Huang L, Sera T, Schultz P G
Department of Chemistry, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3969-73. doi: 10.1073/pnas.91.9.3969.
The cI repressor of bacteriophage 434, known as 434 repressor, binds to 14-bp operator sequences by means of a helix-turn-helix motif. To probe the requirements for selective DNA recognition by this class of DNA binding proteins, as well as to generate new proteins with altered specificities, a library of approximately 3 x 10(6) mutants was generated that contains all permutations of five residues in the recognition helix (helix 3) of the repressor. These mutants were then selected in vivo for their ability to bind both wild-type (WT) and mutant operator sequences. The results of the selection demonstrate that four of these residues--Gln28, Gln29, Ser30, and Gln33--play a critical role in recognition of the WT operator. A number of repressors with mutations at Thr27 showed altered DNA binding affinities and specificities. The approach described here may also prove useful in studies of DNA recognition by other classes of DNA binding proteins.
噬菌体434的cI阻遏蛋白，即434阻遏蛋白，通过螺旋-转角-螺旋基序与14bp的操纵序列结合。为了探究这类DNA结合蛋白对选择性DNA识别的要求，以及产生具有改变特异性的新蛋白，构建了一个约3×10⁶个突变体的文库，其中包含阻遏蛋白识别螺旋（螺旋3）中五个残基的所有排列组合。然后在体内筛选这些突变体，以检测它们结合野生型（WT）和突变操纵序列的能力。筛选结果表明，其中四个残基——Gln28、Gln29、Ser30和Gln33——在识别野生型操纵序列中起关键作用。许多在Thr27处发生突变的阻遏蛋白表现出改变的DNA结合亲和力和特异性。本文所述方法可能在其他类DNA结合蛋白的DNA识别研究中也有用。
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