Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.
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.
Gualerzi
C O
CO
Department of Biology, University of Camerino, Italy.
Severini
M
M
Spurio
R
R
La Teana
A
A
Pon
C L
CL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D020558:GTP Phosphohydrolases