UC Riverside

Translation initiation is a critical step of protein synthesis across all domains of life. In eukaryotes, the first step of this process is formation of a messenger ribonucleoprotein particle between the messenger RNA and the initiation factor complex named eIF4F. This process is required for canonical translation initiation in eukaryotes and is followed by recruitment of the ribosome and downstream initiation processes to form an elongation-competent ribosome. Here, I extensively characterized the formation of the eIF4F complex on yeast mRNAs, using a combination of in vitro reconstitution of this complex and single molecule techniques. A single-molecule FRET-based assay utilizing fluorescently labeled eIF4E and mRNA was used to measure the basal rates of formation and dissociation of the complex between the cap binding subunit eIF4E and using model mRNAs transcribed in vitro, in addition to pools of mRNAs extracted from cells. The eIF4E-mRNA interaction is highly dynamic and dependent on mRNA features, with the rate of binding causing variability between mRNAs. Characterizing the eIF4E binding of different mRNAs revealed that the binding is controlled by mRNA cap-proximal structure and length. The contributions of other eIF4F subunits to cap binding by eIF4F (eIF4A and eIF4G) were elucidated on different mRNAs. eIF4A and eIF4G can both enhance the formation of this complex by accelerating the rate of formation, whereas eIF4G also stabilizes the complex, increasing the bound lifetime of eIF4E to the mRNA cap. Further studies truncating the eIF4G peptide demonstrated that the multiple RNA-binding domains are required to increasing the bound lifetime, whereas the first domain is sufficient to accelerate the rate of formation of the eIF4E-mRNA complex. ATP further increases the rate of formation and lifetime of the complex. Lastly, three and four-color single molecule fluorescence assays were developed, fluorescently labeling eIF4A, and eIF4G. Utilizing these, we revealed the order of disassembly of these factors on mRNA. eIF4E (together with eIF4G) is likely to be ejected from the mRNA cap after formation of the eIF4F complex, which appears to be similar between mRNAs. An approach for automating data analysis on our custom single molecule imaging platform is also discussed. Ultimately, the study elucidated the eIF4E cap binding dynamics on model mRNAs and populations, and elucidated the contribution of other eIF4F subunits, as well as the mechanism of eIF4F assembly and the ATP dependence.