Mutations that alter initiation codon discrimination by Escherichia coli initiation factor IF3

This work describes the isolation of mutations in infC, the structural gene for IF3, using different genetic screens. Among 21 mutants characterised, seven were shown to produce stable variant IF3 proteins unable to fully com plement a strain carrying a chromosomal deletion of the infC gene. The muta nts were also shown to be unable to normally discriminate against several n on-canonical initiation codons such as AUU and ACG. The two mutants with th e strongest complementation or discrimination defects carry changes in the C-terminal domain of IF3, which is responsible for the binding of the facto r to the 30 S ribosomal subunit. We show that the first mutant has an expec ted decreased but the second an unexpected increased capacity to bind the 3 0 S subunit. The in vivo defects of the second mutant are explained by its capacity to bind unspecifically to other targets, as shown by its increased affinity for the 50 S subunit, which is normally not recognised by the fac tor, interestingly, this mutant corresponds to a change of an acidic residu e that might play a negative discriminatory role in preventing interactions with non-cognate RNAs, as has been reported for acidic residues of aminoac yl-tRNA synthetases shown to be involved in tRNA recognition. (C) 1999 Acad emic Press.