Nucleic Acids Research

The interaction between lac repressor and a small operator DNA fragment have been examined by circular dichroism spectroscopy. The binding of Lac repressor on the operator induces a conformational change of the DNA which is different from that observed upon non specific binding on non operator DNA. The CD titration curve indicates that the stoechiometry of interaction is complex. A two operators-one repressor complex was found. This result was confirmed by a gel filtration experiment. The interaction between the Lac repressor and the lac operator of E.coli controls the expression of the structural genes involved in the metabolism of lactose. This system is one of the most suitable for physicochemical studies of specific interaction between nucleic acids and proteins. The sequence of the Lac repressor and of the Lac operator are known (1,2,3) and much information has been obtained from genetic and biochemical studies (for reviews see references 4 and 5). Among the various aspects of this interaction the conformational one is very important. It is clear that to design a model of the interaction between the Lac repressor and the Lac operator it is necessary to know if there is a conformational change of one of the partners in the complex and if possible to characterize it. Recently, Ogata and Gilbert (6,7), using a method based on the chemical modification of the Lac operator by dimethylsulfate in the presence and in the absence of Lac repressor have shown that the contact points between the protein and the nucleic acid lies on the same side of the DNA double helix supposed to be in the classical B form. A similar conclusion was reached by Caruthers and coworkers using synthetic oligonucleotides of sequences related to that of the operator (8-11). It would be of interest to determine whether the conformation of DNA in the complex is such that all the contact points are still on the same side. Until now very little information is available on the conformation of the Lac operator in the complex with Lac repressor. Wang et ac. (12) have X) IRL Pre Umited, 1 Falconberg Court, London W1V 5FG, U.K. 5175 Nucleic Acids Research studied, by filtration on nitrocellulose membranes, the binding of Lac repressor to Xplac DNA with various degrees of negative superhelical twist. They found that Lac repressor unwinds the Lac operator by about 90°. Such a small amount of unwinding is inconsistent with the proposed cruciform structure of DNA in the complex (13). The lack of further information on the structure of the Iac operator in the complex with lac repressor is mainly due to the difficulty to obtain the large amounts of Lac operator which are needed to perform spectroscopic experiments. However the recent cloning of DNA fragments containing the Zac operator sequence in small plasmids has opened the new possibility of obtaining the Lac operator in large amount and thus performing physicochemical measurements (14-16). We wish to report in this article a circular dichroism study on the binding of Lac repressor to a small fragment of DNA containing the Lac operator sequence. It has been previously shown that the binding of Zac repressor to poly d(AT) and to non operator DNA is accompanied by a conformational change which can be followed by circular dichroism (17-19). This technique has been applied to determine the size of the binding site of the Lac repressor and also to quantitate the binding process between the DNA and the Lac repressor-. This led us to undertake this study with the hope that the same type of information could be obtained on the specific interaction between Lac repressor and Lac operator. During the course of this work, the first unexpected information we obtained was that the lac repressor can complex two Lac operators. To confirm this result we performed gel chromatography experiments which are also described in this article. MATERIALS AND METHODS The Lac operator DNA was purified from plasmid pBR 345 (20), a small plasmid containing two identical operator fragments derived from a chemically synthesized DNA, previously cloned in pMB 9. The central 21 base pairs of the fragment have the sequence of the natural Zac operator and are flanked on both sides by Eco RI restriction site sequences. After purification of large amounts of the plasmid, the lac operator fragment was generated by treatment with Eco RI endonuclease and purified away from plasmid vehicle by gel filtration (method to be published). Eco RI endonuclease was prepared according to Greene et ai. (21). Purity of the DNA fragment was checked by electrophoresis on a 15 % polyacrylamide gel. Concentrations of the fragment were determined using the following extinction coefficient at 260 nm : S260 = 377000 M cm . It was calculated, as done by O'Gorman et ac. for the same fragment (22) using the