Hydrolysis of Guanosine 5'-Triphosphate Associated with Binding of Aminoacyl Transfer Ribonucleic Acid to Ribosomes

Abstract

Abstract The binding of aminoacyl transfer RNA to ribosomes is a multistep reaction that is dependent on GTP and on the supernatant fraction called T. An intermediate T-GTP-aminoacyl-tRNA complex can be isolated with the GTP intact. In the work described here, the isolated complex was allowed to interact with ribosomes previously charged with polyuridylic acid and N-acetylphenylalanyl-tRNA; the phenylalanyl-tRNA moiety of the complex bound to the ribosomes, and the GTP was split into GDP and Pi. The same reaction was observed as a GTPase stimulated by T factor, aminoacyl-tRNA, message, and ribosome. This GTPase was not inhibited by sparsomycin, which blocks dipeptide formation, nor by fusidic acid, which blocks the ribosome-dependent GTPase of G factor (the supernatant factor required in addition to the T factor in the polypeptide chain elongation process). The GTPase was not affected by chlortetracycline under conditions in which the binding of aminoacyl-tRNA to the ribosomes was blocked. The 50 S ribosomal subunit alone supported the GTP hydrolysis. Both binding and GTPase paralleled each other under limiting GTP concentrations, were poorly inhibited by GDP and guanylyl 5'-methylene diphosphonate, and paralleled each other in NH4+ and Mg++ requirements and sensitivity to a sulfhydryl inhibitor. It is concluded that, although GTP hydrolysis resulted from the binding of aminoacyl-tRNA to ribosomes in a 1:1 stoichiometry, the two reactions were not tightly coupled under all experimental conditions. An improved procedure for the purification of the T and G factors is described.