Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

About This Grant

PROJECT ABSTRACT: The mechanism and regulation of protein synthesis determines the diversity and capacity of the cellular proteome. At the center of this regulation is the ribosome - a megadalton RNA-protein complex composed of two-subunits – which integrates a wide variety of cellular signals. The ribosome’s exquisite sensitivity to regulatory cues is underscored by the fact that the majority of clinically used antibiotics exert their effect by either dysregulating or blocking specific aspects of the protein synthesis mechanism. Understanding the kinetic and structural basis of protein synthesis promises to elucidate core paradigms of gene expression control and to inform strategies for addressing the global threat posed by drug-resistant and emerging pathogens. Moreover, given that loss of translational control is a hallmark of cancer, a mechanistic understanding of ribosome function holds significant promise for developing novel small-molecule therapies, which are currently lacking in the treatment of human disease. Historically, investigations into structure-function relationships governing the protein synthesis mechanism have focused on bacterial systems approaches. Comparable studies in human systems have been hindered by the demand for large amounts of homogeneous protein synthesis machinery. As a result, the molecular distinctions between bacterial and mammalian protein synthesis - which underpin antibiotic specificity and potential therapeutic windows – remain obscure. Current evidence suggests that the elongation phase of protein synthesis, during which messenger RNA (mRNA) is decoded into protein, is the most time consuming, physiologically regulated and small-molecule sensitive. We and others hypothesize that elongation is particularly susceptible to regulation because it involves transient, repetitive interactions of the ribosome with auxiliary factors, coordinated through finely tuned conformational transitions that are acutely sensitive to perturbation. Small effects on many individual steps compound to exert large impacts on protein production. The proposed research seeks to rigorously and robustly define and quantify the elemental reactions underpinning the elongation phase of protein synthesis in bacteria and human. Our objectives are to: 1) elucidate the conserved mechanistic principles and divergent features of protein synthesis across evolution to define paradigms for selective control; 2] identify novel strategies for more effective antibiotic interventions for the treatment of infectious disease; and 3] evaluate the therapeutic potential of targeting dysregulated translation in cancer. To achieve these goals, we will deploy a suite of advanced biophysical methodologies - including single- molecule fluorescence imaging and state-of-the-art cryo-electron microscopy - to establish comprehensive and integrated kinetic and structural frameworks defining the elemental steps of elongation in bacteria and humans. The insights gained will advance our understanding of translation control, reveal atomic-resolution descriptions of drug actions on bacterial and human ribosomes, and inform new strategies for improving the efficacy of clinical treatments for both microbial and human disease.