DNA-MEMBRANE INTERACTIONS

描述（改编自研究者摘要）：噬菌体T7具有 成为研究DNA跨细胞膜易位的范例。 蛋白质从病毒体喷射到细胞中形成分子马达， 在30 ℃下，使用 质子动力作为它的能量来源。通常只有850 bp的40 kb 基因组被这个马达排出，但突变体的特点是， 通过这种机制将整个基因组转移到细胞中。大部分 噬菌体基因组通过转录进入细胞，并建立 DNA易位测定将用于测量 几种体内的DNA易位酶。这种强大的 试验和建立的遗传系统可用于T7及其宿主 彻底阐明了噬菌体基因组进入 细胞可能是这项研究的结果。将获得的数据 在一般理解机制方面具有广泛的影响， 核酸在疏水脂质双层中的转运能量学 所有的生物系统。病毒粒子马达有缺陷的噬菌体突变体 已被分离，并将用于表征的初始步骤， 感染过程，包括跨膜通道的建立， vivo.相关的蛋白质将被纯化，膜插入， 将测试gp 16的DNA结合和潜在的转糖基酶活性 在体外;这些特性强烈表明从以前的体内 问题研究拟议的实验构成了尝试的初步步骤 在体外重建病毒粒子运动。噬菌体突变体影响步骤或 在感染开始前，已经并将继续被隔离。 他们的分析旨在了解信号转导 从细胞表面进入触发蛋白质的噬菌体头部的途径，以及 然后是DNA，从病毒体中排出

DESCRIPTION (adapted from the investigator's abstract): Bacteriophage T7 has become a paradigm for studying DNA translocation across cell membranes. Proteins ejected from the virion into the cell form a molecular motor that ratchets the phage genome into the cell at about 70 bp/sec at 30oC, using the proton motive force as its source of energy. Normally only 850 bp of the 40 kb genome are ejected by this motor, but mutants were characterized that translocate the entire genome into the cell by this mechanism. Most of the phage genome is brought into the cell via transcription, and the established assay for DNA translocation will be used to measure kinetic parameters of several DNA translocating enzymes in vivo. The combination of this powerful assay and the established genetic systems available for both T7 and its host make the complete elucidation of the mechanism by which a phage genome enters the cell a probable result of this research. The data that will be obtained have broad implications in a general understanding of the mechanisms and energetics of nucleic acid translocation across hydrophobic lipid bilayers in all biological systems. Phage mutants that are defective in the virion motor have been isolated and will be used to characterize the initial steps of the infection process, including establishment of the transmembrane channel in vivo. The proteins involved will be purified, the membrane-insertion, DNA-binding, and potential transglycosylase activities of gpl6 will be tested in vitro; these properties are strongly indicated from previous in vivo studies. The proposed experiments form the initial steps towards attempting to reconstruct the virion motor in vitro. Phage mutants that affect steps at or near the initiation of infection have been, and will continue to be, isolated. Their analysis is designed towards an understanding of the signal transduction pathway from the cell surface into the phage head that triggers protein, and then DNA, ejection from the virion.