Internal structural features of E. coli glycyl-tRNA synthetase examined by subunit polypeptide chain fusions.

In contrast to most aminoacyl-tRNA synthetases which are monomers or oligomers of a single polypeptide, Escherichia coli glycyl-tRNA synthetase has an alpha-2, beta-2 structure. The enzyme requires both subunits for catalysis of either adenylate or aminoacyl-tRNA synthesis. The head-to-tail arrangement of the alpha- and beta-chain coding regions in the genome suggests that the two-subunit protein may be tantamount to a single chain. We fused the carboxyl terminus of the alpha-chain to the amino terminus of the beta-chain, through a short peptide linker. Five different amino acid substitutions were placed in the linker. In all instances, the fusion polypeptide is stable in maxicell extracts. In a glyS null strain, a gene encoding any of the fusion proteins substitutes for the wild-type gene. Assays confirm that, in vitro, the engineered polypeptide fusion is active to within 2- to 3-fold of the wild-type, unfused chains. Oligomers of the fusion protein are observed and may be required for activity. Because the creation and limited manipulation of the artificial peptide linker region does not destroy the activity, we also conclude that the C-terminal part of the alpha-chain and the amino-terminal part of the beta-chain are not important for activity.