In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2.
During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.
Laalami
S
S
URA 1139 du CNRS, Institut de Biologie Physico-Chimique, Paris, France.
Timofeev
A V
AV
Putzer
H
H
Leautey
J
J
Grunberg-Manago
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005838:Genotype; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D039881:Prokaryotic Initiation Factor-2; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate