Goldenberg D, Giladi H, Oppenheim A B
Department of Molecular Genetics, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Biochimie. 1994;76(10-11):941-50. doi: 10.1016/0300-9084(94)90019-1.
Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to DNA at a consensus sequence and bends DNA. HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA sequence. To investigate the protein determinants for IHF specificity we exchanged progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids. Replacement of 11 residues from the C-terminal alpha helix of Hip by the complementary eight residues of HupA (hybrid 1), had only minor effects on the DNA binding activity of the protein. As progressively longer segments of Hip were replaced by HupA, a precipitous decrease in IHF activity was observed. The hybrid with the longest substitution, hybrid 4, was totally inactive in vivo and could not be purified. None of the hybrid proteins could complement HU activity. Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants, led us to conclude that the structural integrity of the C-terminal alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms. This interaction stabilizes the arms to permit DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.
整合宿主因子（IHF）是大肠杆菌中的一种小型异源二聚体DNA结合蛋白，由分别由himA和hip基因编码的α和β两个亚基组成。IHF在共有序列处与DNA结合并使DNA弯曲。由hupA和hupB基因编码的HU蛋白与IHF相似，只是它不与特定的DNA序列结合。为了研究决定IHF特异性的蛋白质因素，我们将Hip C末端逐渐延长的片段与HupA的相应片段进行交换，并对四种这样的IHF/HU杂交体进行了体内和体外活性跟踪。用HupA的互补八个残基替换Hip C末端α螺旋的11个残基（杂交体1），对该蛋白的DNA结合活性只有轻微影响。随着Hip被HupA替换的片段越来越长，观察到IHF活性急剧下降。替换片段最长的杂交体4在体内完全无活性，无法纯化。没有一种杂交蛋白能够补充HU的活性。比较杂交体1、杂交体2和IHF点突变体的活性，使我们得出结论，C末端α螺旋的结构完整性及其空间位置，而非其氨基酸序列，对DNA结合特异性很重要。我们支持这样的假说，即IHF两个亚基的α螺旋3与IHF主体相互作用以固定臂部。这种相互作用稳定了臂部以允许DNA结合特异性。因此，IHF的C末端以间接方式影响对DNA上特定位点的识别。
