In other words, these additional components were not themselves activators, for they did not bind to specific sequences in DNA. Nor were they basal factors, because low, unregulated levels of transcription could be achieved without them. They seemed to constitute a third class of transcription factor, which we called coactivators. We further proposed that coactivators, not TBP, were the targets for the protein binding domains of activators. We envisioned that activators would bind to selected coactivators to speed up the rate at which the basal complex set molecules of RNA polymerase in motion.
We were attracted to this scenario because we had difficulty imagining how a single protein, TBP, would have enough binding sites to accommodate all the activators made by human cells. But if the coactivators that were tightly linked to TBP bore multiple binding domains, the coactivators could collectively provide the docking sites needed to relay messages from hundreds or thousands of activators to the transcription engine.
It was Pugh who originally proposed that coactivators might function as such adapter molecules. His data soon convinced me he was probably correct, but not everyone in our laboratory agreed. Indeed, our weekly meetings in early 1990 were often punctuated by heated discussions. Not surprisingly, when the coactivator concept was presented to other workers in the field, they, too, expressed considerable skepticism. This reaction to an unexpected and complicating result was probably justified at that stage, because our data were only suggestive, not conclusive. We had not yet isolated a single coactivator.
Coactivators: The Missing Links
To satisfy ourselves and the scientific community that we were correct, we had to devise an experimental procedure that would unambiguously establish whether coactivators existed and operated as the relays we envisioned. For approximately two years after Pugh formulated the coactivator hypothesis, we struggled to purify an intact and functional complex containing TBP and all the other associated constituents of factor D. I must admit to some dark moments when it seemed the rather unpopular coactivator hypothesis might be based on some error in our studies.
The breakthrough finally came in 1991, when Brian D. Dynlacht, Timothy Hoey, Naoko Tanese and Robert Weinzierl —graduate students and postdoctoral fellows in our laboratory—found an ingenious way to isolate pure copies of factor D. Subsequent biochemical analyses revealed that, aside from TBP, the complete unit included eight previously unknown proteins. Because we did not yet have proof that these proteins could function as coactivators, we referred to them more generically as TBP-associated factors, or TAFs.
We became convinced that TAFs do indeed convey molecular signals from activators to the basal transcription apparatus after we separated the bound proteins from TBP and completed several more experiments. For instance, we were able to show that mixing of the activator Sp1 with basal factors and RNA polymerase enhanced production of messenger RNA from a gene containing a GC box only when TAFs were added as well. Later, Jin-Long Chen, a graduate student, combined purified TBP and the eight isolated TAFs in a test tube along with a human gene and the rest of the basal transcription machinery. The various proteins assembled on the gene and proved able to respond to several different types of activator proteins. These activators, we later showed, produced their effects by coupling directly with selected TAFs. Together the coactivators in factor D do indeed constitute a kind of central processing unit that integrates the regulatory signals issued by DNA-bound activators.