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Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

The functional properties of the two natural forms of Escherichia coli translation initiation factor IF2 (IF2alpha and IF2beta) and of an N-terminal deletion mutant of the factor (IF2DeltaN) lacking the first 294 residues, corresponding to the entire N-terminal domain, were analysed comparatively. The results revealed that IF2alpha and IF2beta display almost indistinguishable properties, whereas IF2DeltaN, although fully active in all steps of the translation initiation pathway, displays functional activities having properties and requirements distinctly different from those of the intact molecule. Indeed, binding of IF2DeltaN to the 30 S subunit, IF2DeltaN-dependent stimulation of fMet-tRNA binding to the ribosome and of initiation dipeptide formation strongly depend upon the presence of IF1 and GTP, unlike with IF2alpha and IF2beta. The present results indicate that, using two separate active sites, IF2 establishes two interactions with the 30 S ribosomal subunit which have different properties and functions. The first site, located in the N domain of IF2, is responsible for a high-affinity interaction which "anchors" the factor to the subunit while the second site, mainly located in the beta-barrel module homologous to domain II of EF-G and EF-Tu, is responsible for the functional ("core") interaction of IF2 leading to the decoding of fMet-tRNA in the 30 S subunit P-site. The first interaction is functionally dispensable, sensitive to ionic-strength variations and essentially insensitive to the nature of the guanosine nucleotide ligand and to the presence of IF1, unlike the second interaction which strongly depends upon the presence of IF1 and GTP.