The lac operator is DNA.

How repressors act at the molecular level to tmrn off genes is only now beginning to be worked out. Most vital to this understanding is whether the operator, defined genetically as the site for the action of a repressor, would turn out to be part of a DNA molecule, a region of a messenger RNA molecule, or even a protein. Now that two specific repressors (lactose and X) are available," 2 it is possible to attack this problem directly. This was first done by Ptashne,3 who showed that the X phage repressor, a 30,000-mol-wt protein, binds specifically only to that region of a X-DNA molecule where the genetic receptors (operators) lie. Here we report experiments, with the lactose repressor, that further show that the operator is DNA. This repressor binds specifically to DNA molecules that carry the lactose operon, attaching only to that unique region of the DNA molecule where the mutations that characterize the operator lie. Furthermore, this repressor is released from the operator by inducers, such as IPTG (isopropyl-1-thio-,3-D-galactoside). The Principle of the Experiment.-The assay for the lac repressor used the fact that this repressor could bind radioactive IPTG tightly enough to be detected by equilibrium dialysis. Since the relevant affinity is on the order of 106 M, only repressor concentrations in this range are detectable. This assay cannot be used immediately to study the interaction of the repressor with the operator because attainable gene concentrations are so small. Even if one uses lac genes carried on the DNA isolated from a defective phage, one set of genes for each 3 X 107 mol wt, a 3 mg/ml solution of DNA is only 10-7 M. The binding of repressor to such DNA would only be barely visible by the IPTG binding assay. An alternative approach is to prepare radioactive repressor, to follow the molecule directly. The IPTG binding assay has been used to guide a several thousandfold purification of unlabeled repressor. With this knowledge one could try to mimic this purification on a small scale with very highly labeled proteins-a blind purification, since the specific labeling is so high and the physical scale of the radioactive preparation so small that one cannot follow the purification by the IPTG binding assay. A complete purification is unnecessary; all that is required is a sufficient enrichment of the lac repressor so that it represents a reasonable fraction of the labeled material, while other proteins that bind to DNA are removed so that specific effects can be observed. By including a sizing step, isolating only 7-8S material that includes the lac repressor, one can easily distinguish later a small fraction of the label binding to and sedimenting with 35S dlac phage DNA. The details of the purification are given in the experimental methods. Sulfurlabeled proteins from a triploid strain, carrying three copies of the Itc genes, are fractionated with ammonium sulfate and then run on a DEAE Sephadex column using a step elution. The material is then concentrated and run upon a glycerol gradient. Since the repressor, as determined by its binding to IPTG, sediments near 7.68, samples are taken from this region of the gradient, determined by an aldolase marker. When this radioactive material is mixed with phage DNA carry-