Biochemical Properties of gpNu1, a Phage Lambda Protein Required for Viral Assembly

卡塔拉诺9728550在一个空的预先形成的衣壳内包装DNA要经过核蛋白中间体的有序过程，需要端酶，一种所有双链DNA噬菌体共同的酶。酶亚基gpNu1和gpA聚集在一个多元素DNA结合位点上，产生一个核蛋白复合物，从连接体中切除一个基因组并将其插入衣壳中。本研究在分子水平上研究了λ噬菌体1 gpNu1蛋白的生化和生物物理特性，该特性负责核蛋白中间体的协同组装和异常稳定性。目的是了解gpNu1自结合相互作用和gpNu1 gpA蛋白蛋白相互作用在核蛋白复合物组装和稳定性中的作用。这些实验旨在详细了解gpNu1自结合和与DNA的合作结合之间的热力学联系，以及gpA和催化能力强的基因组包装复合物的后续组装。本研究还试图确定gpNu1多肽内的功能域，这些功能域负责观察到的gpNu1寡聚化，gpNu1 - gpA相互作用以及与DNA的合作结合。由于终止酶在许多双链DNA病毒中是常见的，因此本研究的发现可能普遍适用于理解其他病毒组装和包装机制。2. 多种生物过程需要将多种蛋白质组装到细胞和/或病毒染色体内的特定位点上。许多这些复合物最初作为稳定的中间体组装，随后转化为高度移动的生物机器。复杂的核蛋白（DNA和蛋白质）复合物负责关键的生物过程，包括染色体的复制、信使RNA的合成和传染性病毒的组装。本研究旨在阐明病毒在细胞内的组装过程和病毒染色体包装的复杂过程，特别是在终止酶的参与下，终止酶是一种多蛋白机器，它将病毒染色体插入感染细菌大肠杆菌的噬菌体lambda的保护蛋白外壳内。这种酶在许多双链DNA病毒中很常见，是一系列有序的核蛋白复合物的组成部分，对病毒组装至关重要。gpNu1是一种噬菌体1包装蛋白，负责病毒染色体包装过程中核蛋白中间体的组装和异常稳定性，研究了gpNu1的生化和生物物理特性。重要的是，从这些研究中获得的数据将进一步深入了解驱动稳定核蛋白复合物组装的基本原理，这些核蛋白复合物参与对病毒和细胞复制至关重要的各种生物过程。

Catalano 9728550 1. Technical Packaging of DNA within an empty pre-formed capsid proceeds through an ordered progression of nucleoprotein intermediates requires terminase, an enzyme common to all of the double-stranded DNA bacteriophages. The enzyme subunits, gpNu1 and gpA, assemble onto a multi-element DNA binding site generating a nucleoprotein complex, excising a single genome from the concatamer and inserting it into the capsid. This study examines, at the molecular level, the biochemical and biophysical properties of the lambda phage 1 gpNu1 protein which are responsible for the cooperative assembly and unusual stability of the nucleoprotein intermediates involved. The aims are to understand the roles of gpNu1 self-association interactions and gpNu1 gpA protein protein interactions in the assembly and stability of the nucleoprotein complexes critical to the assembly. The experiments are designed to provide a detailed understanding of the thermodynamic linkages between gpNu1 self-association and cooperative binding to DNA, and to the subsequent assembly of gpA and a catalytically competent genome packaging complex. This study also seeks to define functional domains within the gpNu1 polypeptide which are responsible for the observed oligomerization of gpNu1, for gpNu1 gpA interactions, and for cooperative binding to DNA. Since the terminase enzyme is common to many double-stranded DNA viruses, findings from this study may be generally applicable to the understanding of other viral assembly and packing mechanisms. 2. Non-Technical A variety of biological processes require the assembly of multiple proteins onto specific sites within the chromosomes of cells and/or viruses. Many of these complexes initially assemble as stable intermediates which are subsequently transformed into highly mobile biological machines. Elaborate nucleoprotein (DNA and protein) complexes are responsible for critical biological processes including the replication of chromosomes, the synthesis of messenger RNA, and the assembly of infectious viruses. This study is to elucidate the process of viral assembly within the cell and the complex process of viral chromosome packaging, specifically in the involvement of the terminase enzyme, the multi-protein machines which insert the viral chromosome within a protective protein shell in bacteriophage lambda, a virus which infects the bacteria E. coli. This enzyme, which is common to many double-stranded DNA viruses, is an integral part of an ordered series of nucleoprotein complexes which are critical to viral assembly. Experiments are carried out to examine the biochemical and biophysical properties of gpNu1, a phage l packaging protein, which are responsible for the assembly and unusual stability of the nucleoprotein intermediates involved in viral chromosome packaging. Importantly, the data derived from these studies will additionally yield insight into the basic principles driving the assembly of stable nucleoprotein complexes involved in a variety of biological processes critical to both viral and cellular replication.