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 forma tion are considered to be essential for translation initiation in Esch erichia coil in order to clarify the role of certain amino acid residu es believed to be crucial for the GTP hydrolytic activity of E. coil I F2, we have introduced seven single amino acid substitutions into its GTP-binding site (GIy for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were ex pressed in vivo in physiological quantities and tested for their abili ty to maintain the growth of an E. coil strain from which the function al 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 emphas ize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in tran slation initiation. These findings are discussed in relation to a prev iously proposed theoretical model for the IF2 G-domain.