ABSTRACT
The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA(Gln). The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA(Glu) and Glu-tRNA(Gln). The Glu-tRNA(Gln) is then converted to Gln-tRNA(Gln) by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA(Glu) and tRNA(Gln) with an unrelated α-helical cage domain in contrast to the ß-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA(Glu)/tRNA(Gln) discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA(Gln) complex reveals the structural determinants responsible for specific tRNA(Gln) recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.
Subject(s)
Archaeal Proteins/chemistry , Evolution, Molecular , Glutamate-tRNA Ligase/chemistry , Archaeal Proteins/classification , Archaeal Proteins/metabolism , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Glutamate-tRNA Ligase/classification , Glutamate-tRNA Ligase/metabolism , Glutamic Acid/chemistry , Glutamine/chemistry , Methanobacteriaceae/enzymology , Models, Molecular , Phylogeny , Protein Binding , RNA, Transfer, Amino Acyl/metabolism , Substrate SpecificitySubject(s)
Glutamate-tRNA Ligase/chemistry , Glutamate-tRNA Ligase/metabolism , RNA, Transfer, Glu/metabolism , Thermus thermophilus/enzymology , Anticodon/chemistry , Anticodon/genetics , Anticodon/metabolism , Arginine/metabolism , Binding Sites , Crystallography, X-Ray , Evolution, Molecular , Glutamate-tRNA Ligase/classification , Glutamate-tRNA Ligase/genetics , Glutamic Acid/metabolism , Models, Biological , Mutation/genetics , Protein Structure, Tertiary , RNA, Transfer, Glu/chemistry , RNA, Transfer, Glu/genetics , Substrate Specificity , Thermus thermophilus/geneticsABSTRACT
The genes of glutamyl- and prolyl-tRNA synthetases (GluRS and ProRS) are organized differently in the three kingdoms of the tree of life. In bacteria and archaea, distinct genes encode the two proteins. In several organisms from the eukaryotic phylum of coelomate metazoans, the two polypeptides are carried by a single polypeptide chain to form a bifunctional protein. The linker region is made of imperfectly repeated units also recovered as singular or plural elements connected as N-terminal or C-terminal polypeptide extensions in various eukaryotic aminoacyl-tRNA synthetases. Phylogenetic analysis points to the monophyletic origin of this polypeptide motif appended to six different members of the synthetase family, belonging to either of the two classes of aminoacyl-tRNA synthetases. In particular, the monospecific GluRS and ProRS from Caenorhabditis elegans, an acoelomate metazoan, exhibit this recurrent motif as a C-terminal or N-terminal appendage, respectively. Our analysis of the extant motifs suggests a possible series of events responsible for a gene fusion that gave rise to the bifunctional glutamyl-prolyl-tRNA synthetase through recombination between genomic sequences encoding the repeated units.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Evolution, Molecular , Phylogeny , Recombination, Genetic/genetics , Amino Acid Motifs , Amino Acid Sequence , Amino Acyl-tRNA Synthetases/classification , Animals , Arabidopsis , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Cloning, Molecular , Cricetinae , Crossing Over, Genetic/genetics , DNA, Complementary/genetics , Glutamate-tRNA Ligase/chemistry , Glutamate-tRNA Ligase/classification , Glutamate-tRNA Ligase/genetics , Humans , Models, Genetic , Molecular Sequence Data , Protein Structure, Tertiary , Repetitive Sequences, Amino Acid/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , YeastsABSTRACT
The accuracy of protein biosynthesis generally rests on a family of 20 aminoacyl-tRNA synthetases, one for each amino acid. In bacteria, archaea and eukaryotic organelles, the formation of Gln-tRNA(Gln) is prevalently accomplished by a transamidation pathway, aminoacylation of tRNA(Gln) with Glu by glutamyl-tRNA synthetase (GluRS) followed by a tRNA-dependent transamidation of Glu from Glu-tRNA(Gln). A few bacterial species, such as Escherichia coli, possess a glutaminyl-tRNA synthetase (GlnRS), responsible for Gln-tRNA(Gln) formation. Phylogenetic analysis of the GluRS or GlnRS families (GlxRS) suggested that GlnRS has a eukaryotic origin and was horizontally transferred to a restricted set of bacteria. We have now isolated an additional GlnRS gene from the plant Lupinus luteus and analyzed in more details the modular architecture of the paralogous enzymes GluRS and GlnRS, starting from a large data set of 33 GlxRS sequences. Our analysis suggests that the ancestral GluRS-like enzyme was solely composed of the catalytic domain bearing the class-defining motifs of aminoacyl-tRNA synthetases, and that the anticodon-binding domain of GlxRSs was independently acquired in the bacteria and archaea branches of the universal tree of life, the eukarya sub-branch arising as a sister group of archaea. The transient capture of UAA and UAG codons could have favored the emergence of a GlnRS in early eukaryotes.
