Novel features in the tRNA-like world of plant viral RNAs.

tRNA-like domains are found at the 3' end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence similarities with tRNA are those found in valine accepting structures from tymoviruses (e.g. TYMV). All the viral tRNA mimics present a pseudoknotted amino acid accepting stem, which confers special structural and functional characteristics. In this review emphasis is given to newly discovered tRNA-like structures (e.g. in furoviruses) and to recent advances in the understanding of their three-dimensional architecture, which mimics L-shaped tRNA. Identity determinants in tRNA-like domains for aminoacylation are described, and evidence for their functional expression, as in tRNAs, is given. Properties of engineered tRNA-like domains are discussed, and other functional mimicries with tRNA are described (e.g. interaction with elongation factors and tRNA maturation enzymes). A final section reviews the biological role of the tRNA-like domains in amplification of viral genomes. In this process, in which the mechanisms can vary in specificity and efficiency according to the viral genus, function can be dependent on the aminoacylation properties of the tRNA-like domains and/or on structural properties within or outside these domains.

Specific tyrosylation of the bulky tRNA-like structure of brome mosaic virus RNA relies solely on identity nucleotides present in its amino acid-accepting domain.

Residues specifying aminoacylation by yeast tyrosyl-tRNA synthetase (TyrRS) of the tRNA-like structure present at the 3'-end of brome mosaic virus (BMV) RNA were determined by the in vitro approach using phage T7 transcripts. They correspond to nucleotides equivalent to base-pair C1-G72 and discriminator base A73 in the amino acid-acceptor branch of the molecule. No functional equivalents of the tyrosine anticodon residues, shown to be weakly involved in tyrosine identity of canonical tRNA(Tyr), were found in the BMV tRNA-like structure. This indicates a behaviour of this large and intricate molecule reminiscent of that of a minihelix derived from an amino acid-acceptor branch. Furthermore, iodine footprinting experiments performed on a tyrosylable BMV RNA transcript of 196 nt complexed to yeast TyrRS indicate that the amino acid-acceptor branch of the viral RNA is protected against cleavages as well as a hairpin domain, which is possibly located perpendicularly to its accepting branch. This domain without the canonical anticodon loop or the tyrosine anticodon acts as an anchor for TyrRS interaction leading to a better efficiency of tyrosylation.

Major tyrosine identity determinants in Methanococcus jannaschii and Saccharomyces cerevisiae tRNA(Tyr) are conserved but expressed differently.

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1-G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild-type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNA(Tyr) transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1-G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.

Identity of tRNA for yeast tyrosyl-tRNA synthetase: tyrosylation is more sensitive to identity nucleotides than to structural features.

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure. Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discriminator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant for tyrosylation). The three anticodon bases G34, U35, and A36 contribute also to the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect. Mutation of the GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylation efficiency similar to that obtained after mutation of the C1-G72 or A73 determinants. Transplantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannaschii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukaryotic character of the C1-G72 base pair. On the other hand, it was found that tyrosine identity in yeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of the D-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNA(Tyr) with a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pair. Finally, tyrosylation activity is compatible with a +1 shift of the anticodon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.

The peculiar architectural framework of tRNASec is fully recognized by yeast AspRS.

The wild-type transcript of Escherichia coli tRNASec, characterized by a peculiar core architecture and a large variable region, was shown to be aspartylatable by yeast AspRS. Similar activities were found for tRNASec mutants with methionine, leucine, and tryptophan anticodons. The charging efficiency of these molecules was found comparable to that of a minihelix derived from tRNAAsp and is accounted for by the presence of the discriminator residue G73, which is a major aspartate identity determinant. Introducing the aspartate identity elements from the anticodon loop (G34, U35, C36, C38) into tRNASec transforms this molecule into an aspartate acceptor with kinetic properties identical to tRNAAsp. Expression of the aspartate identity set in tRNASec is independent of the size of its variable region. The functional study was completed by footprinting experiments with four different nucleases as structural probes. Protection patterns by AspRS of transplanted tRNASec and tRNAAsp were found similar. They are modified, particularly in the anticodon loop, upon changing the aspartate anticodon into that of methionine. Altogether, it appears that recognition of a tRNA by AspRS is more governed by the presence of the aspartate identity set than by the structural framework that carries this set.