PROJECT SUMMARY (30 lines) Translation is the endpoint of the central dogma and point of temporal and spatial regulation in gene expression. Biochemical, biophysical and structural methods have outlined the general steps of translation, providing a menu of key factors, structures of ribosomes and complexes, and kinetics for the essential steps of initiation, elongation and termination/recycling. Nonetheless, the mechanisms of key steps such as initiation, elongation and termination, and how they are regulated by RNA structures, modification or regulatory proteins remains unclear. A key challenge is that translation is highly dynamic, involving conformational and compositional changes throughout and following heterogeneous mechanistic pathways. During prior funding periods supported by the grants that we will merge in this MIRA, we have developed single- molecule approaches and reagents that observe translation in real time. We combine these dynamic methods with cryoEM structures to gain a temporal and detailed mechanistic view of the process. Our proposed research focuses on key areas translational control: how initiation is achieved in higher organisms—here the pathway by which a small (40S) ribosomal subunit is bound to a mRNA and recognizes a start—will be determined in both yeast and humans, and we will explore how mRNA structure, protein binding and modified nucleotides change the process. We will investigate how long-range RNA interaction between 5’ and 3’ ends of mRNAs may be critical for basal translation initiation and its control. In elongation, we will continue to explore recoding events and co-translational protein folding and develop methods to watch translation elongation in eukaryotic organisms. We will explore the role of ribosomal stalling/pausing and eventual shunting into ribosomal quality control pathways. Finally, we will understand the pathways by which correct stop codons are recognized and ribosomes recycled and determine how correct vs premature stop codons are distinguished in the nonsense mediated decay pathway. Our research leverages decades of reagent and methods development, and a wonderful group of collaborators to explore translational control, and its central linkage to human health and disease.