Biophysical mechanisms of ABC-F proteins

基因组测序通过提供对分子基础的前所未有的洞察而彻底改变了生物学 生命然而，它也提出了新的研究挑战。一个关键的挑战是阐明 没有特征的蛋白质家族的功能，因为至少有一半的蛋白质编码在任何基因组中， 缺乏可靠描述的生物化学功能。我们试图制定系统的方法来解决 通过结合结构生物学和基因组学的工具来解决这个问题。使用这种方法，我们 最近阐明了四个E.属于“ABC-F”序列的大肠杆菌蛋白 该家族在所有真核生物和几乎所有真细菌基因组中都有多个代表编码。没有 其他ABC-F蛋白先前已经详细描述了其功能，尽管已发表的研究 使用基因组规模的分析方法，已经确定了三种人类旁系同源物有助于各种 不同的疾病。此外，约30个真细菌ABC-F旁系同源群中的几个与 介导微生物抗生素抗性。我们最近发表的E.大肠杆菌YjjK蛋白，我们 重命名为EttA（能量依赖平移节流阀A），证明了这个ABC-F家族成员是一个 一种调节翻译因子，介导依赖于ADP/ATP的核糖体起始复合物的冬眠 比例我们确定了一个低温电子显微镜（cryo-EM）结构的EttA被困在ATP结合的 状态结合到70 S核糖体，这确定了其前所未有的翻译控制活性， 通过结合到出口（E）和肽基-tRNA-结合（P）tRNA-之间的新的因子结合位点来介导。 核糖体上的结合位点。这些研究，加上亨特实验室以前对 ABC转运蛋白超家族中同源ATP酶的机械化学，表明EttA使用一种 一种新的分子机制来检测ADP/ATP比率，这是细胞能量状态的关键监测器。的 本申请中提出的研究将利用各种生物化学、生物物理和结构 方法来批判性地评估我们的假设，解释这种新的活动，同时也表征的范围， 由E.杆菌这些研究将提供 了解赋予抗生素的微生物ABC-F旁系同源物的生物化学功能的基础 耐药性和三种人类ABC-F旁系同源物，这对于理解它们在疾病中的作用至关重要。

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.