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Biophysical mechanisms of ABC-F proteins

ABC-F蛋白的生物物理机制

基本信息

批准号：
9339717
负责人：
JOHN Francis HUNT
金额：
$ 30.76万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9339717
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters ATPase Domain Antibiotic Resistance Antibiotics Binding Binding Sites Biochemical Biological Biological Assay Biology Biophysical Process Biophysics Classification Complex Disease Electron Microscopy Family Family member Fluorescence Spectroscopy Foundations Genes Genetic Translation Genetic study Genome Genomics Goals Hibernation Human Image Kinetics Life Mediating Messenger RNA Methods Microbial Antibiotic Resistance Modeling Molecular Molecular Conformation Monitor Names Peptides Process Protein Biosynthesis Protein Family Proteins Publications Publishing Regulation Research Ribosomal Interaction Ribosomes Roentgen Rays Role Specificity Stereotyping Structure Testing Thermodynamics Transfer RNA Translating Translations Transmembrane Domain Variant Work Yeasts biophysical techniques cryogenics deep sequencing experimental study gene function genome sequencing genome-wide human disease insight microbial novel paralogous gene particle peptidyl-tRNA programs reconstruction single molecule structural biology tool transcriptome sequencing translation factor

项目摘要

Genome sequencing has revolutionized biology by providing unprecedented insight into the molecular basis of life. However, it has also established new research challenges. One critical challenge is the elucidation of the function of uncharacterized protein families, because at least half of the proteins encoded in any genome lack reliably described biochemical functions. We have attempted to develop systematic approaches to tackle this problem by combining the tools of structural biology with those of genomics. Using this approach, we recently elucidated the biochemical function of one of four E. coli proteins belonging to the “ABC-F” sequence family, which has multiple representatives encoded in all eukaryotic and almost all eubacterial genomes. No other ABC-F protein had previously had its function characterized in detail, even though published studies using genome-scale profiling methods have identified the three human paralogs as contributing to a variety of different diseases. Furthermore, several of the ~30 eubacterial ABC-F paralog groups have been implicated in mediating microbial antibiotic resistance. Our recently published studies of the E. coli YjjK protein, which we renamed EttA (Energy-dependent Translational Throttle A), demonstrated that this ABC-F family member is a regulatory translation factor that mediates hibernation of ribosome initiation complexes dependent on ADP/ATP ratio. We determined a cryogenic electron microscopy (cryo-EM) structure of EttA trapped in an ATP-bound state bound to a 70S ribosome, which established that its unprecedented translational control activity is mediated by binding to a novel factor-binding site between the exit (E) and peptidyl-tRNA-binding (P) tRNA- binding sites on the ribosome. These studies, together with previous research from the Hunt lab on the mechanochemistry of homologous ATPases in the ABC Transporter superfamily, suggested that EttA uses a novel molecular mechanism to sense ADP/ATP ratio, which is a critical monitor of cellular energy status. The research proposed in this application will harness a wide variety of biochemical, biophysical, and structural methods to critically evaluate our hypothesis explaining this novel activity while also characterizing the range of biochemical functions performed by the four ABC-F paralogs expressed by E. coli. These studies will provide a foundation to understand the biochemical functions of the microbial ABC-F paralogs that confer antibiotic resistance and the three human ABC-F paralogs, which is critical for understanding their roles in disease.

基因组测序通过提供对分子基础的前所未有的深入了解，彻底改变了生物学