2024 Theses Doctoral

Investigating the role of eIF4F during translation initiation

Gentry, Riley

A fundamental, unanswered question in molecular biology is how cells decide which messenger RNAs to translate into proteins at what time. Our current understanding of the protein- and nucleic-acid-features which drive this regulation are the result of decades of genetic, biochemical, and biophysical experiments. One of the earliest factors that these studies showed to be crucial to the mRNA selection is the heterotrimeric protein complex, eIF4F. Yet, despite decades of investigation, the underlying mechanism for how eIF4F functions remains elusive. This is, in part, due to the fact that translation initiation is a multi-component, multi-step process and isolating individual sub steps for careful investigation has proven challenging. Single-molecule methods provide one answer for addressing this heterogeneity: by observing molecules one at a time, individual sub steps can be directly observed even in a heterogenous background of asynchronized molecules.

The primary focus of this thesis is the application of single-molecule fluorescence methods to allow for the unambiguous investigation of eIF4F activity. In chapter 2, novel slide preparation methods were developed which allow for the routine, high-quality data collection at concentrations of fluorophores an order of magnitude above what is conventionally possible. In Chapters 3 and 4, site-specifically fluorophore-labeled variants of each eIF4F subunit are generated. Using these fluorophore-labeled eIF4F variants, experiments are performed on microscope slides which take advantage of the advances in chapter 2 in order to directly visualize the behavior of eIF4F leading up to and during mRNA selection. Collectively, this thesis culminates in new models for how eIF4F recognizes the cap of the mRNA, and its fate after the mRNA is loaded into the ribosome and highlights new avenues by which eIF4F may be regulated.