Alternative designs for construction of the class II transfer RNA tertiarycore

The structural requirements for assembly of functional class II transfer RN A core regions have been examined by sequence analysis and tested by recons truction of alternative folds into the tertiary domain of Escherichia coli tRNA(2)(Gln). At least four distinct designs have been identified that perm it stable folding and efficient synthetase recognition, as assessed by ther mal melting profiles and glutaminylation kinetics. Although most large vari able-arm tRNAs found in nature possess an enlarged D-loop, lack of this fea ture can be compensated for by insertion of nucleotides either 3' to the va riable loop or within the short acceptor/D-stem connector region. Rare pyri midines at nt 9 in the core region can be accommodated in the class II fram ework, 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 unp aired uridines 3' to the variable arm to efficiently aminoacylate several o f the class II frameworks. Because there are no specific enzyme contacts in the tRNA(Gln) core region, these data suggest that tRNA discrimination by GlnRS depends in part on indirect readout of RNA sequence information.