UC Riverside

Regulated protein synthesis is mediated by an intricate ensemble of protein-protein and -mRNA interactions, and is pivotal in allowing for flexible, rapid changes in gene expression in response to cellular conditions. Recognition of the mRNA 5′ m7GTP cap structure by a heterotrimeric factor, eIF4F, is cornerstone step in translation initiation for most eukaryotic mRNAs. The eIF4F complex includes the cap-binding protein, eIF4E, and a “scaffold” protein, eIF4G. Meanwhile, the mRNA 3′ poly(A) tail is bound by a poly(A)-binding protein (Pab1p in Saccharomyces cerevisae). Decades of research led to the proposition of the “closed-loop” model, where simultaneous interactions between eIF4F•mRNA and eIF4F•Pab1p circularize the mRNA, promoting enhanced mRNA stability and synergetic enhancement of translation. However, the kinetic scheme of how the closed-loop factors coordinate on full-length mRNA has not been directly validated experimentally. A barrier to progress in our knowledge of translation initiation is the inherent complexity and dynamism of translation initiation, as all these activities must be coordinated to recognize the mRNA and promote ribosome loading efficiently. Here we attempt to disseminate the interplay between mRNA identity – sequence and structure – and the kinetics of the closed-loop interactions on an initiation timescale. Mounting evidence proposes mRNA end-to-end distance is an intrinsic property of mRNA, and the universality of the closed loop model has come into light in recent years with the discovery that different mRNAs exhibit variable closed-loop dependence in vivo, and also appear to be selected for translation initiation with differential efficiencies. An emerging predictor of in vivo enrichment of circularization factors is coding sequence (CDS) length, which encompasses both nucleotide composition and secondary structures of mRNA. Thus, it’s important to understand the interplay between these elements and to the dynamics that modulate translation initiation efficiency. I hypothesize that mRNA-specific elements control dynamics that differentiate the efficiency of translation initiation for varying mRNAs. Single-molecule methods are well suited to address these fundamental gaps in our knowledge, as they allow for the direct tracking of labeled translation initiation factors as they bind and dissociate. We leverage a single-molecule fluorescence assay to study the dynamics of the closed-loop model, using reconstituted yeast eIF4E, eIF4G and Pab1p on full-length mRNAs. We applied this assay to address how factor-factor and mRNA-factor interactions modulate cap-binding kinetics on individual transcripts in real-time. Our aim was to establish a kinetic model for the closed-loop interactions, which elaborates on how factor-factor interactions modulate cap binding rates. These results will contribute to development of a mechanistic model for the closed-loop interactions in translation initiation, which can be applied to understand a fundamental element of cell biology – translation control in health and disease.