Granston A E, Nash H A
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892.
J Mol Biol. 1993 Nov 5;234(1):45-59. doi: 10.1006/jmbi.1993.1562.
Integration host factor, IHF, is a sequence-specific DNA-binding and DNA-bending protein composed of two related but non-identical subunits. We report the isolation and characterization of hydroxylamine-induced loss-of-function mutations in the genes encoding the IHF subunits. To screen for mutants that preserve proper folding of IHF, clarified extracts were prepared from each mutant and were assayed for production of each subunit by immunoblotting and for formation of heterodimers by chemical cross-linking and subsequent immunoblotting. Extracts from mutants that met these criteria were found to bind a specific IHF site weakly if at all. These alleles therefore identify candidates for residues that may affect the DNA-binding surfaces of IHF. When projected onto the known tertiary structure of the closely related HU protein, these residues are found at the surface; however, with the exception of a single residue, different regions of the protein are implicated in each subunit. This suggests that, despite their homology, each subunit of IHF directs DNA recognition and binding in a distinct manner. To confirm the significance of the differential location of these mutations, we introduced in each subunit alterations that had been isolated as loss-of-function mutations at the corresponding position in the other subunit. In general, the engineered mutants have phenotypes that are strikingly different from those of their hydroxylamine-induced counterparts. In particular, most of the site-directed mutant IHF proteins form or maintain IHF:DNA complexes more readily than mutants that have the same change in the other subunit and were isolated as loss-of-function mutants. We discuss the positions of the mutant amino acid residues as they relate to a proposed molecular model of an IHF:DNA complex.
整合宿主因子（IHF）是一种序列特异性DNA结合和DNA弯曲蛋白，由两个相关但不相同的亚基组成。我们报告了编码IHF亚基的基因中羟胺诱导的功能丧失突变的分离和表征。为了筛选能够保持IHF正确折叠的突变体，从每个突变体中制备了澄清提取物，并通过免疫印迹分析每个亚基的产生情况，以及通过化学交联和随后的免疫印迹分析异二聚体的形成情况。发现符合这些标准的突变体提取物几乎不与特定的IHF位点结合，即使有结合也很微弱。因此，这些等位基因确定了可能影响IHF DNA结合表面的残基候选者。当将这些残基投射到密切相关的HU蛋白的已知三级结构上时，发现它们位于表面；然而，除了一个残基外，每个亚基中涉及蛋白质的不同区域。这表明，尽管它们具有同源性，但IHF的每个亚基以不同的方式指导DNA识别和结合。为了证实这些突变差异位置的重要性，我们在每个亚基中引入了在另一个亚基相应位置作为功能丧失突变分离出来的改变。一般来说，工程突变体的表型与它们的羟胺诱导对应物的表型有显著不同。特别是，大多数定点突变的IHF蛋白比在另一个亚基中有相同变化并作为功能丧失突变体分离出来的突变体更容易形成或维持IHF:DNA复合物。我们讨论了突变氨基酸残基的位置与提出的IHF:DNA复合物分子模型的关系。
