DNA-MEMBRANE INTERACTIONS

描述(改编自研究人员摘要)：噬菌体T7有 成为研究跨细胞膜DNA转移的范例。 从病毒粒子喷射到细胞内的蛋白质形成一个分子马达， 在30oC下，以大约70BP/秒的速度将噬菌体基因组插入细胞，使用 作为其能量来源的质子动力。正常情况下只有40kb中的850bp 基因组是由这个马达喷射出来的，但突变体的特征是 通过这种机制将整个基因组转移到细胞中。大多数 噬菌体基因组通过转录进入细胞，已建立的 DNA易位试验将用于测量动力学参数 体内的几种DNA转位酶。这一强大的组合 T7及其宿主可用的检测和已建立的遗传系统 彻底阐明噬菌体基因组进入的机制 这种细胞可能是这项研究的结果.将获得的数据 有广泛的含义，对机制和一般理解 核酸跨疏水脂双分子层转运的能量学 所有的生物系统。病毒粒子马达有缺陷的噬菌体突变体 已经被分离出来，并将被用来描述 感染过程，包括在体内建立跨膜通道 活着。所涉及的蛋白质将被提纯，膜插入， 将测试gpl6的dna结合和潜在的转糖基酶活性。 在体外；这些特性从以前的体内实验中得到了强烈的指示 学习。拟议中的实验是朝着尝试 在体外重建病毒粒子马达。影响或的步骤的噬菌体突变体 在感染开始附近，已经并将继续被隔离。 他们的分析旨在理解信号转导 从细胞表面进入噬菌体头部触发蛋白质的途径，以及 然后是DNA，从病毒粒子中喷出。

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.