Werner M H, Clore G M, Gronenborn A M, Nash H A
Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Curr Biol. 1994 Jun 1;4(6):477-87. doi: 10.1016/s0960-9822(00)00108-1.
Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction.
We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences.
Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.
大肠杆菌整合宿主因子（IHF）是一种DNA结合蛋白，参与多种生化功能。在其许多活性中，IHF似乎作为一种结构元件，极大地扭曲结合DNA的构象。IHF是由不同亚基组成的二聚体，每个亚基约90个氨基酸长。一个二聚体与一个30碱基对（bp）的靶标特异性相互作用，但在该结合位点的仅一半中发现了保守性良好的序列。因此，长期以来，IHF-DNA系统一直被视为蛋白质-DNA相互作用中不对称性的范例。
我们分离了IHF的亚基，并表明任一亚基都能够特异性识别天然IHF结合位点，并在体外支持λ位点特异性重组。迁移率变动和足迹数据表明，分离的亚基以同型二聚体的形式与DNA相互作用。我们还描述了对称双链体的设计，异源二聚体和同型二聚体的IHF可通过识别特定序列与之结合。
我们对IHF系统的体外操作表明，靶标DNA的结合和弯曲可以对称地完成。自然界中该系统发现的不对称性普遍存在，这表明可能存在其他选择力。我们认为，这些选择力源于IHF保护的DNA大小（30 bp）与蛋白质最初可接触的DNA长度（10 bp）之间的差异。这种差异意味着IHF靶标是分阶段识别的，并且可能使用于初始识别的蛋白质-DNA系统部分与相互作用表面的其余部分不同地进化。我们认为，可最初接触的DNA长度的限制是DNA结合蛋白的一个普遍特性。在这种情况下，可以预期许多蛋白质通过序列元件与DNA结合结构域之间的逐步而非同时接触来识别复杂靶标。
