MEMBRANE TRANSPORT COMPONENTS IN BACTERIA

细菌中的膜运输成分

基本信息

批准号：
3268221
负责人：
DALE L OXENDER
金额：
$ 19.79万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1976
资助国家：
美国
起止时间：
1976-06-01 至 1991-03-31
项目状态：
已结题

项目摘要

The long term objectives of the proposed research is to determine the molecular basis of the transport of amino acids in bacteria. Cloning and DNA-sequencing techniques are being used to isolate and to characterize the five components of the branced chain amino acid transport systems in Escherchia coli. Two of the leucine transport components, livJ and K, are periplasmic binding proteins and three additional components, livH, M, and G, are membrane associated. The current studies will concentrate on three areas. First, M13 cloning and nucleotide sequencing techniques will be applied to determine the DNA sequence of the membrane components. The expression of the membrane components will be amplified by providing high efficiency controllable promoters for the cloned livH, M, and G genes. Gene fusion techniques to produce hybrid proteins are being carried out to facilitate the preparation of antibodies to the membrane components. These antibodies will be used for their purification and characterization. The study of the regulation of leucine transport constitutes a second specific goal of this proposal. The repressor components, livR and 1stR, will be isolated and characterized with the aid of cloning and DNA sequencing techniques. A study of the role of rho factor dependent transcription termination in the regulation of leucine transport will be carried out. A third specific goal is a study of the protein export in bacteria using the periplasmic leucine binding proteins as a model system. The structural and energy requirements for export of proteins into the periplasmic space will be determined using directed mutagenesis techniques. In addition, vectors for obtaining the export of other proteins fused to the leucine specific binding protein will be constructed. Finally, the introduction of oligonucleotide-directed mutagenesis techniques will facilitate structure-function studies with several of the leucine transport components.

拟议研究的长期目标是确定细菌中氨基酸转运的分子基础。克隆和DNA序列技术被用于隔离和隔离表征Branced链氨基酸转运的五个组成部分大肠杆菌的系统。两个亮氨酸传输组件livj 和K是周质结合蛋白和其他三个成分， LIVH，M和G是相关的膜。当前的研究将集中于三个区域。首先，M13克隆和核苷酸测序将应用技术来确定膜的DNA序列成分。膜成分的表达将通过为克隆的LIVH，M，提供高效率可控启动子和G基因。产生混合蛋白的基因融合技术正在进行以促进抗体制备膜成分。这些抗体将用于纯化和表征。亮氨酸转运调节的研究构成了第二秒该提案的具体目标。阻遏物组件，LIVR和1STR，将借助克隆和DNA隔离并表征测序技术。对Rho因子依赖性作用的研究亮氨酸传输调节中的转录终止将是执行。第三个具体目标是研究使用细菌中蛋白质导出的研究周质亮氨酸结合蛋白作为模型系统。结构以及将蛋白质导出到周质空间的能源需求将使用定向诱变技术确定。此外，获取与亮氨酸融合的其他蛋白质出口的向量将构建特定的结合蛋白。最后，引入寡核苷酸指导的诱变技术将有助于结构功能研究与几个亮氨酸转运成分。