Mechanism of ribosomal translocation: Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis

Abstract

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a k app value greater than 30s −1 (20 °C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNA Phe, but not N-acetylphenylalanyl-tRNA Phe ( N-AcPhe-tRNA Phe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNA Phe in the P site, is occupied by N-AcPhe-tRNA Phe, the burst of P i discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNA Phe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.