Aminoacyl-tRNA synthetases attach amino acids to the 3′ termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13–18, 2002) discussed new research into the structure–function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code.

The structural requirements for assembly of functional class II transfer RNA core regions have been examined by sequence analysis and tested by reconstruction of alternative folds into the tertiary domain of Escherichia coli tRNA2Gln. At least four distinct designs have been identified that permit stable folding and efficient synthetase recognition, as assessed by thermal melting profiles and glutaminylation kinetics. Although most large variable-arm tRNAs found in nature possess an enlarged D-loop, lack of this feature can be compensated for by insertion of nucleotides either 3′ to the variable loop or within the short acceptor/D-stem connector region. Rare pyrimidines at nt 9 in the core region can be accommodated in the class II framework, but only if specific nucleotides are present either in the D-loop or 3′ to the variable arm. Glutaminyl-tRNA synthetase requires one or two unpaired uridines 3′ to the variable arm to efficiently aminoacylate several of the class II frameworks. Because there are no specific enzyme contacts in the tRNAGln core region, these data suggest that tRNA discrimination by GlnRS depends in part on indirect readout of RNA sequence information.

Structure-based engineering of the tertiary fold of Escherichia coli tRNA2Gln has enabled conversion of this transfer RNA to a class II structure while retaining recognition properties of a class I glutamine tRNA. The new tRNA possesses the 20-nt variable stem-loop of Thermus thermophilus tRNASer. Enlargement of the D-loop appears essential to maintaining a stable tertiary structure in this species, while rearrangement of a base triple in the augmented D-stem is critical for efficient glutaminylation. These data provide new insight into structural determinants distinguishing the class I and class II tRNA folds, and demonstrate a marked sensitivity of glutaminyl-tRNA synthetase to alteration of tRNA tertiary structure.