Liu Y C, Matthews K S
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
J Biol Chem. 1993 Nov 5;268(31):23239-49.
A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding. Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility. The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex. Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility. Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold. In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers. The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs. In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp. Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound. These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction.
使用一系列化学合成的色氨酸（trp）和突变型操纵基因DNA来检测trp阻遏物的结合情况。尽管如先前报道的那样，大多数DNA仅观察到单一的阻遏物-操纵基因复合物，但不同的DNA序列显示出两个滞后复合物以及一条迁移速度更快的条带。两个滞后条带的相对强度随阻遏物浓度的变化表明，在主要的阻遏物-操纵基因复合物形成过程中，二聚体之间可能发生协同相互作用。直接化学计量测量表明，在主要的阻遏物-操纵基因复合物条带中发现的化学计量比为2:1（每个操纵基因两个二聚体），而对于迁移速度更快的次要阻遏物-操纵基因复合物条带，若存在，则观察到的化学计量比为1:1。对于与TrpEDCBA、aroH和Trp-PL（TrpEDCBA的一种对称性增加的衍生物）操纵基因序列相对应的40碱基对（bp）trp操纵基因，以及包含来自TrpEDCBA的半个结合位点与lac操纵基因序列结合的杂合操纵基因，观察到类似的具有2:1化学计量比的单一复合物的滞后模式，尽管对半个位点DNA的表观亲和力降低了10倍。相比之下，对于Trp-PR（TrpEDCBA的另一种对称性增加的衍生物），1:1二聚体-操纵基因复合物更为突出，这表明序列背景可能会降低二聚体之间的协同作用。观察到的化学计量比还取决于所使用的TrpEDCBA操纵基因DNA的长度，从40 bp和33 bp的TrpEDCBA DNA主要为2:1转变为29 bp、26 bp和20 bp的TrpEDCBA DNA主要为1:1。此外，对于长度为33 bp、29 bp和26 bp的DNA，阻遏物-操纵基因复合物对电泳的稳定性降低。基于两个杂合的40 bp TrpEDCBA/lac半个结合位点DNA的结合数据和足迹模式，似乎阻遏物与特定的trp半个位点紧密结合，而DNA的非特异性半个位点结合较松散。这些结果表明，阻遏物二聚体-二聚体相互作用可能是trp阻遏物-操纵基因相互作用中的一个重要特征。
