Unraveling the landscapes and regulation of scanning, leaky scanning, and 48S initiation complex conformations

Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1 inhibition impact. We provide global views of scanning and leaky scanning, uncovering a central role of eIF4G1-eIF1 in their regulation. We confirm AUG context importance, with non-leaky genes featuring a Kozak context and cytosine at positions −1 and +5. Capturing 48S complexes associated with eIF1, eIF4G1, eIF3, and eIF2 through selective TCP-seq revealed that the eIF3-scanning ribosome is highly vulnerable to eIF4G1-eIF1 inhibition, and eIF1 tends to dissociate upon AUG recognition. Initiation-site footprint analysis revealed a class spanning −12 to +18/19 from the AUG, representing the entire 48S and enriched with eIF2, eIF1, and eIF4G1, indicative of early initiation. Another eIF3-dependent class extends up to +26 and exhibits reduced eIF2 and eIF4G1 association, suggesting a late/alternative initiation complex. Our analysis provides an overview of scanning, initiation, and evidence for conformational rearrangements in vivo.