Expression of bovine mitochondrial elongation factor Ts in Escherichia coli and characterization of the heterologous complex formed with prokaryotic elongation factor Tu

When bovine mitochondrial elongation factor Ts (EF-Ts-mt) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli EF-Tu (EF-Tu(Eco). Ts-mt). This complex is active in poly(U)directed polymerization and this activity is inhibited by kirromycin. The EF-Tu(Eco). Ts-mt complex does not bind guanine nucleotides detectably and is not dissociated to a significant extent by either GDP or GTP. A portion of the EF-Tu(Eco). Ts-mt complex can be dissociated by aa-tRNA in the presence of GTP. The heterologous complex cannot be dissociated completely in the presence of either the 8 M urea or 8 M guanidine hydrochloride, suggesting: that EF-Ts-mt has an unusually tight interaction with E. coli EF-Tu. The EF-Tu(Eco). Ts-mt complex can be dissociated by denaturation using 2 M guanidine thiocyanate. Free EF-Ts-mt can then be purified and renatured. The refolded EF-Ts-mt is active in stimulating the activity of expressed mitochondrial EF-Tu (EF-Tu(mt)) in poly(U)-directed polymerization. Almost ail the EF-Ts-mt molecules appear to refold into a conformation which can interact with EF-Tu(mt). Protease mapping of EF-Ts-mt indicates that the first 54 residues fold into an independent domain. Analysis of deletion derivatives of EF-Ts-mt indicates that extensive regions of this factor are required for its tight interaction with EF-Tu. (C) 1997 Elsevier Science B.V.