FUNCTIONAL DISSECTION OF A PREDICTED CLASS-DEFINING MOTIF IN A CLASS-II TRANSFER-RNA SYNTHETASE OF UNKNOWN STRUCTURE

A core of eight beta-strands and three ar-helices was recently predict ed for the active site domain of Escherichia coli alanyl-tRNA syntheta se, an enzyme of unknown structure [Ribas de Pouplana, L1., Buechter, D. D., Davis, M. W., & Schimmel, P. (1993) Protein Sci. 2, 2259-2262; Shi, J.-P., Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 26, 5312-5318]. A critical part of this predicted structure is two antipa rallel beta-strands and an intervening loop that make up the second of three highly degenerate sequence motifs that are characteristic of th e class II aminoacyl-tRNA synthetases. We present here an in vivo and in vitro analysis of 21 rationally designed mutations in the predicted 34-amino acid motif 2 of E. coli alanyl-tRNA synthetase. Although thi s motif in E. coli alanyl-tRNA synthetase is of a different size than and has only two sequence identities with the analogous motif in yeast aspartyl- and Thermus thermophilus seryl-tRNA synthetases, whose stru ctures are known, the functional consequences of the mutations are exp lainable in terms of those structures. In particular, the analysis dem onstrates the importance of the predicted motif 2 in adenylate formati on, distinguishes between two similar, but distinct, predicted models for this motif, and distinguishes between the functional importance of two adjacent phenylalanines in a way that strongly supports the predi cted structure. The results suggest that similar analyses will be gene rally useful in testing models for active site regions of other class II aminoacyl-tRNA synthetases of unknown structure.