This page is referenced by:

• 1 2025-01-22T09:20:25-05:00 Chapter 14: Crick Enunciates the Central Dogma and Two Research Teams Discover Messenger RNA 6 plain 2025-03-13T15:41:35-04:00

  “…once ‘information’ has passed into protein it cannot get out again.”

  
  
  In his highly impactful treatise “On Protein Synthesis” published in 1958, Francis Crick spelled out two foundational concepts of molecular biology, The Sequence Hypothesis and The Central Dogma.  The former holds that “the specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and that this sequence is a (simple) code for the amino acid sequence of a particular protein.”  Indeed, this foreshadowed his epic publication with Barnett, Brenner and Watts-Tobin in 1961. The second concept states that “once ‘information’ has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein.”  These two concepts posed the question of how information in the sequence of bases is converted into a sequence of amino acids.
  
  The earliest suggestion that information in the sequence of the gene is transmitted via an unstable RNA intermediate was made, albeit hesitatingly, in 1954 by Jacob and Monod’s collaborator in the PaJaMo experiment, Art Pardee. He wrote, “One could visualize an active form of RNA…..which, once used to mold a specific protein molecule, becomes an inert waste product [underlining added] in E. coli.” Based on follow up experiments in which he measured the kinetics of enzyme synthesis after mating in 1958, Pardee more explicitly stated, “A second class of hypotheses assumes that DNA does make an intermediate carrier of information (perhaps RNA). One must then explain why the rate of formation of enzyme remains constant per cell after mating; and a number of possible but not too plausible explanations exist, based on the idea that the cell is saturated with templates in a few minutes, or that the number of templates reaches equilibrium in a few minutes, or that the templates might be unstable and rapidly reach a steady-state concentration [underlining added] as a balance of formation and inactivation. This hypothesis of templates with short lifetimes has been suggested earlier on the basis of the continued requirement of components of nucleic acids for protein synthesis [his 1954 publication].
  
  The unstable messenger RNA hypothesis is most explicitly stated by Jacob and Monod in 1961, “The property attributed to the structural messenger of being an unstable intermediate is one of the most specific and novel implications of this scheme; it is required, let us recall, by the kinetics of induction, once the assumption is made that the control systems operate at the genetic level. This leads to a new concept of the mechanism of information transfer, where the protein synthesizing centers (ribosomes) play the role of non-specific constituents which can synthesize different proteins, according to specific instructions which they receive from the genes through M-RNA.” Notice in the figure from their mating experiment that β-galactosidase synthesis rapidly reaches steady state when inducer (highlighted with red arrow) is added but almost immediately ceases when inducer is absent. Oddly, Jacob and Monod failed to cite the 1954 and 1958 publications by Pardee in their landmark 1961 publication. Perhaps, Pardee had simply been too cautious.
  
  The wider community was slow to accept the concept of an unstable messenger. In his autobiography, Jacob recounts the reaction to a presentation he gave in Copenhagen in 1959 in which he stressed the need for an unstable intermediate, “No one reacted. No one batted an eyelash. No one asked a question”.
  
  
  
  Meanwhile, an important, but slow to be appreciated clue was the discovery by Elliot Volkin and Lazarus Astrachan at the Oak Ridge National Laboratory in 1957 of unstable RNA in phage-infected cells. They carried out a “pulse-chase” experiment in which cells that had been infected with a phage were briefly radioactively labeled with P³² followed shortly thereafter by exposure to an excess of non-radioactive phosphorous (P³¹). As highlighted by the red arrows in the figure, radioactivity was incorporated into an unable RNA species that decreased in abundance during the chase.  The authors and others missed the significance of this unstable RNA. It took until 1960 at an informal gathering at which Jacob, Brenner and Crick were present for the meaning of the Volkin and Astrachan experiment to suddenly become appreciated. As recounted by Jacob, “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 what had suddenly come to mind…What had until then been an abstraction was becoming a molecular species... this RNA of the phage was indeed the unstable intermediary functioning in the synthesis of proteins.”  Voilà!
  
  The pièces de résistance in the discovery of messenger RNA were two back-to-back publications in Nature in 1961 that provided experimental evidence for an unstable intermediate in the transfer of genetic information to the site of protein synthesis, ribosomes.
  
  
  
  It was generally understood that proteins could not be synthesized directly on genes; consider, for example, eukaryotic cells in which chromosomes are segregated from the machinery for protein synthesis, ribosomes, which are located in the cytoplasm. Importantly, these publications distinguished between two competing models. In one model, ribosomes (also referred to as “RNA particles” before their function was fully understood) are each dedicated to the synthesis of a particular protein through their RNA component. In the other model, ribosomes are non-specialized factories that can synthesize at any given time any particular protein as dictated by the messenger RNA with which they are associated.
  
  At the heart of the Brenner, Jacob and Meselson report was a pulse-labeling experiment carried out after phage infection, which was designed to ask whether newly synthesized RNA became associated with ribosomes. The infected cells were pulse-labeled with radioactive (C¹⁴) uridine (Fig. 4 on the left) after phage infection.  After labeling, centrifugation was carried out to separate macromolecules according to density. Note that ribosomes consist of two subunits.  Intact ribosomes (open circles), which are labeled B in Fig. 4, sedimented at about fraction 50. [Ribosomes that had dissociated into their subunits (labeled A in Figure 4) sedimented more rapidly (fractions to the left.] The key result was that newly synthesized RNA (filled circles in Fig. 4) associated with intact ribosomes. Following the pulse of C¹⁴-uridine, infected cells were chased with an excess of non-radioactive (C¹²) uridine (Fig. 5 on the right). It can be seen that the peak of newly synthesized RNA associated with intact ribosomes diminished substantially following the chase, a finding consistent with the idea that the ribosome-associated, pulse-labeled RNA was unstable.  In toto, these results provided support for the existence of an unstable messenger RNA that became associated with ribosomes following phage infection. 
  
  Next, Brenner, Jacob and Meselson went a step further.  To distinguish pre-existing ribosomes from ribosomes synthesized after phage infection, cells of E. coli were grown on “heavy” medium containing the heavy (but not radioactive) isotopes nitrogen-15 and carbon-13 (see historical footnote below) and phosphorous-32 as a radioactive label for ribosomes. The bacteria were then infected with phage and shifted to “light” medium containing nitrogen-14 and carbon-12 and pulse-labeled with S35-suphate to label protein synthesized after phage infection (highlighted in red in Fig. 7).  Finally, after the pulse-labeling, centrifugation was carried out to separate macromolecules according to density. The key result was that newly synthesized protein (filled circles in Fig. 7) was associated with heavy ribosomes. (The expected positions of light, A and B ribosomes is indicated by the two vertical arrows.) The authors concluded that protein in infected cells is synthesized on pre-existing ribosomes. To quote from the authors, “…the experiments with phage-infected cells show unequivocally that information for protein synthesis cannot be encoded in the chemical sequence of the ribosomal RNA. Ribosomes are non-specialized structures which synthesize, at a given time, the protein dictated by the messenger they happen to contain.”  Similar experiments carried out by Gros et al. showed that pulse-labeled RNA also associates with intact ribosomes in uninfected cells. Again, to quote from the authors, “Our working hypothesis is that no fundamental difference exists between protein synthesis in phage-infected and uninfected bacteria. In both cases typical ribosomal RNA does not carry genetic information, but has another function, perhaps to provide a stable surface on which transfer RNA's can bring their specific amino-acids to the messenger RNA template.”
  Finally, we note that it was no accident that the Brenner et al. and Gros et al. manuscripts were published back-to-back in Nature. Watson, the senior author on the Gros et al. publication, had famously sent a telegram to Brenner asking him to hold up (“delay”) his manuscript so that both teams could publish simultaneously. Indeed, Brenner did. 
  An historical footnote. We end this chapter with the story of the source of the highly enriched carbon-13. Nitrogen-15 was readily available in the United States but not highly purified carbon-13. But Meselson wanted both heavy isotopes to maximize separation of pre-existing ribosomes from newly synthesized ribosomes by density gradient centrifugation. Meselson’s mentor, Linus Pauling (Chapter 6), had recently (1958) been elected to the Soviet Academy of Sciences. Pauling asked his colleagues in the Soviet Union if they would purify carbon-13 for Meselson.  It took a year and required a two-story diffusion apparatus. But eventually they produced highly purified carbon-13, which was sent to Meselson. Meselson then fed the carbon-13 in the form of carbon dioxide to algae. The algae were then hydrolyzed and included in the medium for the growth of the E. coli cells. Thus, the historic Brenner, Jacob and Meselson experiment was facilitated by an extraordinarily generous gift of a rare isotope that required an elaborate and time-consuming purification process carried out on behalf of the renowned chemist Linus Pauling!  Oddly, none of this is mentioned in the Nature publication except that carbon-13 was fed to E. coli in the form of an algal hydrolysate. Below are excerpts from Pauling’s 1958 letter to A. N. Nesmeyanov, President of the Academy of Sciences of the Union of the Soviet Socialist Republics:
  
   

• 1 2025-01-22T09:20:25-05:00 Chapter 12: The PaJaMo Experiment Reveals Negative Regulation 4 plain 2025-04-03T11:15:54-04:00

  “…cells with the constitution z⁺i⁺ synthesize enzyme in the presence of inducer only, while z⁺i^- cells synthesize enzyme without induction...”

  The human genome comprises more than 20,000 genes, and the human body comprises large numbers of different cell types and tissues that express different sets of genes at different times.  How does this regulation work? The vast and still evolving story of our understanding of how genes are controlled began with the pioneering discoveries of the French microbiologists François Jacob, Jacques Monod and André Lwoff for which they would share the Nobel Prize in 1965.
  
  André Lwoff (1902-1994) joined the Institut Pasteur in 1921 where he would create a division famously known as the “Attic.” He was later joined in the Attic first by Monod and then Jacob. Lwoff was responsible for the discovery that bacteria can become lysogenic; that is, they can harbor certain phage in a dormant state known as a prophage.  He showed that the prophage can be induced to resume lytic growth.
  
  Jacques Monod (1910-1976) spent a year (1936) before his Ph.D. working in the lab of the famous fly geneticist Thomas Hunt Morgan at CalTech on a grant from the Rockefeller Foundation. Monod obtained his PhD at the Faculty of Science in 1941. He served in the French underground, and after the liberation of France joined Lwoff in the Attic, eventually becoming Director of the Institut Pasteur (1971). Monod’s interest in the regulation of lactose utilization by E. coli stemmed from his Ph.D. thesis on the sequential utilization of sugars by E. coli. Monod liked to generalize and famously said, “What is true for E. coli is true for elephants.” Monod additionally made important contributions to the concept known as allostery that enzymes can exist in alternative conformational states. Monod was also a philosopher and was close to Albert Camus and the French Existentialists. While he could be obstinate, as we will see, Monod was charming as is evident in this video:

  
  
  
  
  François Jacob (1920-2013) entered medical school in 1938 with an interest in surgery but left after two years to join the French resistance, becoming a member of the French 2nd Armored Division in 1940. He was injured in a German air attack in north Africa (Tunisia). He was subsequently severely wounded in Normandy in 1944, thwarting a career as a surgeon. Nonetheless, after the war, he returned to complete his medical degree. Finally, at age 30 he succeeded in joining Andre Lwoff. Jacob recalls repeatedly and unsuccessfully applying to join the Attic until at last Lwoff told him that he had just discovered induction of a prophage and asked him whether he would be interested in working on phage. Jacob did not know what a prophage was but said, “That’s just what I’d like to do.” In the Attic, Jacob began a collaboration with Monod studying the regulation of lactose utilization by the lac operon. Jacob spoke of “day science” and “night science.” He described day science as how we present our discoveries…as a linear progression of observations and scientific design to a “violà” conclusion. Night science is how the discovery process really happens, in its messy progressive, questioning progression, where we construct and then demolish hopeful hypotheses, “fighting a lot with yourself.”  His life’s journey from the battlefield to the laboratory is breathtakingly told in his philosophical autobiography The Statue Within.
  
  Here we will principally focus on a series of historic experiments carried out with postdoctoral fellow Arthur Pardee, famously known as the PaJaMo (Pardee, Jacob and Monod) experiments.  The starting point for these experiments was the observation that the production of the enzyme for the utilization of lactose, -galactosidase, can be induced by the presence of lactose.  Rather than use lactose they used artificial inducers (IPTG and TMG) that are not degraded by the enzyme.
  
  
  The starting point for the PaJaMo experiments was a series of closely linked mutations that exhibited opposite phenotypes.  Mutations of the z gene, z^-, were blocked in production of β-galactosidase whereas mutations of the i gene, i^-, produced the enzyme constitutively. The big question Pardee, Jacob and Monod wanted to answer was whether z^- and i^- were mutations in the same gene or two nearby genes: “The next and most critical problem is whether the z and i factors also belong to the same unit of function (gene or cistron) or not. Let us recall that cells with the constitution z⁺i⁺ synthesize enzyme in presence of inducer only, while z⁺i^- cells synthesize enzyme without induction, and z^-i⁺ or z^-i^- cells do not synthesize enzyme under any condition. The extremely close linkage of z and i mutations suggests that they may belong to the same unit. If this were so, they would not be able to interact through the cytoplasm, but could act together only when in cis position within the same genetic unit. The heterozygote, z⁺i⁺/z^-i^- would then be expected not to synthesize galactosidase constitutively.”
  
  To address this question, they carried out a mating experiment in which they introduced the wildtype, z⁺i⁺, into a doubly mutant strain, z^-i^-, and asked whether the “heterozygote” would produce enzyme in the absence of inducer. On their own the wildtype z⁺i⁺ and the double mutant z^-i^- failed to produce β-galactosidase in the absence of inducer; the wildtype needs inducer and the doubly mutant strain is mutant for z.  The striking result was that the heterozygote did synthesize β-galactosidase and did so in the absence of inducer.  Furthermore, synthesis was transitory, lasting only for about two hours and then shutting off as seen in the figure. This was interpreted as indicating that the zygotes eventually become inducible as the i⁺ gene from the male becomes expressed following mating. Indeed, addition of inducer at hour 2 (highlighted in red) immediately triggered β-galactosidase synthesis.  They concluded that, “The i gene in its active form controls the synthesis of a product which, when present in the cytoplasm, prevents the synthesis of β-galactosidase…”  Hence i produces a “repressor,” but they end their historic publication by leaving two questions unanswered:
  
  “(a) What is the chemical nature of the repressor? Should it be considered a primary or a secondary product of the gene?
  
  (b) Does the repressor act at the level of the gene itself, or at the level of the cytoplasmic gene-product…?”
  
  Pardee, Jacob and Monod went on to propose that the same basic mechanism operates for phage λ in its prophage state, that is, that a phage repressor blocks “synthesis of proteins determined by other genes of the phage…” This was based on the observation in earlier work that when a male bacterium lysogenic for phage λ was mated with a female bacterium lacking the prophage, induction of the phage was triggered. As engagingly described by Jacob, this phenomenon was “immediately baptized ‘erotic induction ’of the prophage; which for purposes of publication was to be changed to ‘zygotic induction’.”
  
  Jacob tells the story that while Monod was on summer vacation, he realized that both the lac operon and Lwoff’s prophage are under negative control by repressors. “Both experiments, conjugation done… on the phage and the PA JA MA are the same! Same situation. Same result. Same conclusion.  In both cases a gene governs the formation of a ….repressor blocking the expression of other genes…”  Monod, who could be obstinate, pushed back against this idea upon his return from vacation but was eventually won over.
  
  Later work would show that the lactose system is an operon that includes z and two other genes y and a that are all transcribed from a common promoter. This is known as the Lac operon.  The repressor gene i is adjacent to the operon and is transcribed from its own promoter.  Also, as we come to in the next chapter, work by Wally Gilbert for the Lac repressor and Mark Ptashne for the phage λ repressor answered the questions posed by Pardee, Jacob and Monod (above): Repressors are proteins that bind to sites called operators to block transcription of genes under their control- the gene for β-galactosidase in the case of the Lac repressor and phage λ genes in the case of the phage repressor. Furthermore, in the case of the lactose system, the inducer works by binding to the repressor, trapping it in a state in which it is unable to bind to the operator.  In the case of phage λ, phage induction is triggered by ultraviolet light which destroys the repressor. 
  
  Monod was also famously obstinate in asserting that negative regulation could explain all gene control. Ironically, later work would show that the lactose system is itself a paradigm for positive control as well as negative control; transcription requires not only the absence of repressor but also the presence of an activator protein called CAP. 
  
  As we will revisit in chapter 14, the PaJaMo experiments set the stage for realizing that genetic information is transferred into protein by an unstable intermediate.

  “The conclusion seems, therefore, inescapable that upon transfer a genetic determinant can, within a few minutes and much before integration as a part of a complete genome, be expressed by the synthesis of a protein at a rate close to the expected maximum…One of the most remarkable features brought out by the preliminary experiments summarized in this paper is the rapidity with which β-galactosidase can be synthesized during conjugation. The time between the introduction into the cell of the genetic determinant controlling enzyme synthesis and the appearance of a detectable amount of β-galactosidase seems to be extremely short, not exceeding a few minutes. Such a lag is rather small for what could be expected a priori for the synthesis of stable macromolecular intermediates.”

  We conclude with an excerpt from video interview (with Stanford Professor Lucy Shapiro) in which Jacob revisits the classic PaJaMo experiments: