In a seminal paper in 1961, Jacob and Monod proposed a general model for bacterial gene regulation. Mainly on the basis of genetic information, they predicted the existence of repressors encoded by a new class of non-structural genes. Repressors controlled the transcription of single or groups of genes. They were regulated by metabolites or other environmental factors, which could promote or antagonize their function. Repressors acted through operators that functioned in cis with the regulated genes (see Milestone 5); however, the precise nature of the repressors and how they interacted with operators was unclear.

The repressor model was sufficient to explain the current knowledge of regulated gene expression without a need to invoke activators (see Milestone 4), and accounted for systems as diverse as the lactose (lac) system of Escherichia coli and lambda phage genes. In the lysogenic state, lambda phage DNA is present in infected bacteria as a prophage with most phage genes not being expressed. Activation of gene expression and a switch to the lytic phase can be triggered by stimuli such as ultraviolet light. Similarly, expression of several genes — organized in a unit known as the lac operon — encoding enzymes that metabolize lactose is induced by external lactose or other β-galactosides, such as isopropyl-β-D-thiogalactoside (IPTG). The repressor model postulated that, in both instances, the respective inducers function by inhibiting the lambda and lac repressors that otherwise repress gene expression.

This visionary paper stimulated a quest for the isolation of repressors, which culminated in the isolation of the lac and the lambda repressors in 1966 and 1967, respectively. Using a mutant strain that was hypersensitive to IPTG, in which they assumed the repressor to bind more tightly to the inducer, Gilbert and Müller-Hill biochemically isolated cell fractions that bound labelled IPTG. They identified a protein of ∼15–20 kDa as the lac repressor. Using phage mutants in which most genes were shut off and by inhibiting host protein synthesis, Ptashne purified differentially labelled proteins from bacteria infected with either wild-type phage or repressor mutants and isolated the lambda repressor as a protein of ∼30 kDa.

The biochemical isolation of repressors paved the way for the elucidation of their mechanism of action. Subsequent work showed that both the lambda and lac repressors bound to DNA. This binding occurred at specific sites in the lac operon and the lambda genome, and, in the case of the lac repressor, DNA binding was shown to be inhibited by IPTG.

Although transcriptional regulation is far more complex and involves activators in addition to repressors, this early work and the astonishing foresight of Jacob and Monod established many of the key principles of gene regulation — most importantly, the existence of trans-acting factors that control gene transcription by binding to control DNA sequences near those genes.