PROJECT SUMMARY (30 lines) Initiation of translation in eukaryotic organisms is rate limiting and highly regulated. The process involves a score of protein factors guiding 40S subunits to the 5’ untranslated region (5’UTR) of an mRNA, and subsequent directional scanning to the first start codon, followed by 60S subunit joining and transition to elongation. The mechanistic basis for this process remains unclear. During the prior funding period we developed single-molecule approaches to provide a real-time dynamic perspective to yeast translation initiation. Here we build on our methodological developments over the past funding period to provide combined structural and dynamic view of how initiation occurs in yeast and humans. In Aim 1 we focus on how 40S subunits are prepared and then join the 5’ end of an mRNA and leverage our labeled factors to understand the dynamics by which the eIF4F complex guides this process in collaboration with mRNA and helicases; we then will explore the directional scanning to the start codon and delineate the role of 5’ UTR length sequence and structure and how 3’ terminal proteins and RNA modulate the process. In Aim 2, we investigate how start codon recognition occurs through 60S subunit joining and the transition to elongation. We determine the conformation/composition signals in start codon recognition, and why eIF5B induced transition to elongation is slow, and whether initiation factors linger during elongation. We will correlate slow events with known in vivo measurements and probe the consequences of queuing in scanning. In Aim 3, we investigate alternative initiation pathways, such as leaky scanning, near cognate start sites and upstream open reading frame translation, termination and re-initiation. For all aims, we merge dynamic and biochemical investigations with structural characterization of intermediates by cryoEM guided by our single-molecule observations. The proposed research is buttressed by strong collaborations on the various systems to support biophysical and structural analysis, reagent preparation, and in vivo correlation. The results of this proposal will provide deep and broad view of the interplay of mRNA sequence and structure and the pathways of translation initiation, providing a foundation to understand translational control and its mis-regulation in disease.