Correlating eIF4A conformational dynamics with eIF4A-, eIF4B-, and eIF4G-dependence and translation efficiencies of yeast mRNAs

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Translation of eukaryotic mRNAs starts with the recognition of the 5’-cap by eIF4F, a complex of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the DEAD-box helicase eIF4A. eIF4A unwinds secondary structures in the 5’-untranslated region (5’-UTR) during recruitment of the pre-initiation complex (PIC) and scanning of the PIC towards the start codon. Its activity is coupled to a conformational cycle, which is stimulated by eIF4B and 4G, as well as the 5’-UTR itself, making eIF4A a regulatory hub in translation initiation. Transcriptome-wide studies identified sets of yeast mRNAs with hyper-dependence on eIF4A, 4B, or 4G, but the molecular basis for the different factor dependence is unclear. We have generated a series of yeast strains that enable us to prepare cell-free extracts lacking translation initiation factors, either individually or in combination. By supplementing these extracts with recombinant factors, we can now perform single-molecule FRET experiments to interrogate eIF4A conformational dynamics and in vitro translation assays to determine translation efficiencies under identical conditions, and over a wide range of concentrations of these factors. Building on this resource, we will test the following hypotheses: In single-molecule experiments and in vitro translation assays using luciferase reporters with 5’-UTRs of mRNAs showing different levels of eIF4A-dependence in vivo, we will test whether the capacity of 5’-UTRs to stimulate eIF4A conformational changes correlates with eIF4A-dependence. Using reporters with 5’-UTRs of mRNAs with different levels of eIF4B- and 4G-dependence, we will map the effects of these factors on eIF4A conformational dynamics and on translation efficiencies. We will also dissect the contributions of individual domains of eIF4B and 4G to eIF4A-dependent and -independent effects. These experiments may provide insights into reprogramming mechanisms of translation in cancer, and may define a minimal system of factors for synthetic biology approaches. Finally, we will test the hypothesis that factor dependence is encoded in the 5’-UTR. Using luciferase reporters fused to 5’-UTRs of selected eIF4A-, 4B- and 4G-dependent mRNAs, we will (1) delete individual elements in these 5’-UTRs and test for loss of factor dependence, and (2) insert these elements into the 5’-UTR of a factor-independent mRNA and test for gain of factor dependence. These studies will identify the structural features that make an mRNA eIF4A-, 4B-, or 4G-dependent, and will demonstrate whether these elements are sufficient to confer factor dependence. Collectively, these studies will define the link between eIF4A conformational dynamics and translation efficiencies, will delineate eIF4A-dependent and -independent effects of eIF4B and 4G, and will uncover the 5’-UTR elements that make an mRNA 4A-, 4B-, or 4G-dependent. The results will provide important insight into the regulation of translation and its dysregulation in disease.