Non-canonical Binding Site for Bacterial Initiation Factor 3 on the Large Ribosomal Subunit

Canonical translation initiation in bacteria entails the assembly of the 30S initiation complex (IC), which binds the 50S subunit to form a 70S IC. IF3, a key initiation factor, is recruited to the 30S subunit at an early stage and is displaced from its primary binding site upon subunit joining. We employed four different FRET pairs to monitor IF3 relocation after 50S joining. IF3 moves away from the 30S subunit, IF1 and IF2, but can remain bound to the mature 70S IC. The secondary binding site is located on the 50S subunit in the vicinity of ribosomal protein L33. The interaction between IF3 and the 50S subunit is largely electrostatic with very high rates of IF3 binding and dissociation. The existence of the non-canonical binding site may help explain how IF3 participates in alternative initiation modes performed directly by the 70S ribosomes, such as initiation on leaderless mRNAs or re-initiation.