Initiation factor IF3 is a small protein of 21 kDa containing two compact α/β domains (IF3C and IF3N) connected by a flexible lysine-rich linker. Most IF3 functions are mediated by the IF3C domain, while IF3N regulates 30S subunit binding. Bacterial initiation factor 3 (infC) is not universally found in all bacterial species but in E. coli it is required for the 30S subunit to bind to the initiation site in mRNA. IF3 is required by the small subunit to form initiation complexes, but has to be released to allow the 50S subunit to bind. IF3 attaches to the platform side of the 30S subunit, close to helices 23, 24, 25, 26 and 45 of 16S rRNA, as well as ribosomal proteins S7, S11, and S12. The IF3C domain interacts with the 30S subunit via its conserved basic residues R99, R116, R147 and R168 . A major function of IF3 is inspecting codon-anticodon pairing at the P-site during start codon selection. It accelerates the dissociation of non-canonical initiation complexes containing mismatched or incorrect tRNAs. IF3 also inspects the initiator tRNA, rejecting elongator tRNAs and it also promotes the dissociation of the 70S ribosome into subunits, providing a pool of free 30S subunits for initiation. Another key role of IF3 is repositioning mRNA on the 30S subunit from a standby site to the P-site decoding site for start codon selection. IF3 works cooperatively with IF1 and IF2 during initiation and modulates IF2 binding and enhances the fidelity of start codon selection.