The primary structure of the elongation factor G from Escherichia coli

Localization of the elongation factor g on escherichia coli ribosome

“…EF-G was isolated according to [13], its 3H-labelling was carried out by reductive methylation [ 141. The preparation and properties of the photoacAbbreviations: IEM, immune electron microscopy; azido(S)-EFG, EFC with the arylazido-residue connected to the single exposed SH-group; azido(N)-EF-G, EFC with the arylazidoresidue connected to the exposed NH,-group(s); anti-EFC, antibody directed against EFG 54 tivated azido(N)-and azido(S)-['H]EF-G derivatives are described in [9,15]; the conditions for the formation of their complexes with the 70 S ribosomes (or isolated 50 S subunits) and for photoaffinity crosslinking are the same as those in [9,15] except for the use of equimolar amounts of ribosomes and EF-G for the complex formation.…”

The primary structure of elongation factor from Escherichia coli

“…The molecular mass of the N-terminal fragment T6 is 6500, that of the following fragment T,, 7500, and that of the C-terminal fragment Ts, 25 000. Traditional chemical and enzymatic methods of polypeptide chain cleavage permitted us earlier to determine the complete primary structures of fragments Tb [4], T, [5,6] and Ts [7] involving a total of >350 amino acid residues.…”

Tyrosine Residues in the C‐Terminal Domain of the Elongation Factor G Are Essential for Its Interaction with the Ribosome

“…Elongation factor G was isolated to homogeneity from Escherichia coli by the method described earlier [5], 50-S and 3 0 3 subunits were kindly provided by Prof. A. S. Spirin. Tetranitromethane, poly(U), tRNA and chloramine T were purchased from Serva, GTP and GDP were obtained from Fluka, trypsin was from Worthington, lactoperoxidase was from Miles Lab, 2-mercaptoethanol, dithiothreitol and p-chloromercuribenzoate were from Pierce and [14C]phenylalanine, [14C]GTP, [y-32P]GTP and K[lZ5I] were purchased from Amersham.…”

“…Electrophoresis was carried out according to Weber and Osborn [20] in the modification described in [5]. Gel rods were frozen at -70 "C and radioautography was carried out at the same temperature for 12 h using a RP Royal X-ray film (Eastman Kodak).…”

“…It is known that EF-G catalyzes the ribosome-dependent GTP hydrolysis [ 1 ] and the binding site of GTP and GDP is located on EF-G [2 -41. Having characterized in detail the fragments formed at limited EF-G trypsinolysis, we arranged them along the protein polypeptide chain and localized the functionally important cysteine residue [5]. This cysteine residue can be easily modified with sulfhydryl reagents which leads to inhibition of both EF-G interaction with the ribosome and the uncoupled GTPase rcaction [l, 6 -81.…”