mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation

mRNA 5′ cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F–mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E–mRNA association rates linearly anticorrelate with mRNA length. eIF4G–mRNA interaction accelerates eIF4E–mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F–mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5′ end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.