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.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

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Çetin B, O'Leary SE

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