Mechanisms of Partial Reactions of the Elongation Cycle Catalyzed by Elongation Factors Tu and G

Abstract

This chapter concentrates on pre-steady-state kinetic work, and provides evidence on how much the interpretation of kinetic results in molecular-mechanistic terms owes to structural information obtained from crystallography and cryo-electron microscopy. Our studies of elongation factor (EF-Tu) function address two main issues: (i) the elucidation of the reaction pathway to identify intermediate steps of A-site binding and (ii) the quantitative evaluation of the pathway in order to understand specificity. Based on measured rates of GTP hydrolysis and peptide bond formation, Thompson and colleagues proposed that the rate of GTP hydrolysis by EF-Tu is independent of the tRNA, thereby providing an internal kinetic standard for translational accuracy. Binding of thiostrepton to the 1070 region of 23S rRNA interferes with translocation, as it strongly inhibits Pi release, translocation, and subsequent turnover of EF-G; in contrast, EF-G binding and GTP hydrolysis are not affected. The GTPase activities of EF-Tu and EF-G intrinsically are very low and are strongly enhanced on the ribosome. Recently, the ability of isolated L12 protein to stimulate GTP hydrolysis by either EF-Tu or EF-G, has been studied. In the fusidic acid-stabilized complex of EF-G with the ribosome, the α- sarcin stem is close to position 196 in the G domain of EF-G, which lies just above the GTP binding site, while the α-sarcin loop region is in the vicinity of position 650 in domain 5 of the factor.