TRANSFER-RNA ANTICODON RECOGNITION AND SPECIFICATION WITHIN SUBCLASS IIB AMINOACYL-TRANSFER-RNA SYNTHETASES

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recog n ize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, res pectively) through an OB-folded N-terminal extension. In the present s tudy, the specificity of constitutive lysyl-tRNA synthetase (LysS) fro m Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing t he anticodon binding site on the other. From this analysis, a tentativ e model is deduced for both the recognition of the cognate anticodon a nd the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 Flay a critical role in this spatial organization This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tR NA complex is further achieved through the accommodation of U34 and U3 6. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L -45). Additional free energy of binding is recovered from the resultin g network of cooperative interactions. Such a mechanism would not depe nd on the modifications 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-cog nate anticodons can be accounted for by a hindrance to the positioning of the L-45 loop. Ln addition, Glu135 would repulse a cytosine base a t position 35. Sequence comparisons show that the composition and leng th of the L-45 loop are markedly conserved in each of the families com posing subclass IIb aminoacyl-tRNA synthetases. The possible role of t he loop is discussed for each case, including that of archaebacterial aspartyl-tRNR synthetases. (C) 1998 Academic Press Limited.