From one end to the other: dynamics of human translation initiation and its control

点击翻译按钮获取中文摘要

Project Summary Protein synthesis begins via a multi-step and highly-regulated process that culminates with a ribosome poised at a start site on a messenger RNA (mRNA). Loss of control is broadly implicated in human disease, including cancers, developmental disorders, neurological diseases, and viral infections. Since translation initiation is rate limiting, an important regulatory strategy involves multi-protein complexes recruited to the opposite end of the mRNA. Recruited regulatory proteins directly enhance or inhibit assembly of the initiation machinery on the mRNA, thereby tuning protein production up or down. Many molecular mechanisms that underlie translation initiation and its long-range control remain unclear. Current paradigms rely on analyses of complexes that are stable for minutes to hours, as the intrinsic dynamics challenge approaches in bulk solutions. Here, I build off my initial postdoctoral research to track the human translation initiation machinery, mRNA, and regulatory complexes as they interact using single-molecule spectroscopy and purified components in vitro, which I complement with structural analyses. In Aim 1 (K99), I focus on how the ribosomal subunits are recruited to and load onto an mRNA, and determine how these landmark initiation events are dictated by mRNA features. In Aim 2 (K99), I examine how the final initiation steps and the transition into active protein synthesis are coordinated and governed by a universally-conserved GTPase, eIF5B. In Aim 3 (R00), I leverage the obtained training and expertise to examine how translation initiation is controlled via the 3’-end of the mRNA by the CCR4-NOT complex, a major regulator with human-health relevance. As my preliminary data demonstrate, my strategy will overcome previous roadblocks to provide a dynamic view of key molecular branchpoints that underlie translation initiation, reveal how they are targeted for control, and may define molecular bases of disease. Aided by strong collaborations and my mentoring team, the proposed research and training plan will provide me with new conceptual and experimental expertise in structural biology and biophysics and enhance my professional development. Together, this proposal will serve as a strong foundation as I transition into independence and continue my investigation of translational control and how it goes awry in human disease.