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) x Ts(mt)). This complex is active in poly(U)-directed polymerization and this activity is inhibited by kirromycin. The EF-Tu(Eco) x 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) x 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) x 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 all 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.
Xin
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Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.
Leanza
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K
Spremulli
L L
LL
D000818:Animals; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism*; D017353:Gene Deletion; D006150:Guanine Nucleotides / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011489:Protein Denaturation; D011728:Pyridones / Q000494:pharmacology; D014357:Trypsin
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D011728:Pyridones; C023512:elongation factor Ts; D014357:Trypsin; D010444:Peptide Elongation Factor Tu; C008536:mocimycin