In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2 - Direct evidence that GTP hydrolysis is necessary for factor recycling

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a st rong negative effect on growth even in the presence of wildtype IF2. We hav e isolated both mutant proteins and performed an in vitro study of their ma in functions. The affinity of both mutant proteins for GTP is almost unchan ged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the V-max values being 11 - and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNA(f)( Met) binding to 70 S ribosomes with similar efficiencies and were as sensit ive to competitive inhibition by GDP as wild-type IF2. Formation of the fir st peptide bond, as measured by the puromycin reaction, was completely inhi bited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that , in contrast to wild-type IF2, both mutant proteins stay blocked on the ri bosome after formation of the 70 S initiation complex. This probably explai ns their dominant negative effect in vivo. Our results underline the import ance of GTP hydrolysis for the recycling of IF2.