Ribosomal translocation assayed by the matrix-bound poly(uridylic acid) column technique

Abstract

The system of translation of cellulose-bound poly(uridylic acid) by Escherichia coli ribosomes has been used for preparation of pre-translocation state ribosomes in columns. Translocation has been induced by passing the elongation factor G (EF-G) with GTP or its non-cleavable analog (guanosine 5'-[beta, gamma-methylene]triphosphate) through the column. A method for quantitative comparison of translocation rates, and thus of effectiveness of translocation-inducing factors, has been proposed. The method is based on an analysis of the profile of deacylated tRNA elution resulting from translocation in the column. The determination of the rate and amount of translocation has been done under different ionic conditions. It has been found that the Mg2+ concentration is a decisive factor of translocation in vitro: at high Mg2+ (30 mM) EF-G cannot induce translocation, and lowering the Mg2+ concentration (to 10 mM) is required for EF-G to become effective. Sufficiently low Mg2+ (3 mM) itself has proved to induce fast and complete translocation, without EF-G.