Nancy nevertheless proved just the tonic Mark needed. With her methodically meticulous presence ensuring that he did not leave out essential experimental reagents, Mark could consistently detect the λ repressor and begin its molecular characterization. They soon showed that it was a protein of molecular weight near 30,000. Though Nancy at first believed Mark could win the repressor race, he knew otherwise. Wally and Benno were already writing up their paper, while he likely needed at least six more weeks in the lab before starting to write. Benno regularly came up to the fourth floor to check on their progress, much to Nancy's annoyance. She perceived his main purpose as gloating. Seeing Mark's always polite reception of Benno, however, Nancy managed an equal courage and grace. As for Wally, Mark so revered him that losing the race to him could never be devastating. Nancy's huge respect for Mark was no less evident: every day she went to the sandwich truck on Divinity Avenue to get his lunch, an egg salad sandwich, accompanied often by a chocolate eclair to fortify his morale.
Wally and Benno's paper “Isolation of the Lac Repressor” was submitted by me to the Proceedings of the National Academy of Science (PNAS) on October 24,1966, just in time for publication in the December issue. If I had delayed its submission to let Mark complete the experiments needed for his paper, Wally and Benno's discovery would bear the next year's publication date. Mark would have preferred this, but I argued that no one would deem his work less important for appearing in print second. On December 27, “Isolation of the λ Phage Repressor” went off to PNAS, to appear in the February 1967 issue. Mark triumphantly announced the λ repressor isolation at a seminar in our lab's tearoom a month before the paper came out. The room was packed and during his moment of triumph (“I did it all alone!”) he never acknowledged Nancy's key role in his success. Afterward, I badly chewed him out. Instantly realizing he had been too full of himself, that evening he called Nancy to apologize and later sent her flowers.
Mark and Nancy went on to test whether their repressor worked by binding to specific DNA sequences. When mixtures of radioactive λ repressor and λ DNA were centrifuged together in sucrose gradients they sedimented together. In contrast, mixtures of λ repressor and DNA from phage λ imm434 did not co-sediment. These much hoped for results were nonreproducible for an awful week until Mark realized that Nancy had inadvertently raised the salt levels in their mixtures. She nervously repeated the experiment using the original salt levels. The new results were just coming off the isotope counter when Nancy and Mark had to attend a seminar on the floor below. Halfway through the talk, the suspense overwhelmed her and she returned to the lab, where she quickly realized all was again well. She and Mark were for the first time ahead of Wally and Benno. Returning from the seminar, Mark shouted for joy, and they went into the halls to spread their good news. Going downstairs, they caught Wally and me about to leave the Biolabs. Upon learning that the experiment was repeatable, Wally's face turned ashen. Mark's overtaking him was not acceptable.
Over the weekend, Wally set about to do the analogous experiment using DNA from a phage Benno obtained from Jon Beckwith of Harvard Medical School that carried the ß-galactosidase gene and its control region. By Monday morning, Wally let it be known that he had preliminary positive results he intended to verify quickly, bringing him again even with Mark and Nancy. Seeing Wally's intense competitiveness made Nancy feel as if she could never survive the male-dominated dog-eat-dog grind of being a scientist. It also was an eye-opener for Mark. His reverence for Wally had taken a big knock.
But Wally's expectation of catching up to Mark and Nancy hit some skids. Holding him up were difficulties in purifying the radioactively labeled lac repressor. Though Mark gallantly held up his submission to Nature, intending to let Wally and Benno publish simultaneously, after two months they were not ready even to start writing, so he sent his paper in without theirs. Having been forewarned about Mark's submission, Nature's editor, John Maddox, sent it to the printer the day it arrived. “Specific Binding of λ Phage Repressor to λ DNA” appeared on April 15,1967, only six days after arriving in Nature's London office. Wally and Benno's paper “The Lac Operator Is DNA” was submitted on October 28 to PNAS, in time at least for publication in the same calendar year.
Nancy became a graduate student again in the fall of 1967 and continued to work in Mark's lab. I had told her to apply to Harvard and a week later she was formally accepted. Mark also encouraged her, saying she was brighter than any of the relatively few women and more than half the men at Harvard. She glowed in the knowledge of the approval of someone she revered. After the misery of Yale, everything had worked out, including her engagement to Brook: soon after becoming Mark's technician she had also become Nancy Hopkins. They moved to Trowbridge Street, their apartment graced by my wedding present of a life-size leather pig, similar to one Brook had long admired in his exclusive “final club” on Massachusetts Avenue.
After his second paper came out, Mark devoted more time to being a visible leftist, joining several South American neurophysiologists in Havana, where he met the cartoonist Jules Pfeiffer. Later he would go to Hanoi and Saigon on a trip organized by a former junior fellow, the MIT linguist Noam Chomsky. Over the past year, our tearoom conversations had centered more on Vietnam than on science, and I remained glued to an issue of the New York Times covering the Tet offensive all through a talk by MIT's Ethan Signer on bacterial-gene-carrying λ phages.
Earlier Benno and Jon Beckwith had marched down Massachusetts Avenue from Cambridge to Boston to demonstrate against America's Southeast Asia policy, discussing λ phages along the route. Immediately following Tet, Benno went with his soon-to-be wife, Barbara, to New York City for a big antiwar rally in Central Park. Equally anti-establishment in outlook, Barbara had first met Benno at one of Mark's parties in his flat north of the Biolabs, on Sacramento Street. Mark's blond-haired, live-in girlfriend, Micky, was from an even more leftist, if not Stalinist, background. Mark frequently escaped with his violin to their cottage on Cape Cod for weekends of heavy fellow-traveler chitchat.
In the summer of 1967, Wally and Celia Gilbert's three-year-old daughter, Elsbeth, was diagnosed with an incurable metastatic sarcoma. They made constant visits to Children's Hospital, compounding Wally's exhaustion when he got back home from late evenings in the lab. His two other children, John and Kate, earlier had thrilled to their father's eureka at finding the repressor. Now the mood of the house on Upland Avenue was grim. Hoping to distract them, in mid-fall I persuaded an ABC-TV producer, Ernie Pendrell, to feature Wally and Mark on a forthcoming TV special about science. Initially he had wanted to do an hourlong feature about my work, sponsored by North American Rockwell. But I told him the race for the repressor would show science at its brightest, making the public aware that ambitious young scientists, like young poets, are more creative when not encumbered by the braking powers of maturity, which I already felt nipping at my own heels. With Harvard's approval, an ABC crew came during the second week of February 1968 and followed me and Wally walking in Harvard Yard with Wally's children eagerly asking on cue, “Did you find it yet, Daddy?” A week later, the crew returned for a raucous Lincoln's Birthday party I threw in my Appian Way flat.
By then, Mark was secure in the knowledge that as of July 1, he would be a member of the Harvard faculty. By appointing him a lecturer in biochemistry instead of an assistant professor, the University could match the salary recently offered him to be an associate professor at Berkeley. In support of his appointment, Francois Jacob wrote to Paul Doty that “Mark is the most gifted young man in his generation of biologists.” Franklin Ford orchestrated swift movement by the administration. The day after Christmas he wrote to tell me Ptashne was confirmed in his decision to stay at Harvard. Several months after, when Mark's appointment formally began, the Harvard Council of Deans honored him and Wally with the prestigious Ledlie Award for 1968, which included a $1,600 honorarium.
Noon seminars from September 1967
Frank Stahl (back to camera)
, Gerald Seher, Mark Ptashne, Wally Gilbert, and Celia Gilbert at the 1968 Cold Spring Harbor symposium
Wally Gilbert and Max Gottesman at Cold Spring Harbor in 1968
By then Benno had returned to Germany as a professor at the Genetics Institute of the University of Cologne. Before going, he again made elegant use of E. coli genetics to generate a mutant that overproduced the lac repressor. Putting this mutant gene on a λ-like phage led to hundredfold amounts of the typical lac repressor output. Just before Benno left Harvard, Jacques Monod was visiting and popped his head in to say hello and comment on Benno's identification of the lac repressor, remarking, “After all, Benno, it was pedestrian.” Then he turned around and left. It was Jacques's sour grapes way of admitting that clever experiments, not a clever idea, had beaten him and his friends at the Institut Pasteur. With Benno, Wally, and Mark's success, the center of gene regulation was now not Paris but Harvard.
An unanticipated by-product of Wally's lac repressor purification effort was helping my graduate student Dick Burgess to optimize his RNA polymerase purification methodologies. More than ever, our lab wanted further insight into how RNA is made from DNA templates. In 1963, I'd had my graduate student John Richardson focus his Ph.D. research on RNA polymerase, the enzyme responsible for making RNA off DNA. RNA polymerase was discovered in 1959, but its molecular form was not easy to pin down. John found that the molecular weight decreases by half when the molecule is suspended in a highly ionized solution. But even this smaller form was larger than any known single polypeptide chain, so it seemed likely that RNA polymerase was constructed from a number of smaller polypeptides. No deep insights, however, came from electron microscopy done at MIT. It revealed approximately spherical particles 120 Ä in diameter, compatible with a molecular weight of almost a million atomic mass units. Just before leaving for Paris to become a postdoc at the Institut Pasteur, John used phage T7 templates to demonstrate the synthesis of RNA products containing up to ten thousand bases. Conceivably their long lengths reflected failure of the purified enzyme to recognize normal T7 stop signals for RNA synthesis.
After John left for France, Dick Burgess, who had just arrived from Caltech, took up the challenge presented by RNA polymerase's large size. Every week Wally's technician Chris Weiss separated the proteins from thirty liters of E. coli cells into several fractions. The one containing the lactose repressor went to Wally and Benno, while Dick took the fraction containing RNA polymerase. The latter still contained many, many other proteins, requiring him to work out a glycerol gradient centrifugation procedure that yielded a highly active RNA polymerase preparation that he called GG. Starting late in 1967, he added a phos-phocellulose column step to his recipe, leading to even purer RNA polymerase, called PC RNA polymerase.
In April 1968, Dick gave a talk at the Federation Society meeting in Atlantic City revealing that PC RNA polymerase particles were made up of small subunits, called a, and large subunits, called ß. Two months later, at the Gordon Conference on Nucleic Acids in New Hampshire, he gave out details of his GG and PC purification methods, later sending written protocols to some ten interested labs, including that of Ekke Bautz at Rutgers. By then Wally's graduate student Jeff Roberts was also studying RNA polymerase. Initially using Dick's GG purified enzyme, he found it very active in transcribing λ DNA. But later, using Dick's even more purified PC preps, he inexplicably found no transcription of λ DNA.
Jeff's unexpected negative result made Dick wonder whether his PC purification procedure had removed an RNA polymerase component necessary for transcription of phage DNA. To test this possibility, he passed a fresh batch of GG enzyme over a PC column to see whether this again led to a loss of phage DNA's transcription ability. After getting the same negative result as Jeff, he showed that the PC enzyme could be activated by adding back a protein that had been extracted in purification. Experiments done the following week by our English postdoc Andrew Travers demonstrated that a single polypeptide chain, soon to be called σ factor, was the GG-enzyme-containing ingredient lacking in PC preps.
Less than a week passed before Dick heard that Ekke Bautz at Rutgers and his student John Dunn had obtained the same results using GG and PC enzymes following the protocol he had sent them in July. At my suggestion, Dick immediately contacted Bautz to see whether they would like their work included in a joint lab paper on the σ factor to be submitted to Nature. Liking the idea, they came up to Harvard on November 6. For me their presence was also a distraction from the unbearable cliffhanger of the presidential elections that day. Richard Nixon's victory over Hubert Humphrey would become clear only late that night. Even the discovery of σ factor did not alleviate the gloom I felt the rest of the week at the prospect of a Nixon presidency.
At the 1970 Cold Spring Harbor symposium: Jeff Roberts with Ann Burgess (left) and John Richardson with Dick Burgess (right)
On Monday morning, November 11, a silver lining briefly presented itself. In that day's Crimson I read the supersize headline “Pusey to Quit Harvard.” The accompanying article reported that Presidentelect Nixon had paid an unannounced visit to Harvard, coming secretively at 9:00 P.M. the night before, to ask Pusey to become his Postmaster General. After a frank and comradely conversation, during which tea and vanilla wafers were served, Pusey accepted later declared to be “the greatest moment of my life.” All too soon, however, the happy glow permeating my being dissipated. Lower on the page was another story reporting that the proposed JFK Library was to be moved from Cambridge to Bayonne, New Jersey, making me realize I was reading one of the hoax issues Crimson editors occasionally created, perhaps to assure themselves that no want of wit had consigned them to the Crimson rather than the Lampoon. After this monumental letdown nothing in the Crimson ever seemed funny.
When the manuscript announcing σ factor was submitted to Nature on December 2, it contained only data obtained at Harvard. The equivalent experiments from Rutgers had been done fewer times and were less complete. “Factor Stimulating Transcription by RNA Polymerase,” by Burgess, Travers, Dunn, and Bautz, speedily appeared as a full article in January 1969. Before publication, I announced our lab's breakthrough in a lecture to Arthur Kornberg's perennially self-congratulatory Biochemistry Department at Stanford. It was a moment I never dreamed would come: our Harvard Biolabs demonstrating the biochemical competence to take on and best mighty Stanford.
In mid-December, Dick and Andrew had heard from Ekke Bautz and John Dunn that σ molecules disappear from cells infected with T4 phage, explaining why host bacterial RNA synthesis stops soon after phage infection. Phage DNA molecules likely coded for phage-specific σ factors that directed core RNA polymerase molecules to specific signals on their respective DNA. Over the next six months, Andrew Travers confirmed this conjecture and sent a paper to Nature in August called “Bacteriophage Sigma Factors for RNA Polymerase.” In its conclusion, he speculated that σ-like transcription factors might be responsible for the massive shifts in RNA transcription patterns underlying the development of higher organisms. Sensing again a major paper on their hands, Nature editors rushed it into print in just over a month.
By early February, Dick had defended his thesis and started a Helen Hay Whitney postdoctoral fellowship that would let him remain at Harvard until his intelligent, blond wife, Ann, finished her Ph.D. thesis. She hailed from a prosperous, hardworking Wisconsin family and did her experiments on bacteriophage ΦX174 down the hall in Dave Denhardt's lab. By then, Dick knew that not one but two ß chains existed in the core (PC) RNA polymerase particle whose structure was α2ββ He and Andrew, furthermore, had preliminary evidence pointing to how σ functioned in the initiation of RNA synthesis. Its role was to direct the core α2ββ1 complex to appropriate starting sites to the DNA for RNA synthesis. RNA polymerase's enzymatic property is solely owing to its α2;ββ1 core component.
The relative importance in controlling gene regulation of repressore and operators versus σ factor shifts still remained open. Bernie Davis at Harvard Medical Sch
ool bet me a case of wine that my group would not discover a second bacterial σ factor over the next two years. Andrew then had let Bernie know of his tentative evidence for a σ factor controlling ribosomal RNA synthesis. Here time was not on his side, costing me a case of cabernet. But Richard Losick, a newly appointed junior fellow, had begun experiments in the Biolabs on how Bacillus subtilis forms spores, soon getting hints of a possible σ factor specific to bacterial sporulation.
The first visible recognition of σ factor's importance came in November 1969 at a meeting in Florence, Italy, on RNA polymerase and transcription. Dick Burgess gave the opening talk, coming from Geneva, where he was now a postdoc in Alfred Tissières's lab. Sponsoring the meeting was Lepetit, the Milan drug company whose rifamycin and rifampicin antibiotics had been shown to inhibit bacteria through binding to the ß subunit of RNA polymerase. At this meeting, Jeff Roberts announced his recent discovery of a protein called p that halts RNA synthesis at specific stop signals on DNA molecules. Just before coming to Italy, he sent off his manuscript “Termination Factors for RNA Polymerase” to Nature, which published it in December 1969.
Avoid Boring People: Lessons from a Life in Science Page 26
