Assembly of the PIC, scanning and start codon selection in WT cells. (i) eIF1 and the scanning enhancer (SEs) elements in the CTT of eIF1A stabilize an open conformation of the 40S subunit to which the TC loads rapidly. (ii) The 43S PIC in the open conformation scans the mRNA for the start codon with Met-tRNAi bound in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis by the TC to produce GDP•Pi, but release of Pi is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1•40S association and the open conformation. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1 and the CTT of eIF1A. Movement of eIF1 and the eIF1A CTT away from the P site disrupts eIF1's interaction with eIF2β-NTT, and the latter interacts with the eIF5-CTD. eIF1 dissociates from the 40S subunit, and the eIF5-NTD disengages from eIF2 and interacts with the eIF1A CTT instead, dependent on the SE elements, thereby facilitating Pi release from eIF2. The eIF5-CTD moves into the position on the 40S subunit previously occupied by eIF1 and blocks reassociation of eIF1. (Below) Arrows summarize that eIF1 and the eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from Hinnebusch and Lorsch, 2012; Nanda et al., 2013).