Probing Structural Dynamics of the Closed-Loop Model in Translation Initiation

Abstract

During translation initiation, communication between the mRNA 5′ and 3′ ends takes on a pivotal role for gene expression regulation. Binding of eukaryotic initiation factor 4F (eIF4F) at the 5′ cap, and of poly(A)-binding protein (Pab1p in yeast) to the 3′ poly(A) tail, is thought to bring the mRNA ends close together, due to a physical eIF4F-Pab1p interaction. Detailed studies have denoted that this “closed loop” arrangement has important functional consequences, including enhanced mRNA stability and synergistic enhancement of translation. A combination of biochemical, genetic, and structural studies from the past four decades have delineated the roles of each closed-loop factor; however, our understanding of the molecular architecture of this multiprotein-mRNA complex remains mostly limited to static snapshots. in addition, little is known on how this architecture is dynamically leveraged to promote and regulate translation initiation. To address this problem, we have developed single-molecule fluorescence assays to visualize when closed-loop factors are bound to the mRNA, while simultaneously probing mRNA end-to-end proximity (when mRNA ends are within ∼ 7 nm of each other). We observed the interaction of recombinant, fluorescently-labeled S. cerevisiae eIF4E and full-length mRNAs fluorescently labeled at their 3′ ends. Our results indicate that mRNA ends are intrinsically close in the absence of factors in vitro, and that a dominant role of Pab1p is to enhance eIF4F-mRNA association kinetics. We are applying this approach to understand the role of mRNA sequence, length, and secondary structure, and our data will provide new insights into the role of the closed loop in translational control.