CHARACTERIZATION OF A SET OF INTEGRATION HOST FACTOR MUTANTS DEFICIENT FOR DNA-BINDING
CHARACTERIZATION OF A SET OF INTEGRATION HOST FACTOR MUTANTS DEFICIENT FOR DNA-BINDING
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DOI：
10.1006/jmbi.1993.1562
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发表时间：
1993-11-05
影响因子：
5.6
通讯作者：
NASH, HA
中科院分区：
文献类型：
--
作者：
GRANSTON, AE;NASH, HA
整合宿主因子（IHF）是一种序列特异性的DNA结合和DNA弯曲蛋白，由两个相关但不相同的亚基组成。我们报告的分离和表征羟胺诱导的功能丧失突变的基因编码的IHF亚基。为了筛选保留IHF正确折叠的突变体，从每个突变体制备澄清提取物，并通过免疫印迹法测定每个亚基的产生，并通过化学交联和随后的免疫印迹法测定异源二聚体的形成。发现符合这些标准的突变体提取物与特定IHF位点的结合较弱（如果有的话）。因此，这些等位基因鉴定可能影响IHF的DNA结合表面的残基的候选者。当投射到密切相关的HU蛋白的已知三级结构上时，这些残基被发现在表面上;然而，除了单个残基之外，蛋白的不同区域涉及每个亚基。这表明，尽管它们的同源性，IHF的每个亚基以不同的方式指导DNA识别和结合。为了证实这些突变的差异位置的意义，我们在每个亚基中引入了在另一个亚基的相应位置上作为功能丧失突变分离的改变。一般来说，工程突变体的表型与羟胺诱导的突变体的表型明显不同。特别是，大多数定点突变IHF蛋白形成或维持IHF：DNA复合物更容易比突变体，具有相同的变化，在其他亚基和分离的功能丧失突变体。我们讨论了突变的氨基酸残基的位置，因为它们涉及到一个拟议的分子模型的IHF：DNA复合物。
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.