Biophysical mechanisms of ABC-F proteins

基因组测序提供了对分子基础的前所未有的洞察力，从而使生物学发生了革命性的变化 生活的一部分。然而，它也带来了新的研究挑战。一个关键的挑战是阐明 未知蛋白质家族的功能，因为在任何基因组中至少有一半编码的蛋白质 缺乏对生化功能的可靠描述。我们试图开发系统的方法来解决 通过将结构生物学和基因组学的工具结合起来解决这个问题。使用这种方法，我们 最近阐明了属于ABC-F序列的四种大肠杆菌蛋白中的一种的生化功能 该家族在所有真核生物和几乎所有真细菌基因组中都有编码的多个代表。不是 其他ABC-F蛋白之前已经对其功能进行了详细描述，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同源动物对各种 不同的疾病。此外，~30个真细菌ABC-F Paralog组中的几个已被卷入 介导微生物的抗生素耐药性。我们最近发表的对大肠杆菌YjjK蛋白的研究，我们 更名为Etta(能量依赖的翻译节流阀A)，证明了这个ABC-F家族成员是一个 介导依赖ADP/ATP的核糖体起始复合体冬眠的调节性翻译因子 比率我们确定了被ATP结合的ETTA的低温电子显微镜(Cryo-EM)结构 国家与70年代的核糖体结合，这确立了其前所未有的翻译控制活动是 通过结合到出口(E)和肽-tRNA结合(P)-tRNA之间的一个新的因子结合位点而介导- 核糖体上的结合部位。这些研究，连同亨特实验室之前关于 ABC转运蛋白超家族中同源ATPase的机械力化学表明ETTA使用一种 新的分子机制来感知ADP/ATP比率，这是一个关键的细胞能量状态监测。这个 在这项应用中提出的研究将利用各种各样的生化、生物物理和结构 方法来批判性地评估我们的假设，解释这一新的活动，同时也表征 大肠杆菌表达的四个ABC-F同源基因的生化功能。这些研究将提供 了解提供抗生素的微生物ABC-F同源基因的生化功能的基础 抗药性和三个人类ABC-F同源基因，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.