CONJUGATIVE MOBILIZATION OF PLASMID R1162

描述(改编自调查人员的摘要)：长期 这个项目的目标是在分子水平上表征DNA 广泛寄主范围的配偶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，编码其配偶所需的三种蛋白质(MOBA-C) 动员。启动之间潜在的生化相似之处 转移(划破双链DNA)和终止(结扎 单链、线状DNA链)将被鉴定和表征。多么 MOBA和MobC导致ORIT DNA在 将对松弛小体进行研究。MOBA的羧基末端区域是 一种底物酶，也是单独合成的，是细胞生长所必需的 R1162的营养复制。启动子结构域在蛋白质合成中的作用 将研究转移后的互补链合成。这个 Mobb蛋白在配偶动员中的作用目前尚不清楚。 我们将探索这种蛋白质调节水平的可能性。 可用于启动一轮转移的镍分子 破坏ORIT DNA和MOBA蛋白之间的联系。纯净的 MOBA蛋白在体外能切割和连接单链ORIT DNA。 这个反应的机理，以及它与一个模型的兼容性 终止转会，将进行调查。实验 这项研究的方法将包括分离第二位点抑制因子 松弛小体的突变、化学探测和重组MOBA ORIT DNA与纯化成分的体外相互作用。

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.