Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

项目简介:

PROJECT ABSTRACT: The mechanism of protein synthesis and its regulation in the cell determines the diversity and capacity of the proteome. The central integration point for this regulatory control is the ribosome: a two-subunit, megadalton RNA-protein assembly. Highlighting the exquisite sensitivity of translation and the ribosome to regulation, the majority of known antibiotics either dysregulate or block ribosome function. Correspondingly, delineation of the protein synthesis mechanism in molecular detail has the potential to inform on paradigms of gene expression control and on how to combat the global health threat of emerging and drug resistant pathogens. As the loss of translation control is a hallmark of cancer, a deeper understanding of the protein synthesis mechanism also holds the promise of targeted therapeutic strategies for human disease treatments that are currently lacking. Investigations into structure-function relationships governing the translation mechanism have been principally conducted in bacteria using traditional ensemble methods. Such studies have revealed that the phase of translation in which protein is synthesized from messenger RNA (mRNA), termed elongation, is the most time intensive and commonly drug-targeted. They have also discerned that elongation entails the ribosome transiently interacting with specific cellular components through an ordered series of events, where the decoding of each mRNA codon is accompanied by large-scale conformational changes within the ribosome and interacting factors, and between the ribosome and its mRNA and transfer RNA (tRNA) substrates. The need for large amounts of homogenous material has thwarted analogous investigations of the human translation mechanism. Hence, conserved and divergent features of the translation mechanism between single-cell organisms and mammals that determine the molecular basis of antibiotic specificity have remained largely obscure. Here, we seek to delineate common and distinct features of bacterial and human protein synthesis — and the translation mechanisms in healthy and cancerous human cells — to: 1] improve the efficacies of existing antibiotics; 2] develop new strategies for antibiotic interventions; and 3] explore the possibility of therapies targeting unchecked proliferative cell growth and metastatic spread. We will do so by establishing quantitative, structural and kinetic frameworks for the elemental steps of elongation in bacteria and humans using an integrated battery of biophysical methods, including single-molecule fluorescence imaging and state-of-the-art cryo-electron microscopy. Our collaborative investigations will delineate the order and timing of conformational events underpinning fidelity in bacterial and human elongation cycles and the structural and mechanistic distinctions that determine the efficacies of clinically relevant antibiotics targeting these processes. These insights will shed light on how translation control is achieved, reveal atomic-resolution descriptions drug action on bacterial and human ribosomes and inform opportunities for new interventions aimed at improving the efficacy and potency of clinical treatments for infectious pathogens and human disease.