[Sequence of events in the process of factor-promoted translocation in the ribosome]

A system of translation of matrix-bound poly(U) by purified Escherichia coli ribosomes was used to obtain pre-translocation state ribosomes in columns and then to induce translocation under controlled conditions by passing the elongation factor G (EF-G) with the non-cleavable GTP analog (guanylyl-methylene diphosphonate). It has been shown that translocation in the ribosome, checked by the release of deacylated tRNA, as well as by the puromycin reaction, is induced by the attachment of EF-G (with the non-cleavable GTP analog) to the ribosome and not by its detachment. In accordance with this, the ionic conditions under which the affinity of EF-G with the GTP analog to the ribosome is increased (NH4Cl instead of KCl, a lowered ionic strength) have been also found to be more effective for translocation. On the other hand, it has been shown that the detachment (removal) of EF-G is a strict pre-requisite for the appearance of competence to bind the next aminoacyl-tRNA, and thus for a continuation of the elongation cycle. A conclusion is made that the mechanical shifts of products and substrates, such as peptidyl-tRNA and deacylated tRNA, within the ribosome in the process of translocation are promoted only by the affinity of EF-G to the ribosome and does not depend on the cleavage of GTP. On the basis of the results obtained, the following sequence of events is deduced for the process of EF-G-promoted translocation: 1) interaction of EF-G.GTP with the pre-translocative ribosome, 2) translocation displacements of products and substrates, including the release of deacylated tRNA (probably conjugated with the shift of mRNA), 3) GTP hydrolysis, 4) release of EF-G and GTP from the post-translocated ribosome.