Abstract:
Elongation factor G (EF-G), a major translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the tip of switch II, which has been implicated in GTPase activation and GTP hydrolysis. To pinpoint the role of the H91 in EF-G's function, we have substituted it with glutamine (Q), alanine (A), glutamic acid (E) and arginine (R). All these EF-G variants were tested in biochemical assays for GTP hydrolysis, inorganic phosphate (Pi) release, mRNA translocation and dissociation of EF-G. Our results show that the mutation of H91 causes much larger defect in Pi release than in GTP hydrolysis. Moreover, we find that the rate of turnover of EF-G gets significantly reduced while translocation is only moderately affected. Thus, our results illustrate the role of the conserved histidine in EF-G's action cycle and in addition, allow us to propose a mechanism for Pi release involving H91.