Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

C.O Gualerzi,M Severini,R Spurio,A La Teana,C.L Pon

doi:10.1016/s0021-9258(18)55305-9

Abstract

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule.

Highlights

From the $Laboratory of Genetics, Department of Biology, University of Camerino, 1-62032 Camerino, Italy and the (Max Planck-Institut fuerMolekulare Genetik, W-1000Berlin (Dahlem) 33, Federal Republic of Germany
This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher K, for GTP and the same Vmaxas compared with intact IF2
We have used C- and G-fragments of IF2 prepared by both proteolysis and genetic engineering and in no circumstance found any difference in their structural and functional properties, it should be mentioned that theresults shown here were obtained exclusively using Cfragment purified following preparative proteolysis and Gfragment purifiedfrom anextract derivedfrom hyperproducing cells

Summary

MATERIALS AND METHODS

Proteins shows that they have approximately the same size Buffers-The buffers used were the following: buffer A Shortened IF2 consisting of the first 514 amino acids was overpro- BindingofIF2andIF2Fragments to 30 and 50 S Ribosomal duced by cells bearing this construct From this con- Subunits-The experiments were carried outessentially as previously struct, the -1.2-kb BanI-ClaI fragment was excised and subcloned described (Pon et al, 1985), except that nativeu, nlabeled protein was into pEV2 (Crow1et al., 1985) via ClaI sites and blunt-endligation of used in place of the i n vitro labeled protein. After 10-fold dilution with buffer A to reduce the NH4C1concentration to 100 mM, this fraction was loaded on a phosphocellulose column (3.5 X 14 cm) equilibrated with the same buffer containing 100 mM NH4C1.The progress of the purificationof the G-fragment was followed by SDS-PAGE (12.5% acrylamide) as indicated above. Seventy p1 of supernatant were withdrawn from the topof each tube andsubjected to SDS-PAGE analysis followed by densitometric determination of the amountof protein remaining unbound

RESULTS

DISCUSSION

I I