Our lab studies one of life’s most fundamental processes: how cells start making proteins. The first step – translation initiation – is tightly regulated to ensure proteins are made in the right quantity at the right time and place. Misregulation of this step is a hallmark of many human diseases, including cancer, neurodegenerative disorders, and inflammatory syndromes. Yet, despite decades of research, we still lack a clear understanding of how translation initiation is carefully controlled.
We use cutting-edge single-molecule approaches to watch translation begin in real time. We reconstituted human translation initiation in vitro using highly-purified components and engineered versions of components that can be specifically labeled with fluorescent tags. When used in tandem with our powerful microscope, we directly monitor individual ribosomes and key helper proteins as initiation occurs in real time. We analyze how the machinery selects mRNA templates for translation, scans for translation start sites, recognizes the correct start site, and assembles into a complete ribosome that can synthesize a protein. This allows us to capture fleeting intermediates and define critical checkpoints with millisecond precision. By also combining high-throughput cellular and structural approaches, we thus build dynamic, high-resolution models invisible to most other approaches.
Our lab has made key discoveries that illuminate how translation initiation is regulated and misregulated in human disease.
We first defined how the large ribosomal subunit is recruited to initiation complexes poised at a translation start site. By combining single-molecule assays with cryo-EM, we demonstrated that two universally conserved factors, eIF1A and eIF5B, work together to guide this step with remarkable precision. These proteins are frequently overexpressed or mutated in cancer, and our findings offer a new framework for understanding how their dysregulation may promote abnormal protein synthesis. More recently, we revealed that recognition of the translation start site is governed by a tunable ‘proofreading’ mechanism: the initiation complex dynamically toggles between eIF1 and eIF5 binding, which respectively reject or commit to potential start sites. This balance is sensitive to changes in factor concentration and start codon identity. Our findings help explain how cells rapidly modulate fidelity and how cancers may exploit alternative start sites to reshape the proteome.
We are now working to uncover how translation initiation is controlled through dynamic processes that occur at both ends of an mRNA. One area explores how initiation complexes scan along the mRNA and make high-fidelity decisions about where to begin protein synthesis. We aim to understand how these decisions are fine-tuned through rapid molecular switches and structural rearrangements, which may go awry in cancer and other diseases. A second area investigates how a regulatory hotspot at the opposite end of the mRNA – far from the start site – controls initiation. This long-range regulation maintains cellular homeostasis and plays outsized roles in neurological, developmental, and immune-related disorders. By directly visualizing these processes in real time, we are building a framework to explain how translation is precisely orchestrated, which could inform design of new therapeutics to treat diseases.
We are grateful for our funding support from the National Institutes of Health (NIGMS), the Damon Runyon Cancer Research Foundation, the Rita Allen Foundation, and the Fred Hutch Obliteride.