Characterization of the translational start site for IF2β, a short form of Escherichia coli initiation factor IF2

The gene for initiation factor IF2, infB, represents one of the few examples in Escherichia coli of genes encoding two protein products in vivo. In a previous work, our group showed that both forms of IF2 (α and β) are closely related and may arise from two independent translational events on infB mRNA. Unambiguous mapping and rigourous determination of the nature of the initiation triplet for IF2β, the smaller form of IF2, is critical for future mutagenesis of this codon, required for investigating the biological importance of both IF2α and IF2β. Three types of experiments were carried out. First, a 77-bp deletion was created at the beginning of the structural gene leading to premature termination of IF2α synthesis. Under these conditions, IF2β is still formed. Second, various Bal31 digests of infB containing the 77-bp deletion were fused to lacZ. Any synthesis of a fused protein with β-galactosidase activity should reflect the occurrence of an initiation event on the messenger corresponding to this DNA segment. It was consequently possible to locate the IF2β initiation site within an 18-base region containing an in-phase GUG codon. Third, to avoid any artefactual reinitiation event possibly occurring under our experimental conditions, we fused to lacZ an infB fragment devoid of IF2α start sequences but containing genetic information for this 18-base region. A hybrid protein with β-galactosidase activity was synthesized. Moreover, its NH2-terminal amino acid sequence coincided with that of IF2β, demonstrating that GUG, located 471 bases downstream from the IF2α external start codon, is the internal start codon for the shorter form of IF2.