These days, the lactose system of Escherichia coli is regarded as the classic example of an operon. It is used by every modern molecular biology or genetics textbook worth its salt to illustrate the basic principles of gene transcription. The regulation of the structural genes lacZ, lacY and lacA through binding of the lacI-encoded repressor to the operator site (lacO) was famously uncovered by Jacob and Monod at the beginning of the 1960s (see Milestone 2). However, it would take the rest of the decade, and some complex bacterial genetics, before their fellow scientists uncovered the key cis-regulatory element of transcription — the promoter.
The lacI-encoded repressor binds to the lacO operator sequence and prevents transcription of the lac structural genes (top). Binding of an inducer prevents the repressor from binding lacO and the structural genes are transcribed (bottom). Studies using the lac operon identified the promoter (p) as a cis controlling element for gene transcription in the 1960s.
Even when they are fully induced, different enzymes are present in bacterial cells in widely varying amounts. So what controls the maximum rate of enzyme production? Scaife and Beckwith subscribed to the idea that gene expression could be controlled by a specific chromosomal site that is able to limit the rate of initiation of either transcription or translation. They therefore tried to identify such a site by searching for mutations that would decrease or abolish the potential of the lac operon to be expressed. Several mutations that resulted in reduced levels of all three lac structural genes were picked up in a mutagenesis screen. The researchers showed that these mutations were cis-dominant by confirming that their effects were not relieved by the introduction of a second lac region into the cell. Furthermore, using gene-repression experiments and detailed genetic analysis, they showed that these mutations were separate from the repressor–operator system, and mapped outside lacI and probably also outside lacO.
By 1968, Ippen and colleagues had built on these initial results using a series of mutants in which increasing amounts of the lac operon and its upstream region had been deleted in a stepwise fashion. Recombination analysis showed that the mutations mapped to just before the beginning of the lac operon. The authors designated the site of the mutations as the 'promoter' (reintroducing a term that had been used previously, with a different meaning, by Jacob, Ullman and Monod). So did the promoter control transcription or translation?
Ippen and co-workers suggested that it is transcription that begins at the promoter. Moreover, as there was no evidence that the lacO region is translated, they proposed that translation begins just downstream of lacO, in a region that Jacob, Ullman and Monod had previously suggested was important for gene expression.
The function of the promoter was elegantly confirmed by Eron and Block in 1971 using an in vitro transcription system. They showed that mutations in the lac promoter altered the levels of transcription initiation, and confirmed the functional significance of transcription initiation in vitro by showing that it required the auxiliary sigma factor (see Milestone 6), was negatively regulated by the lac repressor, and was positively regulated by cyclic AMP (cAMP) and cAMP-binding protein (see Milestone 4). Furthermore, confirming extensive genetic work from the Beckwith group, they showed that the lac promoter had two parts: one that mediated transcription initiation and one that mediated positive regulation. So, by the start of the 1970s, the concept of the promoter, and its potential for complexity of both regulation and structure, had emerged.
