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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^+.

大肠杆菌H-NS蛋白是类核结构的主要成分之一。这种蛋白质不仅与类核结构的紧密组织有关，而且与基因表达的全局调控有关。在这项研究中，对hns的系统突变分析，在H-NS中发现三个不同的功能域，这似乎是负责DNA结合，转录抑制和二聚化，分别。C端结构域的突变导致其DNA结合能力的丧失，表明该结构域直接参与其与DNA的结合，N端结构域被认为参与抑制转录的能力。H-NS的相对中心部分被发现参与蛋白质-蛋白质相互作用，特别是二聚化。还阐明了每个结构域的功能意义。此外，发现bgl操纵子的表达仅以高度等位基因特异性的方式受hns突变的子集的影响。这一发现还涉及bgl操纵子的独特调控机制（即沉默），该机制部分由H-NS介导。大肠杆菌bgl操纵子之所以令人感兴趣，是因为它的表达是沉默的（表型为Bgl^-），至少在标准实验室条件下是如此。为了鉴定推测与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mini-Tn 10的随机插入突变，获得了一种新的突变类型。在这种情况下，插入突变被发现是在前面的leuO基因编码一个推定的LysR样DNA结合蛋白。遗传分析显示，野生型细胞中LeuO的过量产生导致了Bgl^+的表型。