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Functional structure of the repressor protein of the Bacillus subtilis gny operon

枯草芽孢杆菌 gny 操纵子阻遏蛋白的功能结构

基本信息

批准号：
07660125
负责人：
FUJITA Yasutaro
金额：
$ 1.41万
依托单位：
Fukuyama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The head investigator (me) has been studied this subject for last 6years. Among the fruitful achievements on the subject, the results of analysis for the binding domain of the gnt repressor protein (J.Basteriol. 177 : 4813) and those of analysis for interaction of this repressor and the gnt operator (Mol. Gen. Genet. 248 : 583) have been published in 1995after conduct of some additional experiments. In last two years, I have attempted to crystallize the gnt repressor for X-ray analysis under various crystallization conditions. I have also tried to crystallize the purified mutant repressors defective in operator binding. However, I could obtain no crystalline suitable for X-ray analysis but small needle-like one. Therefore, I concluded that the gnt repressor was hard to be crystallized because its tendency to polymerize at the high concentration. Meanwhile, I have analyzed details of kinetics of interaction between the gnt repressor and inducer by means of filter-binding method. I found that although the repressor lost the binding ability to the operator in the presence of the inducer (gluconate or glucono-delta-lacton), it gained a non-specific DNA binding ability. This binding ability was specifically induced in the presence of gluconate or glucono-delta-lacton. I am currently investigating the physiological significance for induction of this non-specific DNA binding. A few years ago, it has been proposed that a new subfamily of bacterial regulatory proteins represented by this gnt repressor protein (GntR family). Nowadays, the GntR family of bacterial regulatory proteins is internationally agreed, the members of which rapidly increased in the recent progress of the sequencing of several bacterial geneomes. I collected information about the functions of the members of the GntR family from the protein databases and classified them according to their functions.

首席研究员（我）已经研究这个课题6年了。在这一课题的丰硕成果中，gnt抑制蛋白结合域的分析结果(J.Basteriol。177: 4813)和该抑制因子与gnt算子相互作用的分析（Mol. Gen. Genet. 248: 583）在进行了一些附加实验后，已于1995年发表。在过去的两年中，我尝试在不同的结晶条件下结晶gnt阻遏物用于x射线分析。我也试着结晶纯化的在操作符结合上有缺陷的突变抑制子。然而，我没有得到适合x射线分析的晶体，只有小针状的晶体。因此，我得出结论，由于gnt抑制因子在高浓度下具有聚合倾向，因此很难结晶。同时，我用过滤器结合法分析了gnt阻遏物与诱导剂相互作用的动力学细节。我发现，虽然阻遏物在诱导剂（葡萄糖酸盐或葡萄糖- δ -内酯）的存在下失去了与操作者的结合能力，但它获得了非特异性的DNA结合能力。这种结合能力是在葡萄糖酸盐或葡萄糖- δ -内酯存在时特异性诱导的。我目前正在研究诱导这种非特异性DNA结合的生理意义。几年前，有人提出了一个以这种gnt抑制蛋白为代表的细菌调节蛋白亚家族（GntR家族）。目前，细菌调控蛋白的GntR家族是国际公认的，该家族成员随着近年来几种细菌基因组测序的进展而迅速增加。我从蛋白质数据库中收集了GntR家族成员的功能信息，并根据其功能进行分类。