PROJECT SUMMARY/ABSTRACT Translation initiation establishes the reading frame for protein synthesis and dedicates the translational machinery to the production of specific mRNAs depending on cellular need. Not surprisingly, translation initiation is the rate-limiting and most highly regulated phase of translation. Misregulation of translation initiation is a causative factor in human cancers; altered levels of translation initiation factors are implicated in cancer development and progression and specific steps of the initiation pathway are altered to enable the rapid proliferation of cancerous cells. Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these initiation factors and plays a role in every step of the initiation pathway. Five essential subunits comprise the eIF3 complex in S. cerevisiae, constituting a core complex conserved in other eukaryotes. Altered expression of each of these subunits provokes cancer development or progression, and several subunits have emerged as proto-oncogenes or therapeutic targets. However, a mechanistic framework for understanding these causal links to cancer does not yet exist. In fact, fundamental gaps in our understanding of eIF3 and its mechanistic contributions to translation initiation remain. In particular, how eIF3 contributes to mRNA recruitment by the ribosome remains a mystery. Recent high-resolution structures have revealed eIF3 binding to the small ribosomal subunit and projecting arms near the mRNA-entry- and exit channels through which mRNA enters and exits the ribosomal pre-initiation complex (PIC). These structures also suggest that a dynamic rearrangement of the eIF3 entry-channel arm occurs in response to mRNA binding by the PIC. However, the mechanistic role of this rearrangement and the specific roles of the individual subunits of eIF3 remain unknown. We are combining powerful genome-scale and in vitro biochemical approaches to address these fundamental questions. Using ribosome profiling, we have identified specific mRNAs whose translation is hypersensitive to disruption of the entire eIF3 complex or its entry-channel arm and will dissect the mechanistic origins of this sensitivity using a reconstituted in vitro system that recapitulates key initiation events (Aim 1). We will also leverage a library of previously-characterized functional variants of eIF3 and the small ribosomal subunit to illuminate the mechanistic collaboration between the entry-channel arm and the PIC using an assay that probes the stability of mRNA binding in the entry- and exit-channels of the PIC (Aim 2). Finally, we will make use of an existing approach for recombinant expression and purification of eIF3 to enable the first in vitro investigation of lethal mutations to the eIF3 complex, thereby removing a key challenge in the field (Aim 3). Together, these efforts will shed light on the mechanism of mRNA recruitment and the role of eIF3 and its subunits. This new understanding wi...