UC Riverside

Faithful and efficient protein synthesis (translation) is critical for cell viability. Translation initiation involves highly coordinated dynamics that play a vital role in regulating gene expression. One of the first translation initiation factors that encounters a eukaryotic mRNA is a trimeric protein complex called eIF4F. eIF4F is composed of eIF4E, which recognizes and binds the mRNA 5ʹ cap (m7G(5ʹ)ppp(5ʹ)N, where N is the +1 nucleotide of the transcript); eIF4G, a multifunctional, multi-domain scaffolding protein, and eIF4A, an RNA helicase (Jackson et al., 2010). These factors in concert enable mRNA recognition and activation for recruitment to the ribosome, but how they coordinate dynamically to define mRNA-specific cap-recognition efficiencies on the initiation timescale remains elusive. Subsequently, the ribosome in complex with other key initiation factors interacts with the eIF4F bound mRNA and moves along the 5' untranslated region (UTR) to locate the start codon. This movement, scanning, was first proposed about 50 years ago (Kozak 1978). With indirect evidence, scanning is thought to proceed linearly with 5'-to-3' directionality (Kozak, 1978; Hinnebusch, 2011; Vassilenko et al., 2011). Although the scanning model was proposed many decades ago, the physical molecular mechanism of the motion, the extent of its variation between different mRNAs under various cellular conditions, and the regulatory role of RNA–binding proteins remain unclear due to the highly dynamic nature of the process. To address these questions, we used single-molecule fluorescence approach to understand how eIF4F dynamically interacts to contribute to cap-recognition efficiency and observe the dynamics of the ribosome complex scanning along various cellular mRNAs. Our study provides insights into these vital processes in translational control and uncovers dynamic interplay of key eukaryotic translation initiation factors.