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首页> 外文期刊>Journal of Molecular Biology >CLARIFICATION OF THE DIMERIZATION DOMAIN AND ITS FUNCTIONAL SIGNIFICANCE FOR THE ESCHERICHIA COLI NUCLEOID PROTEIN H-NS
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CLARIFICATION OF THE DIMERIZATION DOMAIN AND ITS FUNCTIONAL SIGNIFICANCE FOR THE ESCHERICHIA COLI NUCLEOID PROTEIN H-NS

机译：大肠埃希氏菌核蛋白H-NS的二聚化域的澄清及其功能意义

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The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding respectively. However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS. The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not. As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization. This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS. This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor. These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor. (C) 1997 Academic Press Limited. [References: 18]
机译：大肠杆菌核苷蛋白H-NS可以作为表达多种基因的全局调节剂。我们最近分析了H-NS的结构-功能关系，并分别参考了负责转录抑制和DNA结合的结构域。然而，H-NS的假定二聚结构域及其功能意义的鉴定尚不清楚。为了解决这个特定问题，我们首先研究了N-末端或C末端截短形式的H-NS的集合，它们对野生型H-NS的体内功能具有所谓的显性负作用。结果表明，某些截短形式表现出这种显性负效应，而其他截短形式则没有。根据显性负效应的结果判断，假定从残基21至63延伸的H-NS的相对中心部分参与二聚化。通过这些截短形式的H-NS的体外化学交联分析和凝胶过滤分析证实了这一点。此外，由截短形式的H-NS（命名为N91）引起的显性-阴性表型的使用，使我们能够分离出一个错义突变体，预期该突变体在二聚化中特别有缺陷。该突变体在由H-NS的N末端91个氨基酸组成的N91中的第30位（Leu30至Pro）具有氨基酸取代。该突变体确实在体外与野生型H-NS形成异二聚体的能力上存在缺陷。当将此特定的单个氨基酸取代引入全长H-NS时，所得的H-NS突变体丧失了体外形成二聚体和用作转录阻遏物的能力。这些发现共同为我们提供了证据，证明H-NS形成二聚体的能力对于H-NS发挥转录抑制因子的作用至关重要。 （C）1997 Academic Press Limited。 [参考：18]

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