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Imaging Protein Synthesis on the Ribosome using Single-Molecule FRET

使用单分子 FRET 对核糖体上的蛋白质合成进行成像

基本信息

批准号：
10264055
负责人：
Scott C Blanchard
金额：
$ 46.22万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10264055
关键词：
Antibiotic Resistance Antibiotics Bacteria Bacterial Model Bacterial Translocation Behavior Binding Biochemical Biology Biophysics Cancerous Cell Proliferation Cells Chemicals Clinical Clinical Treatment Codon Nucleotides Collaborations Cryoelectron Microscopy Data Data Collection Detection Development Drug Design Drug Targeting Event Funding Future Gene Expression Gene Expression Regulation Health Human Image Individual Infrastructure Intervention Investigation Kinetics Knowledge Label Light Link Malignant Neoplasms Mammals Messenger RNA Methods Modeling Molecular Molecular Conformation Neoplasm Metastasis Organism Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Phase Physiological Process Protein Biosynthesis Proteins Proteome RNA RNA delivery Reaction Regulation Research Resolution Ribosomes Role Saint Jude Children&apos s Research Hospital Sampling Series Site Specificity Structural Models Structure Structure-Activity Relationship System Techniques Therapeutic Intervention Time Transfer RNA Translating Translation Process Translations Work Yeast Model System biological systems biophysical techniques cancer therapy cell growth clinical efficacy clinically relevant combat comparative computerized data processing design drug action drug resistant pathogen efficacious treatment fluorescence imaging fluorophore global health human disease imaging platform improved infectious disease treatment innovation insight kinetic model molecular dynamics novel novel strategies novel therapeutics pathogen polypeptide reconstitution single molecule single-molecule FRET small molecule targeted agent targeted treatment temporal measurement three dimensional structure translation factor treatment strategy

项目摘要

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.

项目简介: