Günes C, Müller-Hill B
Institut für Genetik, Universität zu Köln, Germany.
Mol Gen Genet. 1996 Jun 12;251(3):338-46. doi: 10.1007/BF02172524.
Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.
通过X射线晶体学进行的结构分析表明，色氨酸阻遏物的螺旋-转角-螺旋（HTH）基序第一个螺旋的第二个残基精氨酸69与以α为中心的共有色氨酸操纵基因第9位的鸟嘌呤之间存在直接接触。因此，我们将色氨酸阻遏物的第69位残基替换为甘氨酸、异亮氨酸、亮氨酸或谷氨酰胺，并在体内和体外测试所得阻遏物突变体与合成的以α或β为中心的对称色氨酸操纵基因变体的结合情况。我们提供了遗传和生化证据，表明色氨酸阻遏物第69位的异亮氨酸与以α为中心的色氨酸操纵基因第9位的胸腺嘧啶特异性相互作用。与以α为中心的色氨酸操纵基因第8和9位的其他碱基也存在相互作用。在体外，突变体RI69的色氨酸阻遏物与共有以α为中心的色氨酸操纵基因以及在第9位带有胸腺嘧啶的类似色氨酸操纵基因变体结合。对色氨酸阻遏物突变体RI69与以β为中心的色氨酸操纵基因对称变体相互作用的体内分析表明，与在第5位（对应于以α为中心的色氨酸操纵基因的第9位）由鸟嘌呤替换为胸腺嘧啶的以β为中心的对称色氨酸操纵基因变体的结合特异性发生了变化。
