Characterization of the Ribosome-dependent Guanosine Triphosphatase Activity of Polypeptide Chain Initiation Factor IF 2

Abstract

Abstract 1. Polypeptide chain initiation factor IF 2 exhibits a potent ribosome-dependent GTPase activity. This activity has been characterized with regard to various biochemical parameters. The reaction requires the presence of both 30 S and 50 S ribosomal subunits. The stoichiometry of the reaction is GTP → GDP + Pi. The reaction requires Mg++ and is highly stimulated by NH4+ or K+. Besides GTP, dGTP is also active in the reaction while other nucleoside triphosphates are inactive. K8 for GTP is 7.7 x 10-6 m; GDP is a potent competitive inhibitor with a Ki of 2.0 x 10-6 m. No exchange between GTP and either GDP or Pi can be detected. 2. The GTPase activity of IF 2 is distinct from that of either of the peptide chain elongation factors, T and G. 3. Evidence is presented that the GTPase and fMet-tRNA binding activities exhibited by IF 2 are mediated by the same protein; the GTPase and fMet-tRNA binding activities of IF 2 cochromatograph on phosphocellulose and DEAE-Sephadex and cosediment on glycerol gradient centrifugation. Furthermore, both activities display similar sensitivities to p-hydroxymercuribenzoate, to heat, and to inhibition by GDP. 4. Under conditions of fMet-tRNA binding to ribosomes to form the 70 S initiation complex, stimulation of IF 2-dependent GTP hydrolysis (coupling) can be demonstrated.