanes 1 and 2, wild-type tRNA₄^Arg; and lanes 3 and 4, mutated tRNA₄^Arg/C38/G73. The second blot (lanes 5 and 6) was probed with a tRNA^Asp-specific oligonucleotide. It shows that an aspartate residue attached to a tRNA molecule induces less of a shift than that of an arginine residue.

Figure 5

Yeast aspartate identity elements in the four tRNA^Arg isoacceptors. Nucleotides at position 73 (discriminator base), the anticodon loop and 10–25 bp are indicated. For comparison, the sequence of the single tRNA^Asp isoacceptor is also indicated. Aspartate identity elements are highlighted with gray squares. Note the absence of aspartate determinants from the anticodon loop of each of the four tRNA^Arg isoacceptors. tRNA₄^Arg shares two nucleotides with tRNA^Asp (open squares) in addition to U33 conserved in all tRNAs. Aspartate acceptance of each wild-type tRNA is indicated (+/−). Gains (G) or losses (L) in aspartylation capacity observed after single nucleotide mutations of tRNA₄^Arg and tRNA^Asp are indicated.

Figure 6

Characteristic features of tRNA^Arg and tRNA^Asp isoacceptors in various yeast species. tRNA genes were sought in eight yeast species (see Materials and Methods). Isoacceptors are named according to their anticodon triplets. In each case, the nucleotides at positions 37, 38 and 73 are given. Further, structural information on the nucleotide distribution in the subdomains α and β of the D-loops as well as the size of the variable regions (v) are indicated. Numbers indicate the percentage of identity of a given tRNA^Arg with tRNA^Asp in a same species. Along the left-hand side of the table, the pylogenetic link between the different yeast species is represented in a schematic diagram based on the rRNA sequences.