C OMMUNICATION A Single Mutation in the IF3 N-Terminal Domain Perturbs the Fidelity of Translation Initiation at Three Levels Dianna Maar 1 , Dionysios Liveris 2 , Jacqueline K. Sussman 1 , Steven Ringquist 3 , Isabella Moll 4 , Nicholas Heredia 1 , Angela Kil 1 , Udo Bläsi 4 , Ira Schwartz 2 and Robert W. Simons 1 ⁎ 1 Department of Microbiology, Immunology, and Molecular Genetics, University of California-Los Angeles, 1602 Molecular Science, Los Angeles, CA 90095, USA 2 Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA 3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA 4 Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Vienna, Austria Received 21 May 2008; received in revised form 30 August 2008; accepted 5 September 2008 Available online 16 September 2008 Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal do- main (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3 Y75N protein binds 30S subunits normally, but is defective in start-codon discrimination, inhi- bition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed. © 2008 Elsevier Ltd. All rights reserved. Edited by D. E. Draper Keywords: translation initiation; IF3; accuracy *Corresponding author. E-mail address: bobs@microbio.ucla.edu. Present addresses: D. Maar, Department of Neurobiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; J. K. Sussman, Department of Microbiology, California State University, Long Beach, CA 90840, USA; S. Ringquist, Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; N. Heredia, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94551, USA. Abbreviations used: IF3, initiation factor 3; NTD, N-terminal domain; CTD, C-terminal domain; SD, Shine–Dalgarno; NIH, National Institutes of Health. doi:10.1016/j.jmb.2008.09.012 J. Mol. Biol. (2008) 383, 937–944 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.