Goshima N, Inagaki Y, Otaki H, Tanaka H, Hayashi N, Imamoto F, Kano Y
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Gene. 1992 Sep 1;118(1):97-102. doi: 10.1016/0378-1119(92)90254-m.
Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions). These proteins were synthesized in a hupA-hupB double-deletion mutant. HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo. HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development. In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo. These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding [Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145], plays an important role in the physiological function of this protein. The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.
通过基因工程构建了大肠杆菌类组蛋白HU和整合宿主因子（IHF）之间的嵌合蛋白，其中HU的部分臂区域被IHFα的相应区域（命名为HupANhimA）或IHFβ的相应区域（HupANhimD）取代；或者，α螺旋2-β1区域被IHFα的相应区域（HupAXhimA）或IHFβ的相应区域（HupAXhimD）取代（符号N和X表示NotI和XhoI连接点）。这些蛋白在hupA-hupB双缺失突变体中合成。HupANhimA在体外非特异性DNA结合方面表现出显著降低，并且在体内Mu噬菌体的复制转座中HU活性急剧丧失。HupANhimD在DNA结合能力方面也显示出显著降低，尽管该蛋白支持Mu噬菌体的发育。相比之下，其他两种嵌合HU蛋白在非特异性DNA结合能力方面仅表现出轻微变化：它们在体内保留了Mu噬菌体转座的活性。这些观察结果证实，HU-2的柔性臂（一个被认为用于DNA结合的结构域[Tanaka等人，《自然》310(1984)376 - 381；Goshima等人，《基因》96(1990)141 - 145]）在该蛋白的生理功能中起重要作用。结果表明，HU蛋白臂结构的独特构象，特别是该结构中两条链反平行β-折叠的N端一半，对于该蛋白的DNA结合能力很重要。
