CONJUGATIVE MOBILIZATION OF PLASMID R1162

质粒 R1162 的接合动员

• 批准号: 6080703
• 负责人: RICHARD J MEYER
• 金额: $ 7.55万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6080703
• 关键词: DNA binding protein; DNA primase; DNA replication; Escherichia coli; affinity chromatography; bacterial proteins; biochemical evolution; genetic manipulation; genetic mapping; genetic recombination; host organism interaction; microorganism conjugation; molecular cloning; mutant; nucleic acid sequence; open reading frames; plasmids; point mutation; protein biosynthesis; protein purification; protein sequence; protein transport; site directed mutagenesis; transcription factor; transposon /insertion element

DESCRIPTION (Adapted from the investigator's abstract): The long-term goal of this project is to characterize at the molecular level the DNA processing steps required for the conjugal transfer of broad host-range plasmids. The proposed work will be focussed on the multi-copy plasmid R1162, which encodes three proteins (MobA-C) required for its conjugal mobilization. Underlying biochemical similarities between initiation of transfer (nicking of duplex DNA) and termination (ligation of a single, linear DNA strand) will be identified and characterized. How MobA and MobC bring about the localized melting of oriT DNA in the relaxosome will be investigated. The carboxy-terminal region of MobA is a primase, also synthesized separately, that is required for the vegetative replication of R1162. The role of the primase domain in complementary strand synthesis after transfer will be examined. The function of MobB protein in conjugal mobilization is presently unknown. The possibility will be explored that this protein regulates the level of nicked molecules available to initiate a round of transfer by disrupting the association between oriT DNA and MobA protein. Purified MobA protein can cleave and ligate single-stranded oriT DNA in vitro. The mechanism of this reaction, and its compatibility with a model for the termination of transfer, will be investigated. Experimental approaches in this study will include isolating second-site suppressor mutations, chemical probing of the relaxosome, and reconstituting MobA oriT DNA interactions in vitro with purified components.

描述（根据调查员的摘要改编）：长期 该项目的目标是在分子水平上表征DNA 广泛宿主范围的夫妻转移所需的处理步骤 质粒。 拟议的工作将集中在多拷贝质粒上 R1162，它编码其结合所需的三个蛋白质（MOBA-C） 动员。 启动之间的基本生化相似性 转移（双链DNA的刻痕）和终止（连接A 将识别和表征单个线性DNA链）。 如何 MOBA和MOBC带来了Orit DNA的局部熔化 弛豫组将进行研究。 MOBA的羧基末端区域是 一个原始合成的原始酶，这是 R1162的营养复制。 培养物域中的作用 转移后的互补链合成。 这 目前，MOBB蛋白在结合动员中的功能尚不清楚。 可能会探讨这种蛋白质调节水平的可能性 可用于通过 破坏Orit DNA和MOBA蛋白之间的关联。 纯化 MOBA蛋白可以在体外切割和结合单链的Orit DNA。 该反应的机制及其与模型的兼容性 将调查转移的终止。 实验 这项研究的方法将包括隔离第二站点抑制器 突变，弛豫的化学探测和重建MOBA 在体外与纯化成分的Orit DNA相互作用。