Goshima N, Kohno K, Imamoto F, Kano Y
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Gene. 1990 Nov 30;96(1):141-5. doi: 10.1016/0378-1119(90)90355-u.
We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.
我们通过在M13mp18载体上进行合成寡脱氧核糖核苷酸定向的位点特异性诱变，构建了编码HU-1蛋白的大肠杆菌hupB基因的四个突变体。HupBR45蛋白含有Arg58→Gly和Arg61→Gly的改变，而HupBF3、HupBK2和HupBA1蛋白分别含有Phe47→Thr、Lys37→Gln和Ala30→Asp的改变。HupBF3和HupBR45在42℃下不能在hupA-hupB-himA三重突变体中维持正常细胞生长，不能在hupA-hupB双突变体中进行mini-F或RSF1010增殖，也不能支持Mu噬菌体发育，而HupBA1和HupBK2能支持这些细胞活动。DNA亲和柱层析显示HupBF3和HupBR45对DNA的亲和力降低。这些观察结果表明，HU-1分子C端一半的臂结构中的两个高度保守的Arg残基以及连接分子两半的短β-折叠中的一个Phe残基对于该蛋白的DNA结合能力和生物学功能很重要。
