What was the most “elegant” experiment ever? Many molecular biologists, who were active during the so-called “golden age” of the 1950s and 1960s, might opt for the 1958 experiment of Mathew Meselson and Franklin Stahl, which demonstrated the semiconservative replication of DNA (1). My choice is the 1960 experiment by Sidney Brenner, Francois Jacob, and Matt Meselson, which established the existence of messenger RNA (mRNA) (2). The story behind the discovery is an appropriate topic for the blog since bacteriophage T2 had a key role to play. It is told here, largely through the words of one of its contributors, Pasteur Institute scientist and Nobel laureate, Francois Jacob (3).
Imagine for the moment that we are back in the late 1950s, at a time when the precise role of RNA was not yet known. However, pertinent evidence was accumulating, which implied that RNA had a role in protein synthesis. For example, cellular RNA levels correlated with the levels of protein synthesis.
But what might the role of RNA be? The example of eukaryotic cells seemed to indicate that DNA could not directly serve as the template for protein synthesis. The DNA in those cells is contained within the membrane-bounded nucleus, whereas protein synthesis occurs in the cytoplasm. Might RNA then serve as an intermediate information carrier?
Jacob, and others, knew that protein synthesis took place in the cytoplasm, on tiny granules called ribosomes. Moreover, “for each gene there were corresponding ribosomes specifically charged with producing the corresponding protein (3).” This remark might seem to suggest an accurate view of protein synthesis. Nonetheless, the understanding of ribosomes at the time was fundamentally wrong. Each gene was thought to be transcribed to a unique RNA that became an integral component of a ribosome. Moreover, that integral RNA was thought to confer on the ribosome the specificity to support the synthesis of only the one protein that corresponded to that particular RNA—a scenario under which an entire ribosome needed to be produced de novo to support the translation of a gene.
With that view of ribosomes in mind, Jacob was troubled by the results from an earlier experiment, carried out in 1957 by Arthur Pardee, Jacob himself, and Jacques Monod—the famous (and also quite “elegant”) PaJaMa experiment (4). In this experiment, the Lac gene of an Hfr (male) strain of E. coli is transferred to a Lac-minus, F-minus (female) strain. [This experiment is famous because it was carried out under experimental conditions which enabled the three researchers to demonstrate the existence of a previously unknown regulatory molecule; the “repressor.”] What troubled Jacob was that the Lac gene of the donor E. coli strain was expressed “immediately upon entry of the gene…”—a result not in accord with the thinking of the day about the nature of ribosomes, and the way in which they translated genes into proteins.
It seemed impossible to Jacob that ribosomes, which are complex structures composed of proteins and RNA, could be produced quickly enough to enable the virtually immediate translation of the transferred Lac gene, as had been seen in the PaJaMa experiment. What’s more, the prevailing view of ribosomes also did not fit “with the existence of units of activity recently baptized ‘operons,’ that contained several genes. Nor with a regulation functioning directly on the DNA through the intermediary of a switch, now called an ‘operator.’”
The “perplexity prevailing in the Pasteur group” led to a new line of thought— “either direct synthesis of the protein on DNA itself, with no intermediary; or production of an unstable intermediary, probably an RNA with rapid renewal. But the former hypotheses seemed highly improbable and the latter without a chemical basis, without any trace of a molecule that could substantiate it.”
In 1959 Jacob attended a colloquium on microbial genetics in Copenhagen, where he intended to discuss this conundrum. “A small group attended, including notably Jim Watson, Francis Crick, Seymour Benzer, Sydney Brenner, Jacques (Monod), and even the physicist Niels Bohr. Courteous as ever, Jim Watson spent most of the sessions ostentatiously reading a newspaper. So, when it came time for him to speak, everyone took from his pocket a newspaper and began to read it”
When Jacob’s turn to speak came, he raised the possibility of a need for an unstable intermediary, which he called X. “No one reacted. No one batted an eyelash. No one asked a question. Jim continued to read his newspaper.”
“A new opportunity to discuss protein synthesis arose around Easter 1960 in Cambridge (England), in Sydney’s small apartment in King’s College, where he was a Fellow.” Although the meeting that morning was casual, several heavy hitters were present, including Francis Crick, Leslie Orgel, and Ole Maaloe, in addition to Jacob and Brenner.
Crick and Brenner discussed the results of a recent experiment carried out by Pardee and Monica Riley (Pardee’s student at the time). “They had succeeded in charging the DNA of male bacteria with radioactive phosphorus; in making them transfer to females the gene of galactosidase; in letting it synthesize the enzyme for some minutes; and then in destroying the gene through the disintegration of the radioactive phosphorus. The result was clear: once the gene was destroyed, all synthesis stopped. No gene, no enzyme. Which excluded any possibility of a stable intermediary.” [Recall the thinking that stable ribosomes contained an integral RNA that conferred its specificity.]
“At this precise point, Francis and Sydney leaped to their feet. Began to gesticulate. To argue at top speed in great agitation. A red-faced Francis. A Sydney with bristling eyebrows. The two talked at once, all but shouting. Each trying to anticipate the other. To explain to the other what had suddenly come to mind. All this at a clip that left my English far behind. For some minutes, it was impossible to follow them, just as it would have been impossible for them to follow a discussion in French between Jacques (Monod) and me. What had set off Francis and Sydney was, once again, a connection between the lactose system and phage. After infecting the colon bacillus, certain highly virulent phages blocked the synthesis of new ribosomes. As had been shown by two American Researchers, Elliot Volkin and Lazarus Astrachan, the only RNA then synthesized had two remarkable properties: on the one hand, unlike ribosomal RNA, it had the same base composition as DNA; on the other hand, it renewed itself very quickly. Exactly the properties required for what we called X, the unstable intermediary we had postulated for galactosidase. Why, in Paris, when we were looking for a support material for X, had we not thought of this phage RNA? Why had I not thought of it? Ignorance? Stupidity? Oversight? Misreading of the literature? Failure of judgment? A little of all these, no doubt. A mixture that, as in a detective novel, had made us fail to spot the murderer, the molecule responsible. In the last analysis, however, what mattered was that X, the unstable intermediary, was materializing…it had to be shown that all this was not a dream; that this RNA of the phage was indeed the unstable intermediary functioning in the synthesis of proteins: the issue that we and Sydney immediately decided to take up. …” See Aside 1.
[Aside 1: Volkin and Astrachan, at the Oak Ridge National Laboratory in Tennessee, showed that there actually are two kinds of RNA seen during phage infection—a stable type found in ribosomes (now known as ribosomal RNA, which does not have the same base composition as the DNA ), and an unstable, rapidly turning over type, that has the same base composition as the viral DNA, but not the bacterial DNA (5). Transfer RNA remained to be discovered.]
That afternoon, Jacob and Brenner found out that they each had been invited to spend a month (June) at the California Institute of Technology. Brenner’s invitation came from Matt Meselson, and Jacob’s from Max Delbruck. “A unique opportunity to work together to demonstrate the nature and role of X.” Importantly, Meselson recently developed a technique that would make the discovery possible.
That evening, at a party given by Crick and his wife, Jacob and Brenner discussed the experiment that they were envisioning. But: “It was difficult to isolate ourselves at such a brilliant, lively gathering, with all the people crowding around us, talking, shouting, laughing, singing, dancing. Nevertheless, squeezed up next to a little table as though on a desert island, we went on, in the rhythm of our own excitement, discussing our new model and the preparations for experiments at Caltech.”
In their new concept of protein synthesis: “The ribosomes had lost all specificity. They had become simple machines for assembling amino acids to form proteins of any kind, like tape recorders that can play any kind of music depending on the magnetic tape inserted in them. In protein synthesis, it was X, the unstable RNA copied on a gene, that had to play the role of the magnetic tape, associating with the ribosomes to dictate to them a particular sequence of amino acids corresponding to a particular protein.” Thus, the experiment would be to “show that the unstable RNA, synthesized after infection of a colon bacillus by the virulent phage, associated with pre-existing ribosomes, synthesized before infection, to produce the proteins of the phage.”
A key problem would be to distinguish ribosomes made before infection from any ribosomes that might be made after infection. Their solution would be provided by Matt Meselson’s new technique in which “he marked macromolecules by cultivating bacteria in heavy isotopes before putting them back in a normal environment. Using ultracentrifugation, he could then separate the marked molecules along gradients of density…”
Thus, the plan was to grow cells for several generations in medium containing the heavy isotopes 15N and 13C as the sole nitrogen and carbon sources, respectively. In this way, essentially all ribosomes present in the cells would be “heavy”. Next, the cells would be washed and placed in medium containing the normal isotopes, 14N and 12C. Then, the cells would immediately be infected with the phages. Any new ribosomes made after the infection got underway would be “light”.
Here is a key point. Recall that Volkin and Astrachan showed that the only RNA that is made after infection is the unstable RNA, which has the same base composition as the phage DNA. [That is so because the phage shuts down host transcription and translation.] Consequently, this phage RNA can be specifically labeled by adding 32P to the infected cultures (5). Brenner, Jacob, and Meselson hoped to find this rapidly turning-over phage-specific RNA in the density gradients, in association with the old heavy ribosomes that were made before infection. “If we were right, if our hypothesis was correct, the radioactivity of the RNA had to be associated, in the gradients, with the band of “heavy” ribosomes.”
However: “We were not succeeding.” The problem that was frustrating their efforts was that the ribosomes were unstable in the density gradients. “In vain did we try to check through the experiment, to modify it, to change a detail here and there. It was now three weeks since Sydney Brenner and I had arrived at the California Institute of Technology. We had come for the sole purpose of carrying out this experiment with Matt Meselson. An experiment that we had no doubt was going to change the world. But the gods were still against us. Nothing worked.”
“Our fine confidence at the start had evaporated. Disheartened, Meselson had departed-to get married! Sydney and I talked about going back to Europe. In a burst of compassion, a biologist by the name of Hildegaard had taken us under her wing and, to give us a change of scene, driven us to a nearby beach. There we were, collapsed on the sand, stranded in the sunlight like beached whales. My head felt empty. Frowning, knitting his heavy eyebrows, with a nasty look, Sydney gazed at the horizon without saying a word. Never yet had I seen Sydney Brenner in such a state. Never seen him silent…And our time was running out. For, come what may, Sydney and I had decided to leave at month’s end.”
“Hildegaard tried to tell us stories to lighten the atmosphere. But we were not listening. Suddenly, Sydney gives a shout. He leaps up, yelling, “The magnesium! It’s the magnesium!” Immediately we get back in Hildegaard’s car and race to the lab to run the experiment one last time. We then add a lot of magnesium… Sydney had been right. It was indeed the magnesium that gave the ribosomes their cohesion. But the usual quantities were insufficient in the density gradients used to separate heavy and light compounds. This time we added plenty of magnesium. The result was spectacular. Eyes glued to the Geiger counter, our throats tight, we tracked each successive figure as it came to take its place in exactly the order we had been expecting. And as the last sample was counted, a double shout of joy shook the basement at Caltech…This was merely one experiment, performed in extremis… But we now knew that we had won. That our conception explained the transfers of information in the synthesis of proteins…Scarcely was the experiment over than we gave a seminar at Caltech to demonstrate the existence of X and its role as magnetic tape. No one believed us. The next day we left, each to his own home. The bet had paid off. In the nick of time.”
Apropos our Virology blog, this experiment also showed that viruses subvert the cellular protein synthesis machinery for their own ends.
Nobel laureate Sidney Brenner was the main subject of two earlier posts—The Phage in the Letter, reposted September 8, 2016 and Sidney Brenner: Only Joking, January 5, 2014 (6, 7). Each of these posts highlighted Brenner’s mischievous sense of humor. Jacob offers more insight into Brenner’s personality in his account of the episode on the beach with Hildegaard: “There we were, collapsed on the sand, stranded in the sunlight like beached whales. My head felt empty. Frowning, knitting his heavy eyebrows, with a nasty look, Sydney gazed at the horizon without saying a word. Never yet had I seen Sydney Brenner in such a state. Never seen him silent. On the contrary, he was an indefatigable talker at every opportunity. A tireless storyteller, able to discourse for days and nights on end. Interminable monologues on every conceivable subject. Science, politics, philosophy, literature, anything that cropped up. With stories he made up as he went along. Generously laced with jokes. With nasty cracks, too, at the expense of just about everyone. An excellent actor, he could render a speech in Hungarian, a lecture in Japanese. Mimic Stalin or Franco. Even himself. He went without a break from one register to another. A sort of fireworks whose effects he gauged from the expressions of the people around him.”
In the September 8th reposting I wrote: “While Brenner’s work as a molecular biology pioneer may have justified a Nobel Prize, he received the award in 2002 for his later studies of the nematode Caenorhabditis elegans, in which his research group traced the fate of each cell from the zygote right through to the adult worm. Their work established C. Elegans as a model system that is now studied in hundreds of laboratories all over the world (6).”
Jacob collaborated with Jacques Monod to elucidate the genetic switch that regulates beta-galactosidase synthesis in E. coli. Their collaboration established the concepts of regulator genes and operons, for which they shared in the 1965 Nobel Prize for physiology or medicine.
In 1940, Jacob, who was Jewish, left medical school in occupied France to join Free French Forces in London. He then served as a medical officer in North Africa, where he was wounded, and was wounded again, this time severely, at Normandy in August 1944. Monod too was active in the French Resistance, during the Nazi occupation of Paris. He eventually become chief of staff of the French Forces of the Interior. In that capacity, he helped to prepare for the Allied landings in Normandy. Monod and Jacob each received France’s highest honors for their wartime service. For more on Jacob, see Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob, posted January 28, 2015 (8).
Matt Meselson (still at Harvard at 86 years in age) is best known for showing that DNA replication is semi-conservative and for his part in the discovery of messenger RNA. Jacob tells us that at the time of their collaboration at Cal Tech: “He (Meselson) was haunted by the Cold War, by the need to establish better relations with the Soviet Union. In his soft voice, he could discourse for hours on strategy, tactics, nuclear arms, the Rand Corporation, first strikes, reprisals, annihilation.” Meselson later helped to persuade President Richard Nixon to renounce biological and chemical weapons, and to support an international treaty (the 1972 Biological Weapons Convention) banning the use of biological agents.
- Meselson M and FW Stahl, 1958. The Replication of DNA in Escherichia coli, Proceeding of the National Academy of Sciences USA. 44:671–82.
- Brenner S, F Jacob, and M Meselson, 1961. An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis, Nature 190:576-80.
- Francois Jacob, The Statue Within: An Autobiography, English language translation copyright 1988 Basic Books Inc.
- Pardee, AB, F Jacob, and J Monod, 1959. The genetic control and cytoplasmic expression of ‘inducibility’ in the synthesis of β-galactosidase by coli, Journal of Molecular Biology 1:165–178.
- Volkin E and L Astrachan. 1956. Phosphorus Incorporation in Escherichia coli Ribonucleic Acid after Infection with Bacteriophage T2. Virology 2:149-161.
- The Phage in the Letter. Reposted on the blog September 8, 2016.
- Sidney Brenner: Only Joking. Posted on the blog January 5, 2014 (find under Archives, January 2014).
- Genealogies and a Selective History of Lysogeny: Featuring Friedrich Loeffler, Emile Roux, Andre Lwoff, Elie Wollman, and Francois Jacob. Posted on the blog January 28, 2015.