It is the time of year we revisit the classics: An annual reading of Dickens’s A Christmas Carol perhaps, or maybe Auden’s For the Time Being: a Christmas Oratorio (for you highbrows). Familiar songs of the season are all around us—inescapably so—and movies we’ve practically memorized line by line are watched again as if for the first time. Some traditions have dishes we anticipate at this time of year, whether they actually taste good or not (lutefisk…fried eel…fried eel?). In this spirit of reconsidering timeless things, I reread a classic paper in the field of molecular biology; not just any classic, but THE classic paper on gene regulation in the history of the topic: the blockbuster 1961 publication “Genetic Regulatory Mechanisms in the Synthesis of Proteins,” by François Jacob and Jacques Monod (J. Mol. Biol. 3:318-365).
With Andre Lwoff, Jacob and Monod would go on to win the 1965 Nobel Prize in Physiology or Medicine “for their discoveries concerning genetic control of enzyme and virus synthesis.” Although this paper includes graphs and tables from numerous experiments, it combines simple experimental microbial systems to advance a comprehensive model explaining how genome information is translated into proteins that do the work of the cell. The findings described by Jacob and Monod provided answers to dominant questions in cell and molecular biology at the time. These—and those of experiments that emanated from this paper in the few years following—laid the foundation for developing tools and technology that are still widely used. The paper—and the spirited men and women who carried out the simple but revealing experiments it describes—are memorialized for a broader audience by Horace Freeland Judson in his brilliant history of molecular biology, The Eighth Day of Creation.
Figure from an experiment showing beta-galactosidase expression increase during cell culture growth in the presence of an inducer. Source.
The paper starts with–and really is all about–the question of how cells produce new enzymes only in the presence of the substrates for those enzymes; i.e., how substrates “induce” production of the enzymes that break down those substrates. Of ascendant value in addressing this question was the system encoding beta-galactosidase from the gene lacZ, as well as co-expressed genes for a permease (lacY) and an acetylase (lacA) as well as a regulator lacI and an operator lacO. The whole story was based on a simple observation: that cells grown with a beta-galactoside sugar produce approximately 10,000 times more units of enzyme compared to cells that are grown in its absence. This simple system of inducer-dependent expression had by then become a principal tool for studying gene regulation, spawning clever and cute experiments like the eponymous “PaJaMo” experiment (Arthur Pardee, François Jacob, and Jacques Monod), which firmly established the inducible nature of gene expression. The major question in the field of molecular biology that Jacob and Monod wrestled with in their classic then came down to: how does information transfer from genes to proteins after addition of inducer? For this, they adopted the concept of a “messenger” that conveys gene-encrypted information into protein synthesis. Their hypothesis arose from an emerging consensus among biologists that ribosomes are essentially nonspecific contributors whose role was directed by instructions provided by a messenger.
In contemplating the nature of the messenger, they argued that it is likely not long-lived, citing both published and unpublished research (their own and others’) in which enzyme production—and its cessation—occur very rapidly after addition and removal, respectively, of inducer: if the messenger were long-lived, then it should enable continued production of enzyme synthesis even if inducer were removed. Not incidentally, but of continued relevance today, is that their interest in induction had them examine numerous galactosides as inducers of the lac operon. They concluded that whether or not the beta-galactosidase enzyme could actually bind to and cleave the inducer was irrelevant to the amount of induction observed. In fact, the index compound against which all others were compared was one that was recognized with 10 times less affinity than lactose, but which induced enzyme synthesis nearly 10 times better. This was isopropyl thiogalactoside, or IPTG, which is of course routinely used today to control gene expression in a range of experimental and even therapeutic settings. The Jacob and Monod work on induction is thus exhibit A for the high-value return on basic science investments.
Another then-emerging concept they elaborated on was the operon, which they defined as a “genetic unit of co-ordinate expression”. The year prior to the JMB paper, Jacob and Monod, along with David Perrin and Carmen Sanchez, had discussed the operon in a short publication of the French Academy of Sciences (Jacob et al., Comptes Rendus des Seances de L'Academie Des Sciences, 250:1727); that same paper suggested that these proposed messenger RNAs are “cytoplasmic replicas” of the operon. In the lac system, the three genes responding to inducer (zya) are all physically linked, and this was a feature of other examples Jacob and Monod provided to support the operon theory. Physically linked, multiple open-reading frames on a single transcript like this are common in bacteria and archaea, where transcription and translation are coupled, but not in eukarya. Nevertheless, anticipating a potentially more complex future for this concept, Jacob and Monod concluded their discussion of the operon with this statement: “One may…wonder whether it will be possible experimentally to extend this concept to dispersed (as opposed to clustered) genetic systems.” Of course, today, we routinely encounter examples of complex, unlinked, transcription activity in response to single signals through a specific regulator.
Lest we think these demigods always and ever drew only correct conclusions or foreshadows in their writing, on a couple points they were famously wrong or misguided. In addition to induction of the lac operon, they also raise the concept of “negative adaptation,” which to them meant inhibition, or repression, of enzyme gene expression, as opposed to its induction. They suggested that induction is principally for synthesizing enzymes involved degrading things (i.e., catabolic effects), while repression is used for enzymes that synthesize things (i.e., anabolic effects). They also reviewed work that was being carried out on phage lambda, whose lytic growth is also controlled by a repressor. They thus posit in their models that regulation is essentially the domain of repressors, and that gene expression occurs after some mechanism for inactivating repressors. Their generalized conclusion that protein synthesis is controlled this way held sway for several years, and had an impact on delaying the acceptance of later landmark work by Ellis Englesberg and others on the arabinose operon (Genetics 198:455–460, October 2014 ). Arabinose catabolism is by the AraC protein, which activates ara gene expression in response to arabinose, as those many of us who use the pBAD promoter for expressing genes well know.
Another misconception advanced by Jacob and Monod, albeit softly, and with some trap doors in case it turned out not to be so, came from their pondering about the nature of the regulator gene, which they called “i” (and which we now call lacI). Mutations in lacI led to constitutive expression of beta-galactosidase (hence their conclusion that it is a repressor), but in considering evidence from many experiments about how lacI worked, they suggested that the repressor is an RNA molecule. Even in being flat-out wrong here, they unintentionally foretold a future when RNA as a regulatory molecule has become well established.
Just as frequent re-reading of Dickens is enjoyable because of one focuses on something different each time, what is most interesting about the Jacob and Monod paper changes on each reading. In this visit, their concept of “the messenger” was intriguing. The factor that conveys genetic information from the genetic code outward to functional enzymes had been a hazy notion, but by 1961 was starting to emerge as an understood, identifiable, actual thing. The ideas swirling about the field at that time, based on experiments by many different investigators studying bacteria, phage and even yeast, were that the genetic messenger (i) is of similar base composition as the genes and (ii) may be associated with ribosomes, where proteins were likely synthesized. After reviewing key findings of others, Jacob and Monod describe some “recent observations” made by studying E. coli that had been infected with phage T4. Using radioactive labels for proteins and nucleic acids, and some exceptional experimental design and technique, Sydney Brenner and Jacob, along with Matthew Meselson, demonstrated that, upon T4 infection, labeled phage RNA could be found associated with ribosomes that had been made in the bacterial host before phage infection. Furthermore, labeled phage protein was also found associated with ribosomes, before showing up in the cytoplasm. The experiment provided very strong support to the “messenger RNA” hypothesis, that a copy of the gene was being sent over to the protein synthesizing ribosomes to direct protein production (Brenner et al., Nature 190:576). While Jacob and Monod appropriately describe these findings in academically detached fashion, the history of how this experiment came to be is one of many compelling episodes from the Eighth Day of Creation. Brenner and Jacob had discussed the experiment over several meetings, with great excitement, and were eager to carry it out. Brenner, working in England, and Jacob, in France, arranged to visit the CalTech lab of Matthew Meselson to carry it out (Meselson, working hard to discover the molecular basis of genetic recombination, later indicated that he was basically along for the ride on the messenger experiment, as Brenner and Jacob had done the deep thinking on it without his input).
The story Judson tells of the period leading up to and including the month that Brenner and Jacob were together in Pasadena reads like a science thriller, liberally quoting from interviews with the many legendary scientists who were involved in the effort. At one point we learn that Brenner and Jacob had an epiphany on the experiment while taking a day at the beach near the end of their time in California.
The Jacob and Monod JMB paper remains a vivid account of the most advanced knowledge of that time about the most important molecular processes in the cell. Except for the few points of error or overreach, it is not outlandish to suggest that Jacob and Monod essentially outlined pretty much what there is to know about gene regulation, and we have spent the ensuing 50+ years coloring in the rest. The work they describe induced a historian to enquire more about the amazing people who were making these earth-shattering discoveries, and to write a beautiful story of discovery, insight and personality. The complete joy in uncovering knowledge comes through in their writing, which is also very logical and thought provoking. In their work, one clearly sees what Jacob later said to Judson in discussing the messenger experiment: “Science is almost always done best by [the young]… We were like children playing.”
Which papers and reviews from our era that will have the sort of influence that the Jacob and Monod paper had? Do you recognize one with such far-reaching implications? Or are we somehow, sadly, beyond the point where one paper can have such a lasting impact on our knowledge and on the questions we ask? In any event, take the time to reread the classics in your field, the ones you find compelling and deeply insightful. It is not a chore, or at least don’t make it one. Listen to Jacob…approach the work with the fresh eyes of a child playing at a game.
Biological dogma photo source.