Intragenic suppressor mutation helps bypassing the requirement for GTP hydrolysis by IF2 during translation initiation.

Initiation factor 2 (IF2) is a GTPase implicated in translation initiation in bacteria. Its main function is the recruitment of initiator fMet-tRNA-fMet to the P site of the 30 initiation complex (30S IC). After 50S subunit joining, IF2 hydrolyzes GTP and dissociates from the 70S initiation complex (70S IC), allowing the subsequent peptide bond formation to occur. The role of the GTPase activity of IF2 has been highly controversial. IF2 mutant H448E, lacking the ability to hydrolyze GTP, confers a lethal phenotype on E. coli cells expressing it. The phenotype has been attributed to the inability of the mutant to dissociate from the 70S IC, thereby blocking the process of elongation. In this work we show that a second R847G mutation in IF2, that lowers the affinity to the initiator tRNA, can rescue toxicity caused by the H448E mutation in vivo. Biochemical characterization of the double mutant reveals two mechanisms of rescue. First, the efficiency of the 30S IC formation is significantly lowered for the mutant as compared to wild-type IF2. Hence, the endogenous wild-type IF2 is able to compete efficiently with the mutant IF2 during initiation complex formation in vivo. Second, a faster release of IF2(H448E/R847G) from the 70S IC is observed despite the lack of GTP hydrolysis, allowing translation elongation to occur. We conclude that (i) the GTPase activity of IF2 is not essential for translation initiation and (ii) a lowered affinity of IF2 for 70S IC, promoting its dissociation, can help surpass the requirement of GTP hydrolysis.