Defining mechanisms that underlie well-controlled translation initiation

Fred Hutchinson Cancer Center, Seattle WA

Investigators

Abstract

PROJECT SUMMARY Translation initiation determines the identity and amount of a synthesized protein. Dysregulated initiation occurs broadly in disease, including cancers, inflammatory diseases, and developmental and neurological disorders. The process requires a dozen factors and involves multiple coordinated steps that occur in under a minute. A key commitment step occurs when the initiation machinery selects a translation start site on an mRNA, which canonically is an AUG codon. Proper selection requires single-nucleotide precision to maintain the reading frame and avoid synthesis of truncated and potentially toxic peptides. Yet, genetic, biochemical, and genome-wide studies indicate that translation initiation also occurs at non-AUG start sites. These alternative start sites have critical roles during stress responses. Their differential usage becomes dysregulated in cancers and other human diseases. While we know eIF1, eIF5, and eIF1A play key roles, the molecular mechanisms that not only ensure high-fidelity recognition of the start site, but also enable flexible use of non-AUG start sites remain unclear. A major roadblock has been the inability to monitor the three proteins as they interact and rearrange in real time during initiation. In my postdoctoral research, I pioneered in vitro single-molecule spectroscopy approaches to analyze human translation initiation as it occurs in real time. During the ESI MIRA phase, we will use my versatile and powerful system to examine the molecular events that underlie tunable recognition of the translation start site. In Project 1, we will examine how the initiation machinery flexibly discriminates AUG and non-AUG start sites by developing new FRET-based single-molecule assays to monitor eIF1 and a key conformational rearrangement. In Project 2, we will define how initiation complexes commit to a start site by developing a new FRET-based single-molecule assay to directly monitor eIF5. In both projects, we will combine our single-molecule assays with synergistic biophysical, biochemical, cellular, and structural approaches. Collectively, we should define the kinetics that underlie start site recognition and reveal transient molecular branchpoints that proofread start site selection. We also should uncover the timing of molecular rearrangements that control the progression of initiation and define how cancer-linked mutations in eIF1A disrupt the dynamics. As my research program evolves, our discoveries and innovations will set the stage for us to examine non-canonical initiation modes and how human regulatory proteins control initiation. Our findings thus should illuminate key knowledge gaps in translation and may highlight new interactions or molecular interfaces that could be targeted with therapeutics to treat human disease.