Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties.
Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond. IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes. According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed [Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J. W. B., Hansen, H. F., Petersen, H. U., Clark, B. F. C., Kjeldgaard, M., La Cour, T. F. M., Mortensen, K. K., & Nyborg, J. (1987) Biochemistry 26, 5070-5076]. A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ. The N- and C-terminal sequences of this IF-2 peptide were characterized. Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels. This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain. However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes. It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Vachon
G
G
Laboratoire de Biologie Moléculaire, URA CNRS 1172, Université de Poitiers, France.
Laalami
S
S
Grunberg-Manago
M
M
Julien
R
R
Cenatiempo
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism
D015852:Eukaryotic Initiation Factor-2; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D001616:beta-Galactosidase; D019204:GTP-Binding Proteins