Journal Article: Majors, J. (1975). "Initiation of in vitro mRNA synthesis from the wild-type lac promoter." PNAS. 72:11 pp 4393-4398.
One of the most important discoveries ever made in the history of molecular biology was that of the lac operon. The lac operon taught us about gene regulation and mechanisms of gene activation and repression.
When the operon was being characterized, it was not immediately understood exactly how the lac repressor inhibited transcription of the operon. In this 1975 PNAS paper, John Majors presents evidence that the lac repressor and RNA polymerase compete for binding to overlapping DNA sequences. This data would indicate that the reason repressor prevents transcription is because it inhibits RNAP binding to DNA and therefore inhibits formation of the initiation complex. Majors obtains this data by testing the in vitro transcription rate of different reactions in which repressed DNA is exposed to RNAP and inducer at different times. When the repressor is induced by IPTG (an allolactose analog), transcription can occur in the mixture.
When IPTG is added before RNAP, Majors notices that there is a lag phase in the transcription test, which he interprets as the period during which RNAP binds to DNA (happens fairly slowly). When IPTG induces the repressor, it dissociates from the DNA extremely rapidly. However, when you add RNAP to DNA, it associates with the DNA fairly slowly. Consequently, you can tell exactly when during the reaction the RNAP begins to associate with the DNA by observing the lag phase. Majors adds RNAP to a mixture that has already been incubated with repressor. If the RNAP and repressor were bound at the same time, then when the repressor is induced, there would be no lag phase in transcription because you don't have to wait for the RNAP to associate with the DNA. However, if the RNAP cannot bind to repressed DNA, then only after the repressor is induced can the RNAP begin to associate, so you would see a lag phase in transcription. It turns out that there is indeed a lag phase after induction, so RNAP and repressor compete for binding sites in the regulatory region of the lac repressor.
This discovery, though it seems pretty obvious to us, is actually extremely important to understanding exactly how the lac repressor works. Also, reading through the methods, I feel so spoiled by the conveniences of the modern day lab! It took so many steps to obtain a 200 bp fragment of DNA (purification of DNA, excision, isolation, purification of restriction enzyme, digestion of fragment, etc….). Today you would just amplify the fragment you want from a genomic DNA sample using PCR. Also, in 1975, there was no NEB database of any restriction enzyme you could imagine. Anyhow, it is really cool to see how much work went into every discovery.
Image from: http://www.ebi.ac.uk/biomodels-main/static-pages.do?page=ModelMonth%2F2006-12