Jacob Monod Hypothesis Essay Sample

A combination of genetic and biochemical experiments in bacteria led to the initial recognition of (1) protein-binding regulatory sequences associated with genes and (2) proteins whose binding to a gene’s regulatory sequences either activate or repress its transcription. These key components underlie the ability of both prokaryotic and eukaryotic cells to turn genes on and off, although innumerable variations on the basic process have been discovered. In this section, we first describe some of the early experimental findings leading to a general model of bacterial transcription control. In the next section, we take a closer look at how bacterial RNA polymerase initiates transcription and the mechanisms controlling its ability to do so.

In bacteria, gene control serves mainly to allow a single cell to adjust to changes in its nutritional environment so that its growth and division can be optimized. Thus, the prime focus of research has been on genes that encode inducible proteins whose production varies depending on the nutritional status of the cells. Although gene control in multicellular organisms often involves response to environmental changes, its most characteristic and biologically far-reaching purpose is the regulation of a genetic program that underlies embryological development and tissue differentiation. Nonetheless, many of the principles of transcription control first discovered in bacteria also apply to eukaryotic cells.

Enzymes Encoded at the lac Operon Can Be Induced and Repressed

E. coli can use either glucose or other sugars such as the disaccharide lactose as the sole source of carbon and energy. When E. coli cells are grown in a glucose-containing medium, the activity of the enzymes needed to metabolize lactose is very low. When these cells are switched to a medium containing lactose but no glucose, the activities of the lactose-metabolizing enzymes increase. Early studies showed that the increase in the activity of these enzymes resulted from the synthesis of new enzyme molecules, a phenomenon termed induction. The enzymes induced in the presence of lactose are encoded by the lacoperon, which includes two genes, Z and Y, that are required for metabolism of lactose and a third gene, A (Figure 10-1). The lacY gene encodes lactose permease, which spans the E. coli cell membrane and uses the energy available from the electrochemical gradient across the membrane to pump lactose into the cell (Section 15.5). The lacZ gene encodes β-galactosidase, which splits the disaccharide lactose into the monosaccharides glucose and galactose (see Figure 2-10); these sugars are further metabolized through the action of enzymes encoded in other operons. The lacA gene encodes thiogalactoside transacetylase, an enzyme whose physiological function is not well understood.

Figure 10-1

The lac operon includes three genes: lacZ, which encodes β-galactosidase; lacY, which encodes lactose permease; and lacA, which encodes thiogalactoside transacetylase. Binding of regulatory proteins to sites in the control region immediately upstream (more...)

Synthesis of all three enzymes encoded in the lacoperon is rapidly induced when E. coli cells are placed in a medium containing lactose as the only carbon source and repressed when the cells are switched to a medium without lactose. Thus all three genes of the lac operon are coordinately regulated. The lac operon in E. coli provides one of the earliest and still best-understood examples of gene control. Much of the pioneering research on the lac operon was conducted by Francois Jacob, Jacques Monod, and their colleagues in the 1960s.

Some molecules similar in structure to lactose can induce expression of the lac-operon genes even though they cannot be hydrolyzed by β-galactosidase. Such small molecules (i.e., smaller than proteins) are called inducers. One of these, isopropyl-β-D-thiogalactoside, abbreviated IPTG,is particularly useful in genetic studies of the lac operon, because it can diffuse into cells and, since it is not metabolized, its concentration remains constant throughout an experiment.

Mutations in lacI Cause Constitutive Expression of lac Operon

Insight into the mechanisms controlling synthesis of β-galactosidase and lactose permease first came from the study of mutants in which control of β-galactosidase expression was abnormal. A sensitive colorimetric assay for β-galactosidase uses X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) as substrate:

Hydrolysis of this colorless analog of lactose by β-galactosidase yields an intensely blue product. When wild-type E. coli cells are plated on media containing X-gal plus lactose as the major carbon source, all the colonies that grow appear blue. When the cells are plated on media containing X-gal plus glucose as the carbon source, the resulting colonies appear white; in this case, β-galactosidase synthesis is repressed and there is not sufficient β-galactosidase in the cells to hydrolyze the X-gal to its colored product. However, when the cells are exposed to chemical mutagens before plating on X-gal/glucose plates, rare blue colonies appear. In most cases, when cells from these blue colonies are recovered and grown in media containing glucose, they are found to express all the genes of the lacoperon at much higher levels than wild-type cells in the same medium. Such cells are called constitutive mutants because they fail to repress the lac operon in media lacking lactose and instead continuously, or constitutively, express the enzymes. By recombinational analysis (Section 8.3), these mutations were mapped to a region on the E. colichromosome to the left of the lacZgene, a region called the lacI gene (see Figure 10-1).

Jacob and Monod reasoned that such constitutive mutants probably had a defect in a protein that normally repressed expression of the lacoperon in the absence of lactose. Hence they called the protein encoded by the lacIgene the lac repressor and proposed that it binds to a site on the E. coligenome where transcription of the lac operon is initiated, thereby blocking transcription. They further hypothesized that when lactose is present in the cell, it binds to the lac repressor, decreasing its affinity for the repressor-binding site on the DNA. As a result, the repressor falls off the DNA and transcription of the lac operon is initiated, leading to synthesis of β-galactosidase, lactose permease, and thiogalactoside transacetylase (Figure 10-2).

Figure 10-2

Jacob and Monod model of transcriptional regulation of the lac operon by lac repressor. When lac repressor binds to a DNA sequence called the operator (O), which lies just upstream of the lacZ gene, transcription of the operon by RNA polymerase is blocked. (more...)

Isolation of Operator Constitutive and Promoter Mutants Support Jacob-Monod Model

The model proposed by Jacob and Monod predicted that a specific DNA sequence near the transcriptionstart site of the lacoperon is a binding site for lac repressor. They reasoned that mutations in this sequence, which they termed the operator (O), would prevent the repressor from binding, thus yielding constitutive mutants that could be identified on X-gal/glucose indicator plates. To distinguish between mutations in the lacIgene, which inactivate the repressor, and mutations in the operator, which prevent repressor binding, Jacob and Monod mutagenized cells carrying two copies of the wild-type lacI gene, one on the bacterial chromosome and one on a plasmid. In this system, separate mutations in both copies of lacI in a given cell are required to generate a lacIconstitutive mutant, a low-probability event. In contrast, only a single mutation in the operator of one copy of the lac operon is required to yield a constitutive mutant. Using this approach, Jacob and Monod isolated mutants that expressed the lac operon constitutively even when two copies of the wild-type lacI gene encoding the lac repressor were present in the same cell. These operator constitutive (Oc) mutations mapped to one end of the lac operon, as the model predicted (see Figure 10-2).

Most mutations that prevent expression of β-galactosidase in cells exposed to an inducer such as IPTG map in the lacZgene itself. But a rare class of mutations map to a region between lacI and the operator, in a region termed the promoter (P). Cells carrying these mutations also cannot induce expression of the lacY and lacA genes; that is, these mutations prevent expression of the entire lacoperon. According to the Jacob and Monod model, such promoter mutations block initiation of transcription by RNA polymerase (see Figure 10-2). Consequently, no lac mRNA and therefore no lac proteins are synthesized, even when lac repressor binds IPTG and comes off the lac operator.

Regulation of lac Operon Depends on Cis-Acting DNA Sequences and Trans-Acting Proteins

Subsequent analyses of the effects of various mutations in E. coli cells containing one or two copies of lacDNA provided further insight into regulation of lac-operonexpression. In these experiments, assays for β-galactosidase and lactose permease activity were conducted in the presence and absence of inducer (IPTG). These analyses showed that the Ocmutation is dominant over O+ (the wild-type lac O sequence). In addition, the Oc mutation only affects expression of lac genes on the same DNA molecule (i.e., genes in cis to the mutation). Experimental demonstration of the cis-acting nature of the Oc mutation is illustrated in Figure 10-3.

Figure 10-3

Experimental demonstration that Oc mutations are cis-acting. E. coli cells containing two copies of the lac operon are diagrammed. Diagonal lines indicate genes and control regions carrying mutations. In these cells, the lac operon on the bacterial chromosome has (more...)

As noted earlier, mutations in lacI (in cells with a single lacoperon) cause constitutiveexpression of β-galactosidase and lactose permease because no functional repressor is made. Unlike the Ocmutation, which is dominant, the lacI mutation is recessive to the wild-type lacI+gene. Furthermore, the wild-type lacI+ gene can exert control over the lacZ and lacY genes on a different DNA molecule (i.e., genes in trans to lacI+). The trans-acting ability of lacI+is easy to understand since this gene encodes a protein, which is free to diffuse through the cell and bind to any lacoperator in the cell (Figure 10-4).

Figure 10-4

Experimental demonstration that the lacI+ gene is trans-acting. (Top) Cells carrying a single lacI gene produce an inactive repressor; as a result, they express β-galactosidase and lactose permease constitutively. (Bottom) When a wild-type (more...)

In general, cis-acting mutations are in DNA sequences that function as binding sites for proteins that control the expression of nearby genes. For example, the cis-acting Oc mutations prevent binding of the lac repressor to the operator. Similarly, mutations in the lacpromoter are cis-acting, since they alter the binding site for RNA polymerase. When RNA polymerase cannot initiate transcription of the lacoperon, none of the genes in the operon can be expressed irrespective of the function of the repressor. In general, trans-acting genes that regulate expression of genes on other DNA molecules encode diffusible products. In most cases these are proteins, but in some cases RNA molecules can act in trans to regulate gene expression.

Biochemical Experiments Confirm That Induction of the lac Operon Leads to Increased Synthesis of lac mRNA

The Jacob and Monod model of repressor control of lacoperontranscription, which was based on genetic experiments with E. coli mutants, proposes that addition of inducer causes an increase in transcription of the lac operon. This prediction was tested directly through pulse-labeling experiments that measured the rate of lac mRNA synthesis in E. coli cells grown initially in glucose media and then after addition of IPTG. The results of such experiments showed that little lac mRNA is synthesized before the addition of IPTG, but lac mRNA synthesis is detectable within 1 minute after the addition of IPTG and reaches a maximal rate by 2 minutes (Figure 10-5). At later times, lac mRNA synthesis is maintained at this maximal rate as long as inducer is present. These findings demonstrated directly that inducer does indeed cause an increase in transcription of the lac operon.

Figure 10-5

Biochemical demonstration that inducer leads to an increase in lac operon transcription. Small samples of an E. coli culture growing in glucose medium were removed just before and at short intervals after addition of IPTG. [3H]uridine was added to each (more...)

SUMMARY

  •  Many proteins in bacteria are inducible, that is, their synthesis is regulated depending on the cell’s nutritional status. Differential expression of genes encoding such proteins most commonly occurs at the level of transcription initiation.

  •  According to the Jacob and Monod model of transcriptional control, transcription of the lacoperon, which encodes three inducible proteins, is repressed by binding of lac repressor protein to the operator sequence (see Figure 10-2). In the presence of lactose or other inducer, this repression is relieved and the lac operon is transcribed.

  •  Mutations in the promoter, which binds RNA polymerase, or the operator are cis-acting; that is, they only affect expression of genes on the same DNA molecule in which the mutation occurs.

  •  Mutations in an operator sequence that decrease repressor binding result in constitutivetranscription. Mutations in a promoter sequence, which affect the affinity of RNA polymerase binding, can either decrease (down-mutation) or increase (up-mutation) transcription.

  •  Repressors and activators are trans-acting; that is, they affect expression of their regulated genes no matter on which DNA molecule in the cell these are located.

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.

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