Ogata R, Gilbert W
Proc Natl Acad Sci U S A. 1977 Nov;74(11):4973-6. doi: 10.1073/pnas.74.11.4973.
We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.
我们通过将阻遏物与溴尿嘧啶取代的操纵基因交联，确定了乳糖阻遏物与乳糖操纵基因之间重要的接触点。我们在一个包含乳糖操纵基因的55个碱基长的限制片段中，用溴尿嘧啶取代胸腺嘧啶，并在其5'端之一用32P进行标记。对该片段进行紫外线照射，会在溴尿嘧啶处产生单链断裂。我们通过使DNA变性并在聚丙烯酰胺凝胶上展示紫外线产生的片段，来检测序列中每个溴尿嘧啶处的断裂情况。在乳糖阻遏物存在的情况下，紫外线辐射未能在特定位点断裂。我们将此归因于一种竞争性反应，即DNA与阻遏物发生交联而非断裂。因此，这些可交联位点确定了乳糖阻遏蛋白在DNA大沟中靠近胸腺嘧啶甲基的位置。
