Mapping the active sites of bacterial translation initiation factor IF3

lF3C is the C-terminal domain of Escherichia coli translation initiation factor 3 (IF3) and is responsible for all functions of this translation initiation factor but for its ribosomal recycling. To map the number and nature of the active sites of IF3 and to identify the essential Arg residue(s) chemically modified with 2,3-butanedione, the eight arginine residues of lF3C were substituted by Lys, His, Ser and Leu, generating 32 variants that were tested in vitro for all known JF3 activities. The JF3-30 S subunit interaction was inhibited strongly by substitutions of Arg99, Arg112, Arg116, Arg147 and Arg168, the positive charges being important at positions 116 and 147. The 70 S ribosome dissociation was affected by mutations of Arg112, Arg147 and, to a lesser extent, of Arg99 and Arg116. Pseudoinitiation complex dissociation was impaired by substitution of Arg99 and Arg112 (whose positive charges are important) and, to a lesser extent, of Arg116, Arg129, Arg133 and Arg147, while the dissociation of noncanonical 30 S initiation complexes was preserved at wild-type levels in all 32 mutants. Stimulation of mRNA translation was reduced by mutations of Arg116, Arg129 and, to a lesser extent, of Arg99, Arg112 and Arg131 whereas inhibition of non-canonical mRNA translation was affected by substitutions of Arg99, Arg112, Arg168 and, to a lesser extent, Arg116, Arg129 and Arg131. Finally, repositioning the mRNA on the 30 S subunit was affected weakly by mutations of Arg133, Arg131, Arg168, Arg147 and Arg129. Overall, the results define two active surfaces in lF3C. and indicate that the different functions of IF3 rely on different molecular mechanisms involving separate active sites. (C) 2003 Elsevier Ltd. All rights reserved.