We describe a strategy for tracking Mg²⁺-initiated folding of ³²P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA₁^Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of ³²P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.

Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K _M of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA ^Gln by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA ^Gln by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS–tRNA ^Gln complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid–RNA pairing.

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α₂) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α₂β₂) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α₂β₂ GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α₂β₂ GlyRS, convergent with α₂ GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.