Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

How the eukaryotic 43S preinitiation complex scans along the 5′ untranslated region (5′UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. Once engaged with the mRNA, 43S scanning occurs at >100 nucleotides per second, independent of multiple cycles of ATP-hydrolysis by RNA helicases. The scanning ribosomes can proceed through RNA secondary structures, but 5′UTR hairpin sequences near start codons drive scanning ribosomes at start codons back in the 5′ direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5′UTR structures and upstream near-cognate start codons. One Sentence Summary Direct observation of scanning eukaryotic ribosomes establishes a quantitative framework of scanning and its regulation.