DNA-Membrane Interactions

描述（由申请人提供）：T7噬菌体是研究DNA跨细胞膜易位的范例。一种强大的测定方法，可以定量测定基因组从噬菌体到细胞的运输速率，从而可以解剖潜在的体内机制。该分析还检测抗终止后体内转录率的变化；它将用于了解体内使用T7和噬菌体HK022的转录抗终止。这项工作的长期目标是为病毒感染的初始步骤提供一个完整的机制描述，包括DNA穿过细胞包膜进入细胞质的运输。这些研究对所有活细胞中核酸跨疏水脂质双分子层转运的机制和能量学具有广泛的意义。强大的检测方法和已建立的病毒及其宿主遗传系统相结合，使这一目标得以实现。T7蛋白从病毒粒子喷射到细胞中，不仅在细胞包膜上形成一个用于DNA易位的通道，而且还重组了一种酶，以大约70 bp/秒的速度将40kb的基因组送入细胞。现有的基因16突变体，已知在DNA易位上有缺陷，将允许确定喷射蛋白的细胞内位置，并定义gp16的跨膜结构域并进行化学探测。我们将评估膜电位在通道形成和维持中的作用。更多的突变体将被分离出来，这将有助于确定gp16中DNA易位的活性位点，并确定基因15蛋白在感染开始时的生理功能。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以及T7抗终止系统的生理意义将在体内和体外进行表征。A3启动子及其抗终止元件在T7 DNA包装中的干扰，以及T7 gp2在克服干扰中的作用将通过体内和体外方法进行阐明。

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.