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, plays a role in every step of the initiation pathway, and has emerged as a player in translational regulation. Five essential subunits compose the eIF3 complex in S. cerevisiae, constituting a core complex conserved across 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 to 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 potential rearrangement remains unknown, as do the roles of the eIF3 mRNA-entry- and exit-channel arms or their constituent subunits. We are combining powerful genome-scale technologies with ensemble and single-molecule biochemical approaches to address these fundamental questions. We have developed a recombinantly-reconstituted eIF3 complex that recapitulates the in vitro functions of eIF3, enabling for the first time the study of individual subunits or sub- complexes, as well as lethal eIF3 mutations in vitro. We will leverage this system to dissect the mechanistic contributions of the mRNA-entry-channel arm subunits (Aim 1) and the communication between this arm and the mRNA-exit-channel arm (Aim 2). Using multiple complementary genome-scale tools, we will reveal how specific mutations targeting these two arms affect the translation of specific mRNAs across the transcriptome. Together, these efforts will shed light on the mechanistic roles of eIF3 and its subunits during mRNA recruitment, and connect these to the broader biological roles of eIF3 in living cells. This new understanding will contribute to a framework for interpreting the cri...