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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 10.1Bacterial Gene Control: The Jacob-Monod Model

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 lac operon, 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.

Figure 10-1

The lac operon includes three genes: lacZ, which encodes β-galactosidase; lacY, which encodes lactose permease; and lacA, which encodes (more...)

Synthesis of all three enzymes encoded in the lac operon 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.

Image ch10e1.jpg

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:

Image ch10e2.jpg
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 lac operon 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. coli chromosome to the left of the lacZ gene, 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 lac operon in the absence of lactose. Hence they called the protein encoded by the lacI gene the lac repressor and proposed that it binds to a site on the E. coli genome 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.

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 (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 transcription start site of the lac operon 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 lacI gene, 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 lacI constitutive 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 lacZ gene 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 lac operon. 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 lac DNA provided further insight into regulation of lac-operon expression. 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 Oc mutation 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.

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 (more...)

As noted earlier, mutations in lacI (in cells with a single lac operon) cause constitutive expression of β-galactosidase and lactose permease because no functional repressor is made. Unlike the Oc mutation, 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 lac operator in the cell (Figure 10-4).

Figure 10-4. Experimental demonstration that the lacI+ gene is trans-acting.

Figure 10-4

Experimental demonstration that the lacI+ gene is trans-acting. (Top) Cells carrying a single lacI gene produce an (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 lac promoter are cis-acting, since they alter the binding site for RNA polymerase. When RNA polymerase cannot initiate transcription of the lac operon, 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 lac operon transcription, 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.

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 (more...)


  •  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 lac operon, 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 constitutive transcription. 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.

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Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21683