Shin J A, Ebright R H, Dervan P B
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Nucleic Acids Res. 1991 Oct 11;19(19):5233-6. doi: 10.1093/nar/19.19.5233.
Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.
乳糖阻遏蛋白（LacR）是一种具有螺旋-转角-螺旋基序的序列特异性DNA结合蛋白。基于质子核磁共振光谱研究，卡普泰因及其同事提出，LacR的螺旋-转角-螺旋基序与DNA结合的方向与λ阻遏蛋白、λ cro、434阻遏蛋白、434 cro和CAP的螺旋-转角-螺旋基序相反[博伦斯，R.，谢克，R.，范博姆，J.和卡普泰因，R.，《分子生物学杂志》193，1987，213 - 216]。在本研究中，我们通过亲和切割法确定了LacR - DNA复合物中LacR的螺旋-转角-螺旋基序的方向。将DNA切割部分EDTA.Fe连接到对应于LacR DNA结合结构域的56个残基合成蛋白的N端。我们已经在修饰后的蛋白与LacR的左侧DNA半位点之间形成了复合物。所得DNA切割位置相对于左侧DNA半位点的位置为卡普泰因及其同事的提议提供了有力支持。
