DNA-MEMBRANE INTERACTIONS

DNA-膜相互作用

• 批准号: 6519100
• 负责人: IAN J MOLINEUX
• 金额: $ 26.69万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-04-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519100
• 关键词: DNA binding protein; Escherichia coli; bacteriophage T7; cell membrane; gene mutation; genetic mapping; methylation; molecular cloning; protein structure function; virus DNA; virus infection mechanism

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.

描述（改编自调查员的摘要）：噬菌体T7具有 成为研究跨细胞膜DNA易位的范例。 从病毒粒子向细胞弹出的蛋白质形成了一个分子运动 使用噬菌体基因组在30oC时以约70 bp/sec的范围划定噬菌体基因组，使用 质子动力作为其能源的来源。通常只有40 kb的850 bp 基因组被该电动机驱逐出境，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 通过转录将噬菌体基因组带入细胞，已建立 DNA易位的测定将用于测量动力学参数 几种DNA在体内易位酶。这个强大的结合 测定和既定为T7及其宿主的固定遗传系统 完全阐明噬菌体基因组进入的机制 该研究的可能结果。将获得的数据 在对机制和机制的一般理解中具有广泛的影响 核酸转运的能量，跨疏水脂质双层中的核酸易位 所有生物系统。在病毒座电机中有缺陷的噬菌体突变体 已被隔离，将用于表征 感染过程，包括建立跨膜通道 体内。涉及的蛋白质将被纯化，膜插入， 将测试GPL6的DNA结合和潜在的克糖基化酶活性 体外;这些特性从先前的体内强烈表明 研究。提出的实验构成了尝试尝试的初步步骤 在体外重建病毒粒子电动机。噬菌体突变体影响或 接近感染的启动已经并且将继续孤立。 他们的分析旨在理解信号转导 从细胞表面到触发蛋白质的噬菌体头的途径， 然后DNA，从病毒体中排出。