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首页> 外文期刊>Journal of Molecular Biology >Conformational dynamics of a transposition repressor in modulating DNA binding
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Conformational dynamics of a transposition repressor in modulating DNA binding

机译：易位阻遏物在调节DNA结合中的构象动力学

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摘要

The repressor of bacteriophage Mu functions in the establishment and maintenance of lysogeny by binding to Mu operator DNA to shut down transposition. A domain at its N terminus functions in DNA binding, and temperature-sensitive mutations in this domain can be suppressed by truncations at the C terminus. To understand the role of the C-terminal tail in DNA binding, a fluorescent probe was attached to the C terminus to examine its environment and its movement with respect to the DNA binding domain. The emission spectrum of this probe indicated that the C terminus was in a relatively hydrophobic environment, comparable to the environment of the probe attached within the DNA-binding domain. Fluorescence of two tryptophan residues located within the DNA-binding domain was quenched by the probe attached to the C terminus, indicating that the C terminus is in close proximity to this domain. Addition of DNA, even when it did not contain operator DNA, reduced quenching of tryptophan fluorescence, indicating that the tail moves away from the DNA-binding domain as it interacts with DNA. The presence of the tail also produced a trypsin hypersensitive site within the DNA-binding domain; mutant repressors with an altered or truncated C terminus were relatively resistant to cleavage at this site. Interaction of the wild-type repressor with DNA greatly reduced cleavage at the site. A repressor with a temperature-sensitive mutation in the DNA-binding domain was especially sensitive to cleavage by trypsin even in the presence of DNA, and the C-terminal tail failed to move in the presence of DNA at elevated temperatures. These results indicate that the tail sterically inhibits DNA binding and that it moves during establishment of repression. Such conformational changes are likely to be involved in communication between repressor protomers for cooperative DNA binding.
机译：噬菌体Mu的阻遏物通过与Mu操纵子DNA结合以关闭转座而在溶原性的建立和维持中起作用。在其N末端的一个域在DNA结合中起作用，该域中的温度敏感突变可被C末端的截短所抑制。为了了解C末端尾巴在DNA结合中的作用，将荧光探针连接到C末端以检查其环境以及其相对于DNA结合域的运动。该探针的发射光谱表明，C末端处于相对疏水的环境中，与附着在DNA结合域内的探针的环境相当。位于DNA结合结构域内的两个色氨酸残基的荧光被连接至C末端的探针淬灭，表明C末端紧邻该结构域。即使不包含操作员DNA，DNA的添加也会减少色氨酸荧光的猝灭，这表明尾巴在与DNA相互作用时会从DNA结合域移开。尾巴的存在还在DNA结合域内产生了胰蛋白酶超敏位点。具有改变的或截短的C末端的突变体阻遏物在该位点相对抗切割。野生型阻遏物与DNA的相互作用大大减少了该位点的切割。即使在存在DNA的情况下，在DNA结合域中具有温度敏感突变的阻遏物对胰蛋白酶的切割也特别敏感，并且在存在DNA的情况下，高温下C末端的尾巴无法移动。这些结果表明，尾巴在空间上抑制了DNA结合，并且在建立抑制过程中移动。此类构象变化可能参与阻遏物前体之间的相互作用，以实现协同DNA结合。

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