Williams R M, Rimsky S, Buc H
Unité de Physicochimie des Macromolecules Biologiques (URA 1149 du Centre National de la Recherche Scientifique), Institut Pasteur, Paris, France.
J Bacteriol. 1996 Aug;178(15):4335-43. doi: 10.1128/jb.178.15.4335-4343.1996.
Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.
已在体内筛选并鉴定了12种不同的大肠杆菌类核相关蛋白H-NS的显性负突变体。这些突变体在缺乏H-NS的菌株中，启动子抑制活性均存在严重缺陷，并且它们都破坏了H-NS在其两个靶启动子上正常施加的抑制作用。根据这些突变体中导致大片段截短和氨基酸取代的改变位置，我们提出H-NAS至少包含两个不同的结构域。本报告中给出的体外蛋白质-蛋白质交联数据表明，所提出的H-NS N端结构域在H-NS多聚化中起作用。StpA是一种与H-NS具有已知结构和功能同源性的蛋白质。我们通过构建和研究预测为显性负性的StpA突变体，分析了这些同源性的程度。我们的数据表明，在显性负性H-NS中发现的取代和缺失在StpA的背景下具有相似的作用。我们得出结论，StpA和H-NS中的结构域组织和功能密切相关。此外，显性负性H-NS可以破坏天然StpA的活性，反之，显性负性StpA可以破坏天然H-NS的活性。我们证明H-NS的N端结构域可以与全长H-NS和StpA进行化学交联。我们通过提出H-NS和StpA有能力形成杂交物种来解释这些观察结果。
