Regulatory mechanism of gene expression by Escherichio coli nucleoid protein H-NS

大肠杆菌核蛋白H-NS对基因表达的调控机制

基本信息

批准号：
09680671
负责人：
UEGUCHI Chiharu
金额：
$ 2.05万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680671/
关键词：
H-NS gene regulation global repressor nucleoid protein gene silencing DNA-binding protein LeuO シグマ因子ストレス応答

项目摘要

The Escherichia coil H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and dimerization, respectively. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA.The N-terminal domain was suggested to be involved in the ability to repress transcription. The relatively central portion of H-NS was found to be involved in protein-protein interaction, especially dimerization. The functional significance for each domain was also clarified. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.The Escherichia coli bgl operon is of interest since its expression is silent (phenotypically Bgl^-), at least under standard laboratory conditions. To identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl, I screened mutants exhibiting Bgl+ phenotype. By a random insertion mutagenesis with mini-Tn10, a novel type of mutation was obtained. In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein. Genetic analyses revealed that an overproduction of LeuO in the wild-type cells causes the phenotype of Bgl^+.

大胆管螺旋H-NS蛋白是核苷结构的主要成分之一。该蛋白不仅与核苷结构的紧凑组织有关，还与基因表达的全球调节有关。在这项研究中，关于HNS的系统突变分析，在H-NS中发现了三个不同的功能结构域，它们似乎分别负责DNA结合，转录抑制和二聚化。 C末端结构域中的突变导致其DNA结合能力的丧失，这表明该域直接参与了其与DNA的结合。提示N末端结构域参与抑制转录的能力。发现H-NS的相对中心部分参与蛋白质 - 蛋白质相互作用，尤其是二聚化。还阐明了每个域的功能意义。此外，发现BGL操纵子的表达仅以高等位基因特异性的方式仅受HNS突变的一部分影响。关于BGL操纵子的独特调节机制（即沉默），该发现也得到了解决，BGL操纵子由H-N部分介导。大肠杆菌BGL操纵子非常有意义，因为它的表达是无声的（表型BGL^ - ），至少在标准实验室条件下。为了确定可能与BGL调节相关的反式作用因子，我筛选了表现出BGL+表型的突变体。通过使用Mini-TN10的随机插入诱变，获得了一种新型的突变。在这种情况下，发现插入突变位于编码假定LYSR样的DNA结合蛋白的Leuo基因前面。遗传分析表明，野生型细胞中Leuo的生产过多会导致BGL^+的表型。