...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >STRUCTURE OF THE LEXA REPRESSOR-DNA COMPLEX PROBED BY AFFINITY CLEAVAGE AND AFFINITY PHOTO-CROSS-LINKING
【24h】

STRUCTURE OF THE LEXA REPRESSOR-DNA COMPLEX PROBED BY AFFINITY CLEAVAGE AND AFFINITY PHOTO-CROSS-LINKING

机译：亲和力切割和亲和力光交联的LEXA阻遏物-DNA复合物的结构

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The structure of the complex of full-length Escherichia coli LexA repressor with a consensus operator DNA fragment has been probed by affinity photo-cross-linking and affinity cleavage. These methods allow the determination of approximate intermolecular distances between a given protein residue and a base or sugar moiety within the operator. In a first step unique cysteine residues were introduced in positions 7, 28, 38, or 52 of the protein. In all four cases, the original amino acid was an arginine. The four amino acids in these positions were expected to be situated on the surface of LexA interacting with DNA, as infered from the structure of the LexA DNA binding domain [Fogh et al. (1994) EMBO J. 13, 3936-3944]. In a second step, these unique cysteine side chains of the purified proteins were chemically modified either with 4-azidophenacyl bromide or with S-(2-pyridylthio)cysteaminyl-EDTA. The first set of derivatives gives rise to UV-induced cross-linking which may be revealed by alkali/heat treatment; the second leads to direct DNA cleavage in the proximity of the derivatized amino acid. To reduce hydroxyl radical diffusion, the EDTA iron cleavage reactions were done in the presence of high amounts of glycerol. The results indicate that amino acids 7 and 52 are near nucleotide pairs 8-12 of the operator and that amino acids 28 and 36 of LexA are near nucleotide pairs 5-8 of the operator. The results unambiguously define the orientation of the LexA DNA binding domain relative to the operator and provide support for the model of the LexA-operator complex proposed by Knegtel et al. [(1995) Proteins 21, 226-236]. Ethylation interference experiments further suggest that Arg-7 contacts the phosphate group between nucleotides 8 and 9 as predicted by the model.
机译：全长大肠杆菌LexA阻遏物与共有操纵子DNA片段的复合物的结构已通过亲和光交联和亲和力裂解进行了探测。这些方法允许确定给定蛋白质残基与操纵基因内碱基或糖部分之间的近似分子间距离。第一步，将独特的半胱氨酸残基引入蛋白质的7、28、38或52位。在所有四种情况下，原始氨基酸均为精氨酸。从LexA DNA结合结构域的结构推断，这些位置的四个氨基酸预计位于与DNA相互作用的LexA表面上。 （1994）EMBO J. 13，3936-3944]。在第二步中，用4-叠氮苯甲酰溴或S-（2-吡啶硫基）半胱氨酰-EDTA对纯化的蛋白质的这些独特的半胱氨酸侧链进行化学修饰。第一组衍生物引起UV诱导的交联，这可以通过碱/热处理来揭示。第二个导致衍生氨基酸附近的直接DNA切割。为了减少羟基自由基的扩散，在大量甘油的存在下进行了EDTA铁裂解反应。结果表明氨基酸7和52接近操纵子的核苷酸对8-12，而LexA的氨基酸28和36接近操纵子的核苷酸对5-8。结果明确地定义了LexA DNA结合域相对于操纵基因的方向，并为Knegtel等人提出的LexA-操纵基因复合体模型提供了支持。 [（1995）Proteins 21，226-236]。乙氧基化干扰实验进一步表明，如模型所预测的，Arg-7与核苷酸8和9之间的磷酸基团接触。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号