Hayes J J, Tullius T D
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
Biochemistry. 1989 Nov 28;28(24):9521-7. doi: 10.1021/bi00450a041.
We report a new technique for quickly determining which nucleosides in a DNA molecule are contacted by a sequence-specific DNA-binding protein. Our method is related to the recently reported "missing contact" experiment [Brunelle, A., & Schleif, R. F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6673-6679]. We treat the DNA molecule with the hydroxyl radical to randomly remove nucleosides. The ability of protein to bind to gapped DNA is assayed by gel mobility shift. Nucleosides important to protein binding are identified by sequencing gel electrophoresis. The missing nucleoside experiment can be used to scan a DNA molecule at single-nucleotide resolution in one experiment. The bacteriophage lambda repressor-OR1 and cro-OR1 complexes were analyzed to evaluate the method. For both proteins, the most important contacts are located in the protein monomer that binds to the consensus half of the operator. These contacts correspond well to those found by mutational studies, and in the cocrystal structure of the lambda repressor-operator. The missing nucleoside data show that the amino-terminal arms of lambda repressor make energetically important contacts with positions 7 and 8 and the central dyad base pair of the operator. The amino-terminal arm that makes the most extensive contacts to DNA appears to be the one that emanates from the repressor monomer that binds to the consensus half of the operator, in agreement with the cocrystal structure. The lambda cro protein does not have an amino-terminal arm, and the missing nucleoside experiment clearly shows a lack of contacts to DNA in the central region of the operator in this complex.
我们报告了一种新技术，可快速确定DNA分子中的哪些核苷与序列特异性DNA结合蛋白发生接触。我们的方法与最近报道的“缺失接触”实验[布鲁内尔，A.，&施莱夫，R.F.（1987年）《美国国家科学院院刊》84，6673 - 6679]相关。我们用羟基自由基处理DNA分子以随机去除核苷。通过凝胶迁移率变动分析来检测蛋白质与缺口DNA结合的能力。通过测序凝胶电泳鉴定对蛋白质结合重要的核苷。缺失核苷实验可在一次实验中以单核苷酸分辨率扫描DNA分子。对噬菌体λ阻遏蛋白 - OR1和cro - OR1复合物进行了分析以评估该方法。对于这两种蛋白质，最重要的接触位点位于与操纵基因共有序列一半结合的蛋白质单体中。这些接触与通过突变研究以及λ阻遏蛋白 - 操纵基因共晶体结构所发现的接触位点非常吻合。缺失核苷数据表明，λ阻遏蛋白的氨基末端臂与操纵基因的第7和第8位以及中央二联体碱基对形成了能量上重要的接触。与共晶体结构一致，与DNA形成最广泛接触的氨基末端臂似乎是从与操纵基因共有序列一半结合的阻遏蛋白单体发出的那个。λ cro蛋白没有氨基末端臂，缺失核苷实验清楚地表明在该复合物中操纵基因的中央区域与DNA缺乏接触。
