Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.     

Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.     

tRNA glycylation system from Thermus thermophilus. tRNAGly identity and functional interrelation with the glycylation systems from other phylae.

The systems of tRNA glycylation belong to the most complex aminoacylation systems since neither the oligomeric structure of glycyl-tRNA synthetases (GlyRS) nor the discriminator bases in tRNAGly are conserved in the phylae. To better understand the structure-function relationship in glycylation systems of various origins and the functional peculiarities related to their structural divergences, the elements in tRNA conferring its glycine identity in Thermus thermophilus were characterized and compared to those of other systems. Thermophilic identity is conferred by the G1-C72, C2-G71, G3-C70, and C50-G64 pairs together with the G10, U16, C35, and C36 single residues. In contrast to most other aminoacylation systems, the discriminator base is not directly involved in identity. Transplantation of these elements in tRNAAsp and tRNAPhe converts specificity toward glycine albeit conservation of nucleotide 73. Analysis of the functional interrelation of the identity elements shows coupling in synthetase recognition of the elements from anticodon and G10 whereas those from acceptor arm are recognized independently. Despite nondirect implication in identity, the discriminator base contributes cooperatively with C36 in specificity of glycylation. The link between the structural heterogeneity and the functional divergence of the glycylation systems and the phylogenic interrelation of these systems were approached by comparing the ability of GlyRSs of various phylae to glycylate heterologous tRNAGly. Dimeric GlyRSs from mammalian and archaebacteria acylate efficiently only eukaryotic and archaebacterial tRNAGly with a discriminatory A73, whereas tetrameric Escherichia coli GlyRS acylates only eubacterial tRNAGly with a discriminatory U73. In contrast, dimeric yeast GlyRS acylates efficiently both eukaryotic and archaebacterial tRNAGly as well as peculiar prokaryotic isoacceptors. Species specificity is lost with the dimeric GlyRS from Thermus thermophilus that acylates efficiently eubacterial, archaebacterial, and eukaryotic tRNAGly. These features are discussed in the context of the evolution of the glycylation systems and the phylogenic interrelation of the organisms.     

Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems

The tRNA identity rules ensuring fidelity of translation are globally conserved throughout evolution except for tyrosyl-tRNA synthetases (TyrRSs) that display species-specific tRNA recognition. This discrimination originates from the presence of a conserved identity pair, G1-C72, located at the top of the acceptor stem of tRNA(Tyr) from eubacteria that is invariably replaced by an unusual C1-G72 pair in archaeal and eubacterial tRNA(Tyr). In addition to the key role of pair 1-72 in tyrosylation, discriminator base A73, the anticodon triplet and the large variable region (present in eubacterial tRNA(Tyr) but not found in eukaryal tRNA(Tyr)) contribute to tyrosylation with variable strengths. Crystallographic structures of two tRNA(Tyr)/TyrRS complexes revealed different interaction modes in accordance with the phylum-specificity. Recent functional studies on the human mitochondrial tRNA(Tyr)/TyrRS system indicates strong deviations from the canonical tyrosylation rules. These differences are discussed in the light of the present knowledge on TyrRSs.     

tRNAHis guanylyltransferase adds G-1 to the 5' end of tRNAHis by recognition of the anticodon, one of several features unexpectedly shared with tRNA synthetases

All eukaryotic tRNA(His) molecules are unique among tRNA species because they require addition of a guanine nucleotide at the -1 position by tRNA(His) guanylyltransferase, encoded in yeast by THG1. This G(-1) residue is both necessary and sufficient for aminoacylation of tRNA by histidyl-tRNA synthetase in vitro and is required for aminoacylation in vivo. Although Thg1 is presumed to be highly specific for tRNA(His) to prevent misacylation of tRNAs, the source of this specificity is unknown. We show here that Thg1 is >10,000-fold more selective for its cognate substrate tRNA(His) than for the noncognate substrate tRNA(Phe). We also demonstrate that the GUG anticodon of tRNA(His) is a crucial Thg1 identity element, since alteration of this anticodon in tRNA(His) completely abrogates Thg1 activity, and the simple introduction of this GUG anticodon to any of three noncognate tRNAs results in significant Thg1 activity. For tRNA(Phe), k(cat)/K(M) is improved by at least 200-fold. Thg1 is the only protein other than aminoacyl-tRNA synthetases that is known to use the anticodon as an identity element to discriminate among tRNA species while acting at a remote site on the tRNA, an unexpected link given the lack of any identifiable sequence similarity between these two families of proteins. Moreover, Thg1 and tRNA synthetases share two other features: They act in close proximity to one another at the top of the tRNA aminoacyl-acceptor stem, and the chemistry of their respective reactions is strikingly similar.     

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA^Tyrs. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA^Tyr(GΨA). Structural information for TyrRS–tRNA^Tyr complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs–tRNA^Tyrs pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA^Tyr recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA^Tyr pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA^Tyr. On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.     

Major anticodon-binding region missing from an archaebacterial tRNA synthetase

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.     

Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems

The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.     

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.