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首页> 外文期刊>Journal of Molecular Biology >Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities
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Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities

机译：羧基末端结构域二聚体界面突变体434阻遏物改变了二聚化和DNA结合特异性

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摘要

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain. (C) 1998 Academic Press. [References: 42]
机译：由羧基（C）末端结构域介导的阻遏物的强二聚化是与DNA形成特定复合物和协同DNA结合形成四聚体的先决条件。我们已经根据同源UmuD'蛋白的晶体结构生成了434阻遏物C末端结构域的计算机模型。该模型预测93和168之间的一级序列中的残基有助于二聚体界面。我们改变了位于该区域的几个氨基酸残基。凝胶过滤和交联测定用于表征纯化的阻遏物C-末端结构域二聚体界面突变体的二聚作用的强度和特异性。这些结果表明氨基酸残基K121，H139，D161和N163有助于二聚作用的强度和/或特异性。噬菌体434阻遏物对434个操作员的相对亲和力部分取决于阻遏物在操作员位点中心非接触区域检测序列依赖性结构改变的能力。我们发现，C末端域二聚体界面突变体阻遏物二聚体的相对能力并不一定预测它们结合DNA的相对能力，并且这些蛋白质不足以检测操作员强度的非接触碱基依赖性差异。我们的结果表明，与这些突变蛋白复合的DNA结构与在野生型阻遏物-操纵子复合物中发现的结构不同，即使这些突变的位点位于与DNA接触的结构域之外。这些观察结果表明，需要C-末端结构域二聚体界面的结构完整性来适当地定向DNA接触N-末端结构域中包含的DNA结合信息。 （C）1998年学术出版社。 [参考：42]

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