Abstract
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 strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited 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 ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.
Highlights
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 strong negative effect on growth even in the presence of wildtype IF2
Purification of Mutant Forms of IF2—The E. coli translation initiation factor IF2 exists as two forms in the cell, IF2␣ and IF2, which support bacterial growth well under normal growth conditions [23]
Earlier in vitro studies, using nonhydrolyzable GTP analogs, had suggested that GTP hydrolysis may be important for the release of IF2 from the 70 S initiation complex (30 –33)
Summary
Plasmid Constructs—Plasmids overexpressing infB mutant alleles were constructed by exchanging a 1135-base pair SnaB1-SstI DNA fragment in pSL4 [21] with the corresponding fragments of pB18V400G and pB18H448E [4], giving rise to pSL4V400G and pSL4H448E. Curing of the infB transducing phage was performed as described in Ref. 21 This gave rise to strain SL679R that expresses only the short form of the factor. The strains were grown at 30 °C in LB medium in the presence of ampicillin and chloramphenicol to an OD550 ϭ 1, the temperature was shifted to 42 °C to allow expression of the mutant proteins from the thermoinducible pLpR promoters and incubation was carried on for 2 more hours.
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