A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon (original) (raw)
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Biochimie, 2005
Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypeptide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodonbinding domain. This paralog of glutamyl-tRNA synthetases, the YadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA Glu but instead on tRNA Asp . It has been shown that tRNA Asp is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged by YadB. The functional properties of YadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3′-end of tRNA. Biochemical approaches and mass spectrometry investigations revealed that YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamylqueuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA Asp is substrate of YadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA Asp and the amino acid acceptorstem of tRNA Glu . YadB, renamed glutamyl-Q-tRNA Asp synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA synthetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA Asp synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA and aminoacylates the 3′-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed.
tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases
Journal of Molecular Biology, 1998
Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp-and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the speci®city of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA Lys anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the b-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Speci®cation of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a¯exible seven-residue loop between strands 4 and 5 of the OB-fold (L 45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modi®cations of the anticodon loop of tRNA Lys (mnm 5 s 2 U34 and t 6 A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L 45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L 45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.
Nucleic Acids Research, 2007
Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA Gln formation. To analyze the peculiarities of the structure-function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3 Å and a docking model of the synthetase complexed to tRNA Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA Gln recognition. They demonstrate that Yqey plays the role of an affinityenhancer of GlnRS for tRNA Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.
Biochemistry, 2007
Aminoacyl-tRNA synthetases are normally found in one of two mutually exclusive structural classes, the only known exception being lysyl-tRNA synthetase which exists in both classes I (LysRS1) and II (LysRS2). Differences in tRNA acceptor stem recognition between LysRS1 and LysRS2 do not drastically impact cellular aminoacylation levels, focusing attention on the mechanism of tRNA anticodon recognition by LysRS1. On the basis of structure-based sequence alignments, seven tRNA Lys anticodon variants and seven LysRS1 anticodon binding site variants were selected for analysis of the Pyrococcus horikoshii LysRS1-tRNA Lys docking model. LysRS1 specifically recognized the bases at positions 35 and 36, but not that at position 34. Aromatic residues form stacking interactions with U34 and U35, and aminoacylation kinetics also identified direct interactions between Arg502 and both U35 and U36. Tyr491 was also found to interact with U36, and the Y491E variant exhibited significant improvement compared to the wild type in aminoacylation of a tRNA Lys UUG mutant. Refinement of the LysRS1-tRNA Lys docking model based upon these data suggested that anticodon recognition by LysRS1 relies on considerably fewer interactions than that by LysRS2, providing a structural basis for the more significant role of the anticodon in tRNA recognition by the class II enzyme. To date, only glutamyl-tRNA synthetase (GluRS) has been found to contain an α-helix cage anticodon binding domain homologous to that of LysRS1, and these data now suggest that specificity for the anticodon of tRNA Lys could have been acquired through relatively few changes to the corresponding domain of an ancestral GluRS enzyme.
Proceedings of the National Academy of Sciences, 1996
Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNAGIn in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.
Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates
Proceedings of The National Academy of Sciences, 2003
Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNAGlu or both tRNAGlu and tRNAGln. Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNAGlu, whereas GluRS2 was specific solely for tRNAGln. The A. ferrooxidans GluRS2 preferentially charged . Conversely, A. ferrooxidans GluRS1 glutamylated both tRNAGlu isoacceptors and the species. These three tRNA species have two structural elements in common, the augmented D-helix and a deletion of nucleotide 47. It appears that the discriminating or nondiscriminating natures of different GluRS enzymes have been derived by the coevolution of protein and tRNA structure. The coexistence of the two GluRS enzymes in one organism may lay the groundwork for the acquisition of the canonical glutaminyl-tRNA synthetase by lateral gene transfer from eukaryotes.