Mechanism of bacteriophage DNA packaging initiation and DNA translocation.

 描述（由申请人提供）：该资助提出了解决关键病毒DNA包装起始和易位问题的实验。噬菌体T4 DNA包装机制在通过可比较的马达蛋白（2）通过前头部门户包装有dsDNA的可比较的噬菌体中广泛共享，其中大末端酶亚基（TerL）负责易位，小末端酶（TerS）负责包装起始切割多联体。特异性目的1将建立大gp 17（TerL）易位机制。我们提出了一个“DNA嘎吱嘎吱”的线性电机机制，采用了抓地力和释放瞬态弹簧般的压缩B-A型DNA。我们的FRET测量直接支持这种机制，在包装停滞的Y-DNA底物在体外显示出减少的距离从末端酶门户网站，此外，有一个减少的距离之间的紧密定位的染料对在Y-茎DNA，符合B-和A-结构。在正常易位中，TerL马达排出所有不能结合A型的B型紧密结合YOYO-1染料。马达不能包装A型dsRNA或A型DNA：RNA异源双链体。我们的工作表明，添加辅助B型DNA：DNA（D：D）20聚体允许（D：R）包装异源双链A型DNA：RNA 20聚体（D：R），这是B型到A型弹簧马达的额外证据。此外，动力学分析的荧光染料的释放，TerL交联的光致变色染料，和高分辨率的结构数据将提供支持和洞察这一拟议的B型到A型电机机制。对接到前体、门户和门户的剪辑区域的TerL结构域的晶体学和冷冻-EM将确认终止酶的C-末端核酸酶结构域对接到门户，如FRET和SDM分析所示。具体目标2将建立的作用的小末端酶亚基gp 16（TerS）的噬菌体T4在DNA包装位点的相互作用和包装起始的双TerS环机制。FRET测量和超分辨率显微镜将确认T4 TerS蛋白以双环形式起作用以启动包装。体外和体内的功能性TerS-GFP和TerS-mCherry融合蛋白用作标准。FRET工作表明，TS突变形式的TerS蛋白在低温下形成环，但在高温下不形成环，表明环的形成是功能所必需的。在TerS纯蛋白制剂中发现的双22聚体和单11聚体环与DNA包装有何关系？强有力的遗传学证据支持两个同源pac DNA的突触由一个双环形式的TerS，反对一个四链pac DNA结构判断霍利迪连接链交换DNA串联体成熟包装启动。

 DESCRIPTION (provided by applicant): This grant proposes experiments to resolve critical viral DNA packaging initiation and translocation issues. Bacteriophage T4 DNA packaging mechanisms are widely shared among comparable phages packed with dsDNA through a prohead portal by comparable motor proteins (2), where the large terminase subunit (TerL) is responsible for translocation and the small terminase (TerS) for packaging initiation-cutting of the concatemer. Specific Aim 1 will establish the large gp17 (TerL) translocation mechanism. We propose a "DNA crunching" linear motor mechanism that employs a grip-and-release transient spring-like compression of B- to A-form- DNA. Our FRET measurements directly support this mechanism in a packaging stalled Y-DNA substrate in vitro that show a decrease in distance from terminase to portal; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem DNA that conforms to B- and A- structure. In normal translocation the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The motor cannot package A-form dsRNA or A-form DNA:RNA heteroduplexes. Our work shows that addition of helper B- form DNA:DNA (D:D) 20mers allows (D:R) packaging of heteroduplex A-form DNA:RNA 20mers (D:R), additional evidence for a B- to A-form spring motor. Additionally, kinetic analyses of fluorescent dye release, TerL cross-linking of photo-linkable dye, and high resolution structural data will provide support and insight into this proposed B-form to A-form motor mechanism. Crystallography and cryo-EM of TerL domains docked to proheads, portals, and to a clip region of the portal will confirm that the C-terminal nuclease domain of the terminase docks to the portal, as shown by FRET and SDM analysis. Specific Aim 2 will establish the role of the small terminase subunit gp16 (TerS) of phage T4 in DNA pac site interaction and in packaging initiation by a twin TerS ring mechanism. FRET measurements and superresolution microscopy will confirm that the T4 TerS protein acts in a double ring form to initiate packaging. Functional TerS-GFP and TerS-mCherry fusion proteins in vitro and in vivo serve as standards. FRET work shows that a ts mutant form of the TerS protein forms rings at low but not high temperature, showing ring formation is required for function. How do the double 22mer and single 11mer rings found in TerS protein-only preparations relate to DNA packaging? Strong genetic evidence supports synapsis of two homologous pac DNAs by a twin ring form of the TerS that opposes a four stranded pac DNA structure to judge by Holliday junction strand swapping DNA concatemer maturation for packaging initiation.