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 was 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 T. 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 the two cysteine residues at position 190. The modification of Cys19O by 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 protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-TU . EF-Ts)2 complex, a ternary EF-TU . EF-Ts2 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 capable of forming homodimers.
Blank
J
J
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Nock
S
S
Kreutzer
R
R
Sprinzl
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics*; D014357:Trypsin
D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C023512:elongation factor Ts; D014357:Trypsin; D010444:Peptide Elongation Factor Tu