Albert Lasker
Basic Medical Research Award

Every field of science has one big problem that defies solution. In mathematics, the great unsolved problem is the Reimann hypothesis, which attempts to describe the irregular distribution of prime numbers. In physics, the Holy Grail is The Final Theory, which is supposed to explain all the known forces of nature with a single mathematical equation. In chemistry, the grand challenge is to enhance the reactivity of the carbon-hydrogen bond so that the Earth's vast quantities of hydrocarbons can be converted into cheap chemicals and drugs.

Biology may not have the exalted status of its three sister sciences, but its big unsolved problem may be the most challenging of all, and for sure it will be the most fun to solve. How does a single cell, the fertilized egg, give rise to a complex organism, a human being, which is made up of 250 types of cells, all having the same genetic instructions, yet each performing a different function? Red blood cells, for example, manufacture hemoglobin; pancreatic ß-cells produce insulin; skin cells make keratin.

For a cell to produce a protein, two things must happen. First, the gene must be copied from DNA into messenger RNA, and second, the mRNA must be translated into a protein. The first step—the copying of DNA into messenger RNA—is called gene transcription. In bacteria and other prokaryotic organisms, transcription is simple. A single enzyme called RNA polymerase, together with only one accessory protein, attaches to the promoter, which is a short stretch of DNA located just in front of the gene. This attachment of the polymerase is sufficient to begin the transcription process.

As many of you know, there is a current fascination with race horses, owing to the popular film Seabiscuit. I saw the movie and was struck by the analogy between horseracing and RNA transcription. After the RNA polymerase attaches to the promoter of a gene, it unwinds the double helix of the DNA, and then it slides along one strand of the unwound DNA like a thoroughbred horse racing around the track at Hialeah. As it gallops along the DNA, the polymerase constructs an RNA copy of the DNA. In bacteria, there is only one RNA polymerase. The same enzyme produces all three types of RNA: messenger RNA, which is the template for making proteins; ribosomal RNA, which is the machine that synthesizes the protein from amino acids; and transfer RNA, which delivers the amino acids to the ribosome.

The crucial step in bacterial gene transcription is the very first step—the binding step—in which the RNA polymerase attaches to the promoter. This binding is the only step in the entire process that can be regulated by an activator or a repressor protein. These regulators turn transcription on or off, depending on signals that the cell receives from the environment.

The conceptual framework for understanding prokaryotic transcription and its regulation was formulated over a 20-year period from 1955 to 1975, beginning with the classic studies of Jacob and Monod in bacteria. For reasons that are totally mystifying to me, Jacob and Monod never received a Lasker Award. Maybe the Francophobia that is so rampant today in the White House also afflicted the Lasker Jury 40 years ago. A more likely explanation is that the Nobel Committee jumped the gun, leaving the Lasker Jury at the starting gate. The Lasker Jury did, however, give its Basic Award in 1997 to Mark Ptashne, who worked out the detailed mechanism for gene regulation in bacteria.

Unlike bacteria, eukaryotic organisms such as humans encase their DNA in a nuclear compartment, which makes transcription in eukaryotes immensely more complex than in bacteria. As you'll see in a moment, the eukaryotic transcriptional apparatus is a horse of a different color. There was no simple and direct path to connect the bacterial discoveries of Jacob, Monod, and Ptashne to the complex machinery of eukaryotic cells. Scientists studying eurkaryotic transcription had to make a fresh start. This start was accomplished single-handedly by this year's recipient of the Lasker Basic Award, Robert Roeder of The Rockefeller University.

Roeder's work in eukaryotic transcription began in 1965 when he was a graduate student with William J. Rutter at the University of Washington in Seattle. Stimulated by the Jacob-Monod paradigm, Roeder set out to identify and purify the eukaryotic version of the prokaryotic RNA polymerase. But, to his surprise, he found not one but three different RNA polymerases, each devoted to one of the three types of RNA. RNA polymerase I forms ribosomal RNA, polymerase II forms messenger RNA, and polymerase III forms transfer RNA. The three RNA polymerases are referred to by the cognoscenti as Pol I, Pol II, and Pol III. Here you see the first level of complexity in eukaryotic transcription. For a 23-year-old graduate student to discover three eukaryotic RNA polymerases all at one time is analogous to the feat of the 18-year-old jockey Steve Cauthen, who won the Kentucky Derby, the Preakness, and the Belmont in a single year—1978.

In 1969, Roeder established his own laboratory at the Carnegie Institution in Baltimore and later at Washington University in St. Louis. There, he vigorously pursued the purification of the three RNA polymerases. He found that each enzyme was not a single protein, but a large complex of 8–10 different proteins. In a dazzling display of the biochemist's art, Roeder purified all three polymerases in their active forms. Remember, the early 1970s was the Ice Age of protein purification. It was all done in ice buckets and cold rooms. There were no His tags, Flag tags, GST-fusions, or fancy fast performance purification machines.

With pure polymerases in hand, Roeder developed, again single-handedly, the first "cell-free" test-tube reactions in which eukaryotic genes were transcribed in a faithful manner outside of cells. This accomplishment in the late 1970s opened the starting gates of RNA transcription in much the same way that Arthur Kornberg had opened the gates of DNA replication by synthesizing DNA in the test tube. Roeder's methods provided the basic system that scientists have used for all subsequent studies of gene transcription in test tubes.

Early in his studies—in 1979—Roeder made a crucial discovery: Unlike the situation in bacteria, none of the three eukaryotic RNA polymerases by themselves were capable of properly initiating transcription. Accurate transcription ensued only when the test-tube reactions were supplemented with protein extracts derived from cells. In the case of Pol II, which produces messenger RNA, Roeder discovered six factors that are required for Pol II to initiate transcription. These six general transcription factors and the polymerase itself are known today as the "general transcription machinery" because their action is required for the initiation of transcription of all protein-encoding genes in all eukaryotic organisms—from yeast to flies to humans. Each one of these six general transcription factors and Pol II itself is a multisubunit complex composed of 5–8 proteins. All together, the eukaryotic general transcription machinery consists of 44 different proteins. This is in striking contrast to bacteria where only one accessory protein is needed to assist the single polymerase in initiating transcription.

One of the six general transcription factors that Roeder discovered, called TFIID, lies at the very heart of the transcription process. Despite its arcane name, TFIID has become deeply embedded in the vernacular of molecular biology and appears in all textbooks of biochemistry, cell biology, and genetics. Unlike bacterial RNA polymerase, eukaryotic Pol II does not bind to promoter regions directly. Instead, it is TFIID that binds to the promoter, and it does this by recognizing a DNA sequence called the TATA box that is found in the promoters of most eukaryotic genes. After TFIID is bound to the TATA box, it recruits the other five general transcription factors, which assemble on the DNA in an ordered fashion. Once the six general transcription factors are properly assembled, they recruit Pol II to the DNA, and transcription proceeds.

With the discovery of TFIID, the gates of transcription were now open to all. A bevy of molecular biologists, including Philip Sharp at MIT and Pierre Chambon in Strasbourg, now entered the field. They began a lively race to purify the TATA-binding subunit of the TFIID complex. At the beginning of the race, the academic bookies gave the best odds to Roeder since he had already won the Triple Crown by discovering the three polymerases. But midway in the race, the odds changed in favor of Sharp when the bookies learned that Sharp was a Kentucky-born farm boy who grew up near Churchill Downs. In the end, Sharp and his young trainer Lenny Guarente won the race. Sharp, of course, received a previous Lasker Award and Nobel Prize—not for TFIID but for the discovery of split genes. Roeder was not deterred by Sharp's success and was "champing at the bit" to enter the next race. By this time, the transcription field had heated up, and eight labs took the starting line in the race to clone the gene for TFIID. The result was a dead heat. Roeder's paper was submitted to Nature on July 19, 1989, accepted on August 30, and published on September 28. Sharp's paper was submitted to Cell on July 20 (one day after Roeder's), accepted 4 days later, and published on September 22 (6 days before Roeder's). Arnie Berk, a dark horse, submitted his paper to the PNAS on July 19 (the same day as Roeder and one day before Sharp), but his paper was not published until October. The moral of this story is: fast jockeys publish in Cell!

Soon after the TATA-binding subunit of TFIID was cloned, its three-dimensional structure was solved in 1992 by Stephen Burley at Rockefeller in collaboration with Roeder and by the late Paul Siegler at Yale. The results were stunning. The protein is shaped like a saddle that sits like a jockey astride the TATA box, inducing a sharp bend in the DNA. The protruding upper surface of the TFIID serves as a docking site for the other general transcription factors and Pol II. In its explanatory potential, the vision of TFIID straddling the TATA box is one of the most informative crystal structures ever determined.

With this beautiful structure in hand, you might think Roeder would have been content to take a victory lap and just ride high in the saddle, but I can assure you it is totally out of character for Bob to rest on his laurels—either sitting in a saddle or relaxing in his cushy endowed chair at The Rockefeller.

By now, you may feel a bit overwhelmed by the immense complexity of eukaryotic transcription, and I have not even told you about three other layers of complexity in the system. One is the 3000 transcription factors that activate or repress the general transcription machinery. These factors were originally discovered by talented scientists like Robert Tjian and Steven McKnight. A second complexity is the multiprotein co-activator complexes that link the activators and repressors to the general transcription machinery. And finally there are the nucleosomes and the chromatin remodeling complexes, revealed by Roger Kornberg and David Allis, that determine how tightly the DNA is wrapped around histones and how accessible it is to the transcription apparatus. All that complexity is for another day—and perhaps even a few more Lasker Prizes.

There are two things that I hope you will remember about the eukaryotic transcription apparatus—its beauty and its complexity. The beauty, arising from the elegance of Robert Roeder's experiments and his remarkable artistry in combining biochemistry with molecular biology. The complexity, arising from multiple layers of multiprotein complexes interacting with each other in a multitude of diverse combinations that will ultimately, when it's all figured out, solve biology's big mystery.

In his 35 years in science, Bob Roeder and his colleagues have purified, cloned, and characterized an astounding number of proteins—almost 300 at last count. Obviously all of them are important (at least to Bob Roeder), but the one that stands out is TFIID, the centerpiece of the general transcription machinery. As I mentioned earlier, TFIID straddles the DNA-like a saddle, bending it so that Pol II can gallop along the unwound DNA to make RNA—much like a thoroughbred races along the track at Saratoga.

The resemblance of Pol II to a thoroughbred is reminiscent of William Faulkner's classic article on horse racing in which he wrote that nothing affects the emotional nature of man as much as the thoroughbred race horse. According to Faulkner, and I quote: "Horse racing is not just betting. It is a sublimation, a transference: man, with his admiration for speed, strength, and physical power far beyond what he himself is capable of, projects his own desire for physical supremacy, victory, onto the agent—the baseball team, the football team, or the prize fighter. Only the horse race is more universal because the brutality of the prize fight is absent, as is the long time needed in baseball and football for the orgasm of victory to occur, whereas in the horse race it is a matter of minutes, never over two or three, repeated six or eight or ten times in one afternoon."

If ever there were an "orgasm of victory," it is Bob Roeder's test-tube system for gene transcription. Bob tells me that his test-tube system can transcribe any gene in a matter of minutes, never over two or three, and he can repeat it six or eight or ten times every afternoon! If Bob Roeder were a racehorse, his name would be Transcribed.