Mapping the Active Sites of Bacterial Translation Initiation Factor IF3 Dezemona Petrelli 1 , Cristiana Garofalo 1,2 , Matilde Lammi 1 Roberto Spurio 1 , Cynthia L. Pon 1 , Claudio O. Gualerzi 1 * and Anna La Teana 2 1 Laboratory of Genetics Department of Biology MCA University of Camerino, 62032 Camerino, Italy 2 Institute of Biochemistry University of Ancona, 60131 Ancona, Italy IF3C is the C-terminal domain of Escherichia coli translation initiation fac- tor 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) chemi- cally modified with 2,3-butanedione, the eight arginine residues of IF3C were substituted by Lys, His, Ser and Leu, generating 32 variants that were tested in vitro for all known IF3 activities. The IF3 – 30 S subunit inter- action 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. Pseudo- initiation 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 non- canonical 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 IF3C, and indicate that the different functions of IF3 rely on different mol- ecular mechanisms involving separate active sites. q 2003 Elsevier Ltd. All rights reserved Keywords: RNA – protein interactions; translation initiation; mRNA translation fidelity; Arg modification/substitution *Corresponding author Introduction Translational initiation factor 3 (IF3), an RNA- binding protein of 180 amino acid residues (in Escherichia coli), is an essential component of the bacterial translational apparatus. Upon binding to 30 S ribosomal subunits, IF3 interferes with subunit association, thereby promoting dissociation of the 70 S ribosomes and supplying a pool of free 30 S subunits. In addition, IF3 influences the kinetics of codon – anticodon interaction in the P-site-stimulat- ing 30 S initiation complex formation 1–4 and ensur- ing at the same time initiation fidelity in vitro 5,6 and in vivo. 7,8 The latter function entails the kinetic dis- crimination between correct initiation complexes and incorrect complexes such as pseudo-initiation complexes, non-canonical complexes and leader- less initiation complexes. 3,4 The pseudo-initiation complexes are those containing an elongator ami- noacyl-tRNA (with or without a blocked a-NH 2 group) bound to the 30 S subunit in response to a cognate triplet, while the non-canonical initiation complexes are those containing fMet-tRNA decoded by a triplet other than the canonical initiation codons AUG, GUG and UUG. Finally, the leaderless complexes are those containing fMet-tRNA bound to the 30 S subunit in response to the 5 0 AUG of a leaderless mRNA. 9 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved E-mail address of the corresponding author: claudio.gualerzi@unicam.it Abbreviations used: IF3, translational initiation factor 3; IF3N, and IF3C, the IF3N and C domains, respectively; wtIF3, wild-type IF3; BD, 2,3-butanedione. doi:10.1016/S0022-2836(03)00731-9 J. Mol. Biol. (2003) 331, 541–556