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Updated: Aug 23, 2025

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
Published on: May 10, 2018
Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation
Jinfan Wang1, Byung-Sik Shin2, Carlos Alvarado1
1Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA.
None:
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. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward 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.
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