MULTIPLE MOLECULES OF INTEGRATION HOST FACTOR (IHF) AT A SINGLE DNA-BINDING SITE, THE BACTERIOPHAGE-LAMBDA COS-I1-SITE
MULTIPLE MOLECULES OF INTEGRATION HOST FACTOR (IHF) AT A SINGLE DNA-BINDING SITE, THE BACTERIOPHAGE-LAMBDA COS-I1-SITE
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DOI：
10.1016/0300-9084(94)90025-6
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发表时间：
1994-01-01
期刊：
影响因子：
3.9
通讯作者：
KOSTURKO, LD
中科院分区：
文献类型：
--
作者：
MORSE, BK;MICHALCZYK, R;KOSTURKO, LD
整合宿主因子（IHF）是一种大肠杆菌蛋白质，它与DNA序列特异性结合，并在许多细胞内过程（包括λ DNA包装）中充当辅因子。在凝胶移位实验中，cos DNA，一种含有λ DNA包装识别信号的DNA片段，当与纯IHF结合时形成多个蛋白质-DNA复合物。铜（II）-1，10邻二氮杂菲足迹的个人IHF-cos DNA复合物表明，多个复合物的形成不会导致IHF结合到连续的网站上的cos DNA片段。相反，来自两个IHF-cos复合物的DNA足迹显示仅在一个位点处具有保护作用。第一个复合物中的DNA仅部分地受到保护而不被核酸裂解，而第二个移动较慢的复合物中的DNA在相同的结合位点处受到完全保护。定量Western印迹实验确定了两种复合物中IHF与DNA的相对化学计量。结果证实，两个分子的IHF结合在一个单一的网站在cos片段。这个位点，cos I1，与IHF共有序列有两个匹配，但这两个匹配有十三个核苷酸中的八个重叠。使用扩展的IHF共有序列搜索cos周围的DNA序列，发现了与这些序列相邻的额外的低亲和力共有匹配。铜（II）-1，10邻二氮杂菲足迹和IHF-cos I1配合物的化学计量的程度表明，无论是两个分子的IHF结合重叠的网站，或IHF结合到一个网站的低亲和力毗邻强网站。应用热力学模型与IHF和COS DNA的凝胶位移实验的结果表明，多个复杂的形成需要两个IHF结合位点之间的合作相互作用。
Integration host factor (IHF) is an E coli protein that binds DNA sequence-specifically and serves as a cofactor in many intracellular processes including lambda DNA packaging. In gel shift experiments, cos DNA, a DNA fragment containing the recognition signal for lambda DNA packaging, forms multiple protein-DNA complexes when combined with pure IHF. Copper(II)-1,10 orthophenanthroline footprinting of individual IHF-cos DNA complexes shows that multiple complex formation does not result from IHF binding to successive sites on the cos DNA fragment. Instead, the footprinting of DNA from two IHF-cos complexes shows protection at one site alone. DNA in the first complex is only partially protected from nucleolytic cleavage, while DNA in the second, slower-moving, complex is completely protected at the same binding site. Quantitative Western blotting experiments determined the relative stoichiometry of IHF to DNA in the two complexes. The results confirm that two molecules of IHF bind at a single site in the cos fragment. This site, cos I1, has two matches to the IHF consensus sequence, but the two matches overlap by eight of thirteen nucleotides. A search of the DNA sequence around cos, using an expanded IHF consensus sequence, has revealed additional, low-affinity consensus matches, contiguous to these. The extent of the copper(II)-1,10 orthophenanthroline footprint and the stoichiometry of the IHF-cos I1 complexes suggest that either two molecules of IHF bind to overlapping sites, or IHF binds to a site of low affinity contiguous to a strong site. Application of a thermodynamic model to the results of gel shift experiments with IHF and cos DNA suggests that multiple complex formation requires cooperative interaction between the two IHF binding sites.