Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation

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.

Automated summary

The substitution of a conserved histidine in the guanosine nucleotide binding domain of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity, leading to severe protein synthesis inhibition and a dominant lethal phenotype. Reduced affinities of IF2 for ribosomes and fMet-tRNA were found to suppress this lethality, indicating that the conformational changes during translation initiation are thermally driven and energy from IF2-dependent GTP hydrolysis is not necessary for successful initiation. The dissociation of the interaction between IF2 C2 and fMet-tRNA was identified as the rate-limiting step for productive P site alignment and IF2 release from the ribosome.