Wang S, Cosstick R, Gardner J F, Gumport R I
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Biochemistry. 1995 Oct 10;34(40):13082-90. doi: 10.1021/bi00040a020.
The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.8 x 10(8) M-1 (Kd = 1.5 nM), and the nonspecific binding constant is 5.8 x 10(5) M-1 (Kd = 1.7 microM), giving a selectivity of approximately 1,000-fold for a specific site over random sequences. To study the molecular determinants specifying IHF binding, we used a series of 41 oligonucleotides containing adenine analogues that modified the surfaces of the major and minor grooves of the DNA. Many of the analogue substitutions within the previously defined consensus region caused decreased binding. Replacement with various analogues outside the consensus domain had little effect. Quantifying the binding constants for those sites with reduced affinities indicated an interaction with the minor groove within the consensus sequence. The binding constants of sites with 2-aminopurine and an inosine substitution within the same region suggest that IHF may also interact with the major groove. Thus, the specific interaction of IHF with its H' site likely involves interactions with both the minor and major grooves of the DNA.
大肠杆菌的整合宿主因子（IHF）是一种小型的、序列特异性DNA结合蛋白。通过凝胶阻滞试验估算了IHF的特异性和非特异性结合常数。IHF与λ附着位点（attP）中H'位点的平衡缔合常数为6.8×10⁸ M⁻¹（Kd = 1.5 nM），非特异性结合常数为5.8×10⁵ M⁻¹（Kd = 1.7 μM），这表明对特定位点的选择性比对随机序列高约1000倍。为了研究决定IHF结合的分子决定因素，我们使用了一系列41种含有腺嘌呤类似物的寡核苷酸，这些类似物修饰了DNA的大沟和小沟表面。在先前定义的共有区域内的许多类似物取代导致结合减少。在共有结构域外用各种类似物取代影响很小。对那些亲和力降低的位点的结合常数进行定量分析表明，其与共有序列内的小沟存在相互作用。在同一区域内具有2-氨基嘌呤和肌苷取代的位点的结合常数表明，IHF也可能与大沟相互作用。因此，IHF与其H'位点的特异性相互作用可能涉及与DNA的小沟和大沟的相互作用。
