Structure and Function of Essential Nucleoprotein ComplexesAlong a Viral Genome Packaging Pathway

项目摘要。 噬菌体在细菌进化中发挥着重要作用，在介导细菌致病性和抗生素方面发挥着重要作用。 它们在调节人类微生物群方面具有很大的抗药性，在纳米治疗方面具有巨大的潜力。 了解这些与人类疾病有关的问题，并利用它们作为鼻咽癌的潜力 代理人需要对病毒的发展有基本的了解。基因组包装途径 在大型双链DNA(DsDNA)病毒中高度保守，包括原核生物和 真核生物。在这一大类病毒中，终止酶负责(I)切除 来自连体底物的个体基因组(基因组成熟)和(Ii)DNA转位到 Proapsid外壳(基因组包装)。这些功能是由终止酶催化的。 组装成离散的成熟和包装马达复合体。终端酶由一个 催化亚单位和DNA识别亚单位，这两个都是基因组包装所必需的 活着。结构和单分子研究为包装马达复合体提供了洞察力 孤立地由催化亚单位组成；然而，关于运动复合体的信息很少 包含两个基本亚基的。此外，缺乏同样的结构性信息。 必需成熟复合体前体。这在一定程度上是由于缺乏具有良好特征的 全酶制剂和缺乏体外测试来全面评估该途径。我们 已经开发出严格的分析方法，其中生化、生物物理和结构特征 Lambda基因组包装途径可以非常详细地定义。使用这些工具，我们建议 表征细胞的结构(冷冻电子显微镜)和功能(生物物理、动力学)特征 成熟复合体，表现出与四聚体IIE/F限制性内切酶的机制相似 核酸内切酶。我们直接解决了关于DNA体系结构的一个新的争议 成熟的复合体，调节复合体的稳定性。接下来我们要测试的假设是波长马达 也以四聚络合物的形式发挥作用，马达对ATP的水解具有很强的协同性； 这些特点与目前公认的模式有很大的不同。最后，我们 描述了一种假定的“核苷酸开关”机制，该机制控制着从稳定状态的转变 成熟复合体到与衣壳结合的动态运动复合体，我们严格定义 搬运电机的能量平衡。拟议的研究将提供结构和机械方面的 两个序列包装复合体的详细信息及其在基因组包装中的过渡 路径。所有打包基因组的dsDNA病毒都有这些特征。 前驱病毒(噬菌体、疱疹)及其结果将具有广泛和普遍的意义。

Project Summary. Bacteriophages play a major role in bacterial evolution, in mediating bacterial pathogenicity and antibiotic resistance, in modulating the human microbiome and they have great potential as nanotherapeutics. Understanding these issues with respect to human disease and harnessing their potential as theranostic agents requires a fundamental understanding of virus development. The genome packaging pathways are strongly conserved in the large double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic. In this broad class of viruses, a terminase enzyme is responsible for (i) excision of an individual genome from concatemeric substrate (genome maturation) and (ii) translocation of DNA into a procapsid shell (genome packaging). These functions are catalyzed by terminase enzymes assembled into discrete maturation and packaging motor complexes. Terminases are composed of a catalytic subunit and a DNA recognition subunit, both of which are essential for genome packaging in vivo. Structural and single-molecule studies have provided insight into packaging motor complexes composed of the catalytic subunit in isolation; however, there is little information on motor complexes containing both essential subunits. Further, there is a dearth of structural information on the equally essential maturation complex precursor. This is due, in part, to the absence of well-characterized holoenzyme preparations and a dearth of in vitro assays to comprehensively assess the pathway. We have developed rigorous assays in which the biochemical, biophysical and structural features of the lambda genome-packaging pathway can be defined in great detail. Using these tools, we propose to characterize the structural (cryo-electron microscopy) and functional (biophysical, kinetic) features of the maturation complex, which show mechanistic similarity to the tetrameric type IIE/F restriction endonucleases. We directly address an emerging controversy relating to the DNA architecture in the maturation complex that mediates complex stability. We next test the hypothesis that the lambda motor also functions as a tetrameric complex and that ATP hydrolysis by the motor is strongly cooperative; these features represent a significant departure from currently accepted paradigms. Finally, we characterize a putative "nucleotide switch" mechanism that controls the transition from the stable maturation complex to the dynamic motor complex bound to the capsid and we rigorously define the energy budget of the translocating motor. The proposed studies will provide structural and mechanistic detail on two sequential packaging complexes and their transition through the genome-packaging pathway. These features are shared by all of the dsDNA viruses that package genomes from concatemeric precursors (phage, herpes) and the results will be of broad and general significance.