Molecular mechanisms of ribosome biogenesis by force-producing enzymes

PROJECT SUMMARY Ribosomes are molecular machines made of protein and RNA that synthesize proteins. The biogenesis of new ribosomes is amongst the most energetically costly processes in the cell, accounting for ~60% of all ATP consumed. Ribosome biogenesis is dysregulated in a series of diseases known as ribosomopathies, and upregulated in proliferative cancers. However, the molecular mechanisms underlying ribosome biogenesis – how ATP-driven mechanoenzymes (force-producing enzymes) sequentially convert ribosomal precursors into mature particles – remain poorly understood. The knowledge gap is especially prominent in human systems, as most studies on ribosome biogenesis to date have focused on yeast model systems using top-down approaches. The overall vision for my research program is to fill this gap in knowledge; to mechanistically characterize the enzymology of human ribosome biogenesis, to reconstitute ribosome biogenesis steps in vitro, and to integrate the knowledge and assays developed to enable new treatments for ribosomopathies and cancer. Over the next five years, we will focus on the subset of mechanoenzymes from the AAA (ATPase associated with diverse cellular activities) superfamily. Building upon our expertise in single-molecule optical tweezers, we will develop assays to determine how these mechanoenzymes use ATP-dependent conformational changes to generate force. Using bottom-up biochemical reconstitution, we will determine how autoinhibited AAA mechanoenzymes become activated by cofactor binding, how AAA activity is targeted at specific pre-ribosomal substrates, and how force-producing conformational changes are coupled to pre-ribosomal maturation. We will furthermore use small molecule inhibitors of these AAA enzymes to stabilize transient conformational states and to identify how the several ATPase active sites present per molecule coordinate their activities. Initial assays will use yeast AAA orthologs, before advancing to the human orthologs to study how AAA cofactors unique to the human system integrate into the overall mechanisms. We will also study how ribosomopathy-associated mutations in human AAA enzymes affect their function. Ultimately, this work will characterize the core enzymology of AAA-driven ribosome biogenesis steps, will build a molecular understanding of associated ribosomopathies, and will pave the way towards developing AAA-targeting anti-cancer therapeutics.