In 1988 when we began searching for the target of Sp1, we had some idea of where it lay. Our guess was based on an emerging understanding of the so-called basal transcription complex, one part of which seemed to be a likely target.
Closing in on a Target
In the mid-1980s Robert G. Roeder and his colleagues at the Rockefeller University had shown that RNA polymerase cannot transcribe eukaryotic genes unless several other transcription factors—now called basal factors—also collect on the core promoter. And over the course of the 1980s, Roeder's laboratory and others had identified at least six of those essential factors, called A, B, D, E, F and H.
In a test tube, this assembly of factors enabled RNA polymerase to transcribe a bound gene at a basal—low and invariant —rate, but it could not by itself modulate that rate. It was as if someone had constructed and switched on the engine of a car but had lost use of the steering wheel, the accelerator and the brakes. For instance, when my group mixed the components of the complex (including RNA polymerase) with a gene containing a GC box, we obtained a low, unchanging level of transcription. We saw a marked increase in transcription only when we incorporated Sp1 into the mixture.
By the late 1980s it was apparent that human cells harbor at least two separate classes of transcription factors. Basal factors are required for initiation of transcription in all genes; other proteins —activators and repressors—dictate the rate at which the basal complex initiates transcription. Different genes are controlled by distinct combinations of activators and repressors. We now suspect that in the body the basal complex arises spontaneously only rarely; most of the time, cells depend on activators to initiate its construction.
These various discoveries suggested that the glutamine-rich domain of Sp1 enhanced transcription by contacting a basal factor. More specifically, we suspected that Sp1 latched on to factor D, and facilitated its attachment to the promoter. We focused on this subunit because Phillip A. Sharp and Stephen Buratowski of the Massachusetts Institute of Technology had shown that it can land on the core promoter before all other basal factors and can facilitate assembly of the complete basal engine. In fact, factor D is the only basal component able to recognize DNA. It binds selectively to a sequence called the TATA box, found in the core promoters of many eukaryotic genes.
To pursue our hypothesis, we needed to know more about the composition of factor D, which we assumed was a solitary protein. Other investigators also wanted to know its makeup, and so the race was on to attain pure copies. Isolation from human cells proved more challenging than anyone anticipated. Consequently, many groups eventually tried their luck with yeast cells. Finally, in 1989, several laboratories independently succeeded in isolating a yeast protein that displayed the expected properties of factor D. The protein, named TBP (for TATA binding protein), recognized and bound selectively to the TATA box and led to a low level of transcription when it was joined at the core promoter by RNA polymerase and other constituents of the basal machinery.
Believing that the TBP protein was factor D itself, we undertook to test this idea in additional studies. Once we did that, we intended to determine exactly which regions of TBP were contacted by Sp1 and other regulators. Little did we know that we were about to be completely thwarted—and to make a critical discovery.
When B. Franklin Pugh in our laboratory replaced the impure preparations of factor D previously used in our test-tube reactions with purified molecules of TBP, he had no trouble replicating the earlier finding that such substitution in no way disrupted basal transcription. To our surprise and consternation, though, he found that Sp1 was no longer able to influence the basal machinery. We had to conclude that factor D and TBP were not, in fact, equivalent and that factor D actually consisted of TBP plus other subunits. (It is now known that many transcription factors consist of more than one protein.) Apparently, those subunits were not needed for operation of the basal machinery, but they were essential to regulation of that machinery by activators.