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

摘要

When bovine mitochondrial elongation factor Ts (EF-Tsmt) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli EF-Tu (EF-TuEcoTsmt). This complex is active in poly(U)-directed polymerization and this activity is inhibited by kirromycin. The EF-TuEcoTsmt 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-TuEcoTsmt 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-Tsmt has an unusually tight interaction with E. coli EF-Tu. The EF-TuEcoTsmt complex can be dissociated by denaturation using 2 M guanidine thiocyanate. Free EF-Tsmt can then be purified and renatured. The refolded EF-Tsmt is active in stimulating the activity of expressed mitochondrial EF-Tu (EF-Tumt) in poly(U)-directed polymerization. Almost all the EF-Tsmt molecules appear to refold into a conformation which can interact with EF-Tumt. Protease mapping of EF-Tsmt indicates that the first 54 residues fold into an independent domain. Analysis of deletion derivatives of EF-Tsmt indicates that extensive regions of this factor are required for its tight interaction with EF-Tu. ? 1997 Elsevier Science B.V. All rights reserved.