A Human Factor Is Discovered
Nevertheless, we were hard-pressed to explain how proteins that bound to DNA sequences far from the core promoter of a gene could influence transcription of that gene. As is true of other laboratories, we began attacking this puzzle by trying to isolate human transcription factors, none of which had yet been found (with the exception of RNA polymerase itself). We assumed that once we had pure copies of the factors we would be able to gain more insight into exactly how they function.
Because many proteins that bind to DNA play no role in reading genes, we could not find transcription factors efficiently by screening nuclear proteins solely according to their ability to associate with DNA. My group therefore adopted a more discriminating strategy, looking for proteins that in a test-tube reaction both combined with DNA and stimulated transcription.
In 1982 William S. Dynan, a postdoctoral fellow in my laboratory, determined that some protein in a mixture of nuclear proteins fit all the requirements of a transcription factor. It bound to a regulatory element common to a select set of genes—an enhancer sequence known as the GC box (because of its abundance of G and C nucleotides). More important, when added to a preparation of nuclear proteins that included RNA polymerase, the substance markedly increased the transcription only of genes carrying the GC box. Thus, we had identified the first human transcription factor able to recognize a specific regulatory sequence. We called it speci- ficity protein 1 (Sp1).
We immediately set out to purify the molecule. One daunting aspect of this work was the fact that transcription factors tend to appear only in minuscule quantities in cells. Typically, less than a thousandth of a percent of the total protein content of a human cell consists of any particular factor. In 1985 James T. Kadonaga in my laboratory found a way to overcome this substantial technical barrier—and in the process introduced a powerful new tool that has since been used to purify countless transcription factors and other scarce DNA binding proteins.
Because Sp1 selectively recognized the GC box, Kadonaga synthesized DNA molecules composed entirely of that box and chemically anchored them to solid beads. Then he passed a complex mixture of human nuclear proteins over the DNA, predicting that only Sp1 would stick to it. True to plan, when he separated the bound proteins from the synthetic DNA, he had pure Sp1.
From studies carried out by Mark Ptashne and his colleagues at Harvard University, we knew that bacterial transcription regulators are modular proteins, in which separate regions perform distinct tasks. Once we learned the sequence of amino acids in Sp1, we therefore looked for evidence of distinct modules and noted at least two interesting ones.
One end of the molecule contained a region that obviously folded up into three "zinc fingers." Zinc-finger structures, in which parts of a protein fold around a zinc atom, are now known to act as the "hooks" that attach many activator proteins to DNA. But at the time Sp1 was only the second protein found to use them. Aaron Klug and his colleagues at the Medical Research Council in England had discovered zinc fingers, in a frog transcription factor, just a short time before [see "Zinc Fingers," by Daniela Rhodes and Aaron Klug; SCIENTIFIC AMERICAN, February 1993].
The other end of Sp1 contained a domain consisting of two discrete segments filled with a preponderance of the amino acid glutamine. We strongly suspected that this region played an important role during transcription because of a striking finding. In test-tube experiments, mutant Sp1 molecules lacking the domain could bind to DNA perfectly well, but they failed to stimulate gene transcription. This outcome indicated that Sp1 did not affect transcription solely by combining with DNA; it worked by using its glutamine-rich segment—now known as an activation domain—to interact with some other part of the transcription machinery. The question was, which part?