PDF(Pubmed)
Abstract：
In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.
摘要：
在所有尾噬菌体中，通过末端酶马达复合物将双链基因组包装到头部是病毒体形成的重要步骤。尽管进行了广泛的研究,在理解这种高度动态的过程和负责DNA易位的机制方面仍然存在很大的差距.在过去的十五年里，单分子荧光技术已应用于研究病毒核酸包装，使用强大而灵活的T4体外包装系统与遗传，生物化学，和结构分析。在这次审查中,我们讨论这些研究的新发现，包括通过在门户结构上方的染料标记的DNA末端的共定位，确定T4基因组被包装为细长的环。TerL电机的封装效率被证明与基板结构固有地联系在一起，包装在DNA分支上停滞。后者导致了多个实验的设计，其结果均支持提出的扭转压缩移位模型来解释衬底封装。底物压缩的证据来自相对于电机组件的停滞与分解酶释放的染料标记的Y-DNA和其他染料标记的底物的FRET和/或smFRET测量。此外,活性体内T4TerS荧光融合蛋白促进了先进的超分辨率光学显微镜对包装启动可视化的应用。形成孪生的TerS环配合物，每个直径预计为~15纳米，支持用于控制包装起始的双蛋白质环-DNA突触模型，该模型可能有助于解释在pac位点噬菌体中报告的各种环结构。在这些研究中，在单分子水平上对T4包装马达的动力学的检查证明了最先进的荧光工具对于复杂病毒复制机制的未来研究的价值。
