Oertel-Buchheit P, Porte D, Schnarr M, Granger-Schnarr M
Institut de Biologie Moléculaire et Cellulaire, CNRS UPR 6201 affiliated to INSERM, Strasbourg, France.
J Mol Biol. 1992 Jun 5;225(3):609-20. doi: 10.1016/0022-2836(92)90389-2.
The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.
来自大肠杆菌的LexA阻遏物是一种序列特异性DNA结合蛋白，与任何已知的参与DNA结合的结构基序均无明显的序列同源性。由于对该蛋白如何与DNA相互作用了解甚少，我们筛选并鉴定了大量基因内的第二位点突变，这些突变至少部分恢复了缺乏DNA结合能力的LexA突变阻遏物的活性。在47个案例中，这些突变的抑制作用是由于Ind-表型，推测这导致了突变蛋白的稳定。除了一个例外，这些第二位点突变均位于一个小区域（氨基酸残基80至85）内，该区域包括氨基酸残基84和85之间的LexA切割位点，既包括已知的Ind-突变，也包括新的变体，如GN80、GS80、VL82和AV84。其余26个独立分离的第二位点抑制突变均位于LexA的氨基末端DNA结合结构域内，位于第22位（位于螺旋1和螺旋2之间的转角处）以及第57、59、62、71和73位。这些氨基酸残基均位于螺旋3之后，在我们之前鉴定出一组LexA（Def）突变阻遏物的区域。在一些案例中，通过亚克隆或定点诱变去除了亲本LexA（Def）突变。除了一个例外，这些LexA变体在体内表现出比LexA野生型阻遏物更强的抑制作用。纯化了体内抑制率提高最显著的变体（LexA EK71，即Glu71----Lys），并使用改良的硝酸纤维素滤膜结合试验研究了其与短共有操纵基因DNA片段的结合。正如体内数据所预期的那样，LexA EK71与操纵基因以及（更显著地）与非操纵基因DNA的相互作用更强。测定LexA EK71和LexA野生型的平衡缔合常数与单价盐浓度的函数关系表明，与LexA野生型阻遏物相比，LexA EK71可能与操纵基因DNA形成额外的离子相互作用。LexA与非操纵基因DNA片段结合的比较进一步表明，在接近生理盐浓度条件下，LexA与共有操纵基因的相互作用具有非常高的选择性，特异性因子Ks/Kns为1.4×10(6)。
