Translation initiation is a crucial regulatory step in protein synthesis. Three factors (IF1, IF2 and IF3) are involved during this phase and are responsible for the selection and the quantity of the protein produced. IF3 manages the fidelity of translation and acts upon various kinetic regulatory checkpoints. Nevertheless, the relation between this function and the conformational variability of the ribosome-bound factor is unknown. We used intramolecular FRET and rapid kinetics to study the structural changes of IF3 during the formation of the initiation complex. The binding of IF1 and IF2 results in a reduced distance between IF3’s domains, while the binding of ARNt provokes an increase in the distances. The velocities of these movements were between 0.55 and 4.87 s-1. Kinetic assays in absence of the N-terminal domain resulted in a decreased binding affinity of the initiator ARNt an...