The coupling with polypeptide synthesis of the GTPase activity dependent on elongation factor G.
The coupling with polypeptide synthesis of the ribosome-elongation factor G (EF-G)-dependent GTPase activity was studied in a highly purified system with well characterized NH4Cl-washed ribosomes which were from 55 to 67% active in poly(U)-directed polyphenylalanine synthesis. The lowest stoichiometries of total GTP hydrolysis to polyphenylalanine incorporation (2.4 to 2.8) were observed at concentrations of MgCl2 (4 to 6.5 mM) slightly lower than the Mg2+ optimum for polyphenylalanine synthesis (7 to 8 mM), in a system containing 80 mM NH4Cl or KCl. For minimal stoichiometry, the concentration of EF-G should be rate-limiting, whereas that of EF-T (EF-Tu.EF-Ts) and aminoacyl-tRNA should be in excess, since the coupling of the EF-G GTPase activity depends on ribosomes in pretranslocative state. Under this condition, the apparent Km values for GTP of GTPase activity and polyphenylalanine synthesis are identical, and they are about an order of magnitude lower than the Km of the ribosome-EF-G-dependent GTPase activity uncoupled from polypeptide synthesis. The stoichiometry was calculated without the usual correction fro GTP hydrolysis obtained in the same system lacking elongation factor T or aminoacyl-tRNA. Such a correction causes overestimation of the uncoupled EF-G GTPase activity still present in the complete system, leading to artificially low stoichiometric values.
D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010452:Peptide Biosynthesis; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010652:Phenylalanine-tRNA Ligase; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011072:Poly U; D014176:Protein Biosynthesis / Q000187:drug effects; D012270:Ribosomes / Q000378:metabolism
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D011072:Poly U; D006160:Guanosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010652:Phenylalanine-tRNA Ligase; D008274:Magnesium