THE LEUCY PHENYLALANYL-TRANSFER-RNA-PROTEIN TRANSFERASE - OVEREXPRESSION AND CHARACTERIZATION OF SUBSTRATE RECOGNITION, DOMAIN-STRUCTURE, AND SECONDARY STRUCTURE/

Previous work has shown that, in the bacterium Escherichia coli, the a at gene is essential for the degradation of proteins bearing amino-ter minal Arg and Lys residues via the N-end rule pathway of protein degra dation. We now show that the aat gene encodes directly the leucy/pheny lalanyl-tRNA-protein transferase (L/F-transferase). This enzyme cataly zes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned oat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the p reviously purified wild type enzyme and contains no detectable RNA com ponent. We have used the recombinant enzyme to demonstrate that both t he solubility and substrate specificity, for aminoacyl-tRNA substrates , of the L/F transferase are dependent on ionic strength conditions an d that the modified nucleotides found in natural tRNAs are not essenti al for recognition by the enzyme. Limited digestion of the L/F-transfe rase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the LIF-transferase and the NH2-termi nal protein acetylases has been identified.