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 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.
Luchin
S
S
UPR9073 du CNRS, Institut de Biologie Physico-Chimique 13, rue Pierre et Marie Curie, 75005 Paris, France.
Putzer
H
H
Hershey
J W
JW
Cenatiempo
Y
Y
Grunberg-Manago
M
M
Laalami
S
S
D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2
D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D020558:GTP Phosphohydrolases