Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

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Abstract
A complex between elongation factor Tu (EF-Tu), GTP, Phe-tRNA, oligo(U), and the 30S ribosomal subunit of Escherichia coli was formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent on the presence of both the aminoacyl-tRNA and the cognate mRNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex (30S-EF-Tu complex) causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome. These data also suggest that crucial parts of the 70S binding site for the EF-Tu complex may be on the 30S ribosomal subunit in locations where they do not significantly interfere with subunit association. Because this 30S-EF-Tu complex can be isolated from sucrose gradient, 3-dimensional immune mapping of the EF-Tu binding site directly on 30S subunits is feasible.