ELONGATION-FACTOR TS FROM THERMUS-THERMOPHILUS - OVERPRODUCTION IN ESCHERICHIA-COLI, QUATERNARY STRUCTURE AND INTERACTION WITH ELONGATION-FACTOR TU

The gene encoding the elongation factor Ts from Thermus thermophilus w as sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from ir: thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures , which facilitates its separation from E. coli proteins. Purified T. thermophilus EF-Ts forms a homodimer with a disulfide bridge between t he two cysteine residues at position 190. The modification of Cys190 b y iodoacetamide affects neither the dimerization nor the ability of EF -Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF-Ts, but not in prot ein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the qua ternary (EF-Tu . EF-Ts)(2) complex, a ternary EF-Tu . EF-Ts(2) complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capabl e of forming homodimers.