DNA-Membrane Interactions

描述(申请人提供)：噬菌体T7是研究跨细胞膜DNA转移的范例。一种强大的分析方法，可以量化从噬菌体病毒粒子到细胞的基因组传输速率，从而可以剖析体内潜在的机制。该方法还可以检测抗终止后体内转录速率的变化；它将被用来理解使用T7和噬菌体HK022的体内转录抗终止。这项工作的长期目标是对病毒感染的初始步骤提供完整的机械性描述，包括DNA跨越细胞膜和进入细胞质的运输。这些研究对核酸在所有活细胞中通过疏水脂质双层运输的机制和能量学具有广泛的意义。强大的检测和针对病毒及其宿主的已建立的遗传系统相结合，使这一目标得以实现。T7蛋白从病毒粒子喷射到细胞内，不仅形成了一个横跨细胞膜的通道，用于DNA易位，而且还重组了一种酶，以大约70BP/秒的速度将40kb的基因组送入细胞。现有的基因16突变体在DNA易位方面存在缺陷，它将允许确定喷射蛋白在细胞内的位置，并定义和化学探测gp16的跨膜结构域。将评估膜电位在通道形成和维护中的作用。将分离出更多的突变体，这些突变体将有助于确定gp16中DNA易位的活性部位，并确定基因15蛋白在启动感染中的生理功能。正常情况下，转录内化了T7基因组的大部分。每个早期启动子在基因组进入中的作用和T7抗终止系统的生理意义将在体内和体外表征。A3启动子及其抗终止元件在T7DNA包装中的干扰，以及T7GP2在克服干扰中的作用将通过体内和体外方法阐明。

DESCRIPTION (provided by applicant): Bacteriophage T7 is the paradigm for studying DNA translocation across cell membranes. A powerful assay, that quantitates rates of genome transport from the phage virion into the cell, allows dissection of the underlying in vivo mechanisms. The assay also detects changes in the in vivo rates of transcription following antitermination; it will be used to understand transcription antitermination in vivo using T7 and phage HK022. The long-term goal of this work is to provide a complete mechanistic description of the initial steps of viral infection, including DNA transport across the cell envelope and into the cytoplasm. These studies have broad implications for the mechanisms and energetics of nucleic acid transport across hydrophobic lipid bilayers in all living cells. The combination of a powerful assay and established genetic systems for both the virus and its host make this goal achievable. T7 proteins, ejected from the virion into the cell, not only form a channel across the cell envelope used for DNA translocation but also reconstitute an enzyme that ratchets the 40 kb genome into the cell at approximately 70 bp/sec. Existing gene 16 mutants, known to be defective in DNA translocation, will allow the intracellular location of the ejected proteins to be determined, and the transmembrane domains of gp16 to be defined and chemically probed. The role of the membrane potential in channel formation and maintenance will be evaluated. Additional mutants will be isolated that will help define the active site for DNA translocation in gp16 and to determine the physiological function of the gene 15 protein in the initiation of infection. Normally, transcription internalizes most of the T7 genome. The role of each early promoter in genome entry and the physiological significance of the T7 antitermination system will be characterized in vivo and in vitro. The interference of the A3 promoter and its antitermination element in T7 DNA packaging, and the role of T7 gp2 in overcoming interference will be elucidated using in vivo and in vitro approaches.