LACA Awards

The ''lactose'' operon (''lac'' operon) is an operon required for the transport and metabolism of lactose in ''E. coli'' and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the ''lac'' operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the ''lac'' operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the ''lac'' operon won them the Nobel Prize in Physiology in 1965.

Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as a sugar source for the bacterium, the three genes of the ''lac'' operon can be expressed and their subsequent proteins translated: ''lacZ'', ''lacY'', and ''lacA''. The gene product of ''lacZ'' is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. ''lacY'' encodes Beta-galactoside permease, a membrane protein which becomes embedded in the cytoplasmic membrane to enable the cellular transport of lactose into the cell. Finally, ''lacA'' encodes Galactoside acetyltransferase.

It would be wasteful to produce enzymes when no lactose is available or if a preferable energy source such as glucose were available. The ''lac'' operon uses a two-part control mechanism to ensure that the cell expends energy producing the enzymes encoded by the ''lac'' operon only when necessary. In the absence of lactose, the ''lac'' repressor, ''lacI,'' halts production of the enzymes encoded by the ''lac'' operon. The lac repressor is always expressed, unless a co-inducer binds to it. In other words, it is transcribed only in the presence of small co-inducer molecules. In the presence of glucose, the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and EIIA^Glc shuts down lactose permease to prevent transport of lactose into the cell. This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxie.

Structure

*The ''lac'' operon consists of 3 structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: ''lacZ'', ''lacY'', and ''lacA''.
**''lacZ'' encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.
** ''lacY'' encodes Beta-galactoside permease (LacY), a transmembrane symporter that pumps β-galactosides including lactose into the cell using a proton gradient in the same direction. Permease increases the permeability of the cell to β-galactosides.
** ''lacA'' encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to thiogalactoside.

Only ''lacZ'' and ''lacY'' appear to be necessary for lactose catabolic pathway.

Genetic nomenclature

Three-letter abbreviations are used to describe phenotypes in bacteria including ''E. coli''.

Examples include:
* Lac (the ability to use lactose),
* His (the ability to synthesize the amino acid histidine)
* Mot (swimming motility)
* Sm^R (resistance to the antibiotic streptomycin)

In the case of Lac, wild type cells are Lac⁺ and are able to use lactose as a carbon and energy source, while Lac⁻ mutant derivatives cannot use lactose. The same three letters are typically used (lower-case, italicized) to label the genes involved in a particular phenotype, where each different gene is additionally distinguished by an extra letter. The ''lac'' genes encoding enzymes are ''lacZ'', ''lacY'', and ''lacA''. The fourth ''lac'' gene is ''lacI'', encoding the lactose repressor—"I" stands for ''inducibility''.

One may distinguish between ''structural'' genes encoding enzymes, and regulatory genes encoding proteins that affect gene expression. Current usage expands the phenotypic nomenclature to apply to proteins: thus, LacZ is the protein product of the ''lacZ'' gene, β-galactosidase. Various short sequences that are not genes also affect gene expression, including the ''lac'' promoter, ''lac p'', and the ''lac'' operator, ''lac o''. Although it is not strictly standard usage, mutations affecting ''lac o'' are referred to as ''lac o''^c, for historical reasons.

Regulation

Specific control of the ''lac'' genes depends on the availability of the substrate lactose to the bacterium. The proteins are not produced by the bacterium when lactose is unavailable as a carbon source.
The ''lac'' genes are organized into an operon; that is, they are oriented in the same direction immediately adjacent on the chromosome and are co-transcribed into a single polycistronic mRNA molecule. Transcription of all genes starts with the binding of the enzyme RNA polymerase (RNAP), a DNA-binding protein, which binds to a specific DNA binding site, the promoter, immediately upstream of the genes. Binding of RNA polymerase to the promoter is aided by the cAMP-bound catabolite activator protein (CAP, also known as the cAMP receptor protein). However, the ''lacI'' gene (regulatory gene for ''lac'' operon) produces a protein that blocks RNAP from binding to the operator of the operon. This protein can only be removed when allolactose binds to it, and inactivates it. The protein that is formed by the ''lacI'' gene is known as the lac repressor. The type of regulation that the ''lac'' operon undergoes is referred to as negative inducible, meaning that the gene is turned off by the regulatory factor (''lac'' repressor) unless some molecule (lactose) is added. Because of the presence of the ''lac'' repressor protein, genetic engineers who replace the ''lacZ'' gene with another gene will have to grow the experimental bacteria on agar with lactose available on it. If they do not, the gene they are trying to express will not be expressed as the repressor protein is still blocking RNAP from binding to the promoter and transcribing the gene. Once the repressor is removed, RNAP then proceeds to transcribe all three genes (''lacZYA'') into mRNA. Each of the three genes on the mRNA strand has its own Shine-Dalgarno sequence, so the genes are independently translated. The DNA sequence of the ''E. coli'' ''lac'' operon, the ''lacZYA'' mRNA, and the ''lacI '' genes are available from GenBankbr>(view)

The first control mechanism is the regulatory response to lactose, which uses an intracellular ''regulatory protein'' called the ''lactose repressor'' to hinder production of β-galactosidase in the absence of lactose. The ''lacI'' gene coding for the repressor lies nearby the ''lac'' operon and is always expressed (''constitutive''). If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of ''lacZ'' called the ''lac operator''. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels. When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose, made from lactose by the product of the ''lacZ'' gene, binds to the repressor, causing an allosteric shift. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the ''lac'' genes and thereby leading to higher levels of the encoded proteins.

The second control mechanism is a response to glucose, which uses the catabolite activator protein (CAP) homodimer to greatly increase production of β-galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below, about 60 bp upstream of the transcription start site), which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase, enabling the cell to hydrolyse lactose and release galactose and glucose.

More recently inducer exclusion was shown to block expression of the ''lac'' operon when glucose is present. Glucose is transported into the cell by the PEP-dependent phosphotransferase system. The phosphate group of phosphoenolpyruvate is transferred via a phosphorylation cascade consisting of the general PTS (phosphotransferase system) proteins HPr and EIA and the glucose-specific PTS proteins EIIA^Glc and EIIB^Glc, the cytoplasmic domain of the EII glucose transporter. Transport of glucose is accompanied by its phosphorylation by EIIB^Glc, draining the phosphate group from the other PTS proteins, including EIIA^Glc. The unphosphorylated form of EIIA^Glc binds to the ''lac'' permease and prevents it from bringing lactose into the cell. Therefore, if both glucose and lactose are present, the transport of glucose blocks the transport of the inducer of the ''lac'' operon.

Repressor structure

The lac repressor is a four-part protein, a tetramer, with identical subunits. Each subunit contains a helix-turn-helix (HTH) motif capable of binding to DNA. The operator site where repressor binds is a DNA sequence with inverted repeat symmetry. The two DNA half-sites of the operator together bind to two of the subunits of the repressor. Although the other two subunits of repressor are not doing anything in this model, this property was not understood for many years.

Eventually it was discovered that two additional operators are involved in ''lac'' regulation. One (O₃) lies about −90 bp upstream of O₁ in the end of the ''lacI'' gene, and the other (O₂) is about +410 bp downstream of O₁ in the early part of ''lacZ''. These two sites were not found in the early work because they have redundant functions and individual mutations do not affect repression very much. Single mutations to either O₂ or O₃ have only 2 to 3-fold effects. However, their importance is demonstrated by the fact that a double mutant defective in both O₂ and O₃ is dramatically de-repressed (by about 70-fold).

In the current model, ''lac'' repressor is bound simultaneously to both the main operator O₁ and to either O₂ or O₃. The intervening DNA loops out from the complex. The redundant nature of the two minor operators suggests that it is not a specific looped complex that is important. One idea is that the system works through tethering; if bound repressor releases from O₁ momentarily, binding to a minor operator keeps it in the vicinity, so that it may rebind quickly. This would increase the affinity of repressor for O₁.

Mechanism of induction

The repressor is an allosteric protein, i.e. it can assume either one of two slightly different shapes, which are in equilibrium with each other. In one form the repressor will bind to the operator DNA with high specificity, and in the other form it has lost its specificity. According to the classical model of induction, binding of the inducer, either allolactose or IPTG, to the repressor affects the distribution of repressor between the two shapes. Thus, repressor with inducer bound is stabilized in the non-DNA-binding conformation. However, this simple model cannot be the whole story, because repressor is bound quite stably to DNA, yet it is released rapidly by addition of inducer. Therefore, it seems clear that an inducer can also bind to the repressor when the repressor is already bound to DNA. It is still not entirely known what the exact mechanism of binding is.

Role of non-specific binding

Non-specific binding of the repressor to DNA plays a crucial role in the repression and induction of the Lac-operon. The specific binding site for the Lac-repressor protein is the operator. The non-specific interaction is mediated mainly by charge-charge interactions while binding to the operator is reinforced by hydrophobic interactions. Additionally, there is an abundance of non-specific DNA sequences to which the repressor can bind. Essentially, any sequence that is not the operator, is considered non-specific. Studies have shown, that without the presence of non-specific binding, induction (or unrepression) of the Lac-operon could not occur even with saturated levels of inducer. It had been demonstrated that, without non-specific binding, the basal level of induction is ten thousand times smaller than observed normally. This is because the non-specific DNA acts as sort of a "sink" for the repressor proteins, distracting them from the operator. The non-specific sequences decrease the amount of available repressor in the cell. This in turn reduces the amount of inducer required to unrepress the system.

Lactose analogs

A number of lactose derivatives or analogs have been described that are useful for work with the ''lac'' operon. These compounds are mainly substituted galactosides, where the glucose moiety of lactose is replaced by another chemical group.

* Isopropyl-β-D-thiogalactopyranoside (IPTG) is frequently used as an inducer of the ''lac'' operon for physiological work. IPTG binds to repressor and inactivates it, but is not a substrate for β-galactosidase. One advantage of IPTG for '' in vivo'' studies is that since it cannot be metabolized by ''E. coli''. Its concentration remains constant and the rate of expression of ''lac p/o''-controlled genes is not a variable in the experiment. IPTG intake is dependent on the action of lactose permease in ''P. fluorescens'', but not in ''E. coli''.
* Phenyl-β-D-galactose (phenyl-Gal) is a substrate for β-galactosidase, but does not inactivate repressor and so is not an inducer. Since wild type cells produce very little β-galactosidase, they cannot grow on phenyl-Gal as a carbon and energy source. Mutants lacking repressor are able to grow on phenyl-Gal. Thus, minimal medium containing only phenyl-Gal as a source of carbon and energy is selective for repressor mutants and operator mutants. If 10⁸ cells of a wild type strain are plated on agar plates containing phenyl-Gal, the rare colonies which grow are mainly spontaneous mutants affecting the repressor. The relative distribution of repressor and operator mutants is affected by the target size. Since the ''lacI gene'' encoding repressor is about 50 times larger than the operator, repressor mutants predominate in the selection.
* Thiomethyl galactoside MGis another lactose analog. These inhibit the lacI repressor. At low inducer concentrations, both TMG and IPTG can enter the cell through the lactose permease. However at high inducer concentrations, both analogs can enter the cell independently. TMG can reduce growth rates at high extracellular concentrations.
* Other compounds serve as colorful indicators of β-galactosidase activity.
** ONPG is cleaved to produce the intensely yellow compound, orthonitrophenol and galactose, and is commonly used as a substrate for assay of β-galactosidase '' in vitro''.
** Colonies that produce β-galactosidase are turned blue by X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) which is an artificial substrate for B-galactosidase whose cleavage results in galactose and 4-Cl,3-Br indigo thus producing a deep blue color.
* Allolactose is an isomer of lactose and is the inducer of the ''lac'' operon. Lactose is galactose-β(1→4)-glucose, whereas allolactose is galactose-β(1→6)-glucose. Lactose is converted to allolactose by β-galactosidase in an alternative reaction to the hydrolytic one. A physiological experiment which demonstrates the role of LacZ in production of the "true" inducer in ''E. coli'' cells is the observation that a null mutant of ''lacZ'' can still produce LacY permease when grown with IPTG, a non-hydrolyzable analog of allolactose, but not when grown with lactose. The explanation is that processing of lactose to allolactose (catalyzed by β-galactosidase) is needed to produce the inducer inside the cell.

Development of the classic model

The experimental microorganism used by François Jacob and Jacques Monod was the common laboratory bacterium, ''E. coli'', but many of the basic regulatory concepts that were discovered by Jacob and Monod are fundamental to cellular regulation in all organisms. The key idea is that proteins are not synthesized when they are not needed—''E. coli'' conserves cellular resources and energy by not making the three Lac proteins when there is no need to metabolize lactose, such as when other sugars like glucose are available. The following section discusses how ''E. coli'' controls certain genes in response to metabolic needs.

During World War II, Monod was testing the effects of combinations of sugars as nutrient sources for ''E. coli'' and ''B. subtilis''. Monod was following up on similar studies that had been conducted by other scientists with bacteria and yeast. He found that bacteria grown with two different sugars often displayed two phases of growth. For example, if glucose and lactose were both provided, glucose was metabolized first (growth phase I, see Figure 2) and then lactose (growth phase II). Lactose was not metabolized during the first part of the diauxic growth curve because β-galactosidase was not made when both glucose and lactose were present in the medium. Monod named this phenomenon diauxie.

Monod then focused his attention on the induction of β-galactosidase formation that occurred when lactose was the sole sugar in the culture medium.

Classification of regulatory mutants

A conceptual breakthrough of Jacob and Monod was to recognize the distinction between regulatory substances and sites where they act to change gene expression. A former soldier, Jacob used the analogy of a bomber that would release its lethal cargo upon receipt of a special radio transmission or signal. A working system requires both a ground transmitter and a receiver in the airplane. Now, suppose that the usual transmitter is broken. This system can be made to work by introduction of a second, functional transmitter. In contrast, he said, consider a bomber with a defective receiver. The behavior of ''this'' bomber cannot be changed by introduction of a second, functional aeroplane.

To analyze regulatory mutants of the ''lac'' operon, Jacob developed a system by which a second copy of the ''lac'' genes (''lacI'' with its promoter, and ''lacZYA'' with promoter and operator) could be introduced into a single cell. A culture of such bacteria, which are diploid for the ''lac'' genes but otherwise normal, is then tested for the regulatory phenotype. In particular, it is determined whether LacZ and LacY are made even in the absence of IPTG (due to the ''lactose repressor'' produced by the mutant gene being non-functional). This experiment, in which genes or gene clusters are tested pairwise, is called a ''complementation test''.

This test is illustrated in the figure (''lacA'' is omitted for simplicity). First, certain haploid states are shown (i.e. the cell carries only a single copy of the ''lac'' genes). Panel (a) shows repression, (b) shows induction by IPTG, and (c) and (d) show the effect of a mutation to the ''lacI'' gene or to the operator, respectively. In panel (e) the complementation test for repressor is shown. If one copy of the ''lac'' genes carries a mutation in ''lacI'', but the second copy is wild type for ''lacI'', the resulting phenotype is normal—but lacZ is expressed when exposed to inducer IPTG. Mutations affecting repressor are said to be ''recessive'' to wild type (and that wild type is ''dominant''), and this is explained by the fact that repressor is a small protein which can diffuse in the cell. The copy of the ''lac'' operon adjacent to the defective ''lacI'' gene is effectively shut off by protein produced from the second copy of ''lacI''.

If the same experiment is carried out using an operator mutation, a different result is obtained (panel (f)). The phenotype of a cell carrying one mutant and one wild type operator site is that LacZ and LacY are produced even in the absence of the inducer IPTG; because the damaged operator site, does not permit binding of the repressor to inhibit transcription of the structural genes. The operator mutation is dominant. When the operator site where repressor must bind is damaged by mutation, the presence of a second functional site in the same cell makes no difference to expression of genes controlled by the mutant site.

A more sophisticated version of this experiment uses ''marked'' operons to distinguish between the two copies of the ''lac'' genes and show that the unregulated structural gene(s) is(are) the one(s) next to the mutant operator (panel (g). For example, suppose that one copy is marked by a mutation inactivating ''lacZ'' so that it can only produce the LacY protein, while the second copy carries a mutation affecting ''lacY'' and can only produce LacZ. In this version, only the copy of the ''lac'' operon that is adjacent to the mutant operator is expressed without IPTG. We say that the operator mutation is ''cis-dominant'', it is dominant to wild type but affects only the copy of the operon which is immediately adjacent to it.

This explanation is misleading in an important sense, because it proceeds from a description of the experiment and then explains the results in terms of a model. But in fact, it is often true that the model comes first, and an experiment is fashioned specifically to test the model. Jacob and Monod first imagined that there must be a ''site'' in DNA with the properties of the operator, and then designed their complementation tests to show this.

The dominance of operator mutants also suggests a procedure to select them specifically. If regulatory mutants are selected from a culture of wild type using phenyl-Gal, as described above, operator mutations are rare compared to repressor mutants because the target-size is so small. But if instead we start with a strain which carries two copies of the whole ''lac'' region (that is diploid for ''lac''), the repressor mutations (which still occur) are not recovered because complementation by the second, wild type ''lacI'' gene confers a wild type phenotype. In contrast, mutation of one copy of the operator confers a mutant phenotype because it is dominant to the second, wild type copy.

Regulation by cyclic AMP

Explanation of diauxie depended on the characterization of additional mutations affecting the ''lac'' genes other than those explained by the classical model. Two other genes, ''cya'' and ''crp'', subsequently were identified that mapped far from ''lac'', and that, when mutated, result in a decreased level of expression in the ''presence'' of IPTG and even in strains of the bacterium lacking the repressor or operator. The discovery of cAMP in ''E. coli'' led to the demonstration that mutants defective the ''cya'' gene but not the ''crp'' gene could be restored to full activity by the addition of cAMP to the medium.

The ''cya'' gene encodes adenylate cyclase, which produces cAMP. In a ''cya'' mutant, the absence of cAMP makes the expression of the ''lacZYA'' genes more than ten times lower than normal. Addition of cAMP corrects the low Lac expression characteristic of ''cya'' mutants. The second gene, ''crp'', encodes a protein called catabolite activator protein (CAP) or cAMP receptor protein (CRP).

However the lactose metabolism enzymes are made in small quantities in the presence of both glucose and lactose (sometimes called leaky expression) due to the fact that the LacI repressor rapidly associates/dissociates from the DNA rather than tightly binding to it, which can allow time for RNAP to bind and transcribe mRNAs of ''lacZYA''. Leaky expression is necessary in order to allow for metabolism of some lactose after the glucose source is expended, but before ''lac'' expression is fully activated.

In summary:

* When lactose is absent then there is very little Lac enzyme production (the operator has Lac repressor bound to it).
* When lactose is present but a preferred carbon source (like glucose) is also present then a small amount of enzyme is produced (Lac repressor is not bound to the operator).
* When glucose is absent, CAP-cAMP binds to a specific DNA site upstream of the promoter and makes a direct protein-protein interaction with RNAP that facilitates the binding of RNAP to the promoter.

The delay between growth phases reflects the time needed to produce sufficient quantities of lactose-metabolizing enzymes. First, the CAP regulatory protein has to assemble on the ''lac'' promoter, resulting in an increase in the production of ''lac'' mRNA. More available copies of the ''lac'' mRNA results in the production (see translation) of significantly more copies of LacZ (β-galactosidase, for lactose metabolism) and LacY (lactose permease to transport lactose into the cell). After a delay needed to increase the level of the lactose metabolizing enzymes, the bacteria enter into a new rapid phase of cell growth.

Two puzzles of catabolite repression relate to how cAMP levels are coupled to the presence of glucose, and secondly, why the cells should even bother. After lactose is cleaved it actually forms glucose and galactose (easily converted to glucose). In metabolic terms, lactose is just as ''good'' a carbon and energy source as glucose. The cAMP level is related not to intracellular glucose concentration but to the rate of glucose transport, which influences the activity of adenylate cyclase. (In addition, glucose transport also leads to direct inhibition of the lactose permease.) As to why ''E. coli'' works this way, one can only speculate. All enteric bacteria ferment glucose, which suggests they encounter it frequently. It is possible that a small difference in efficiency of transport or metabolism of glucose v. lactose makes it advantageous for cells to regulate the ''lac'' operon in this way.

Use in molecular biology

The ''lac'' gene and its derivatives are amenable to use as a reporter gene in a number of bacterial-based selection techniques such as two hybrid analysis, in which the successful binding of a transcriptional activator to a specific promoter sequence must be determined. In LB plates containing X-gal, the colour change from white colonies to a shade of blue corresponds to about 20–100 β-galactosidase units, while tetrazolium lactose and MacConkey lactose media have a range of 100–1000 units, being most sensitive in the high and low parts of this range respectively. Since MacConkey lactose and tetrazolium lactose media both rely on the products of lactose breakdown, they require the presence of both ''lacZ'' and ''lacY'' genes. The many ''lac'' fusion techniques which include only the ''lacZ'' gene are thus suited to X-gal plates or ONPG liquid broths.

See also

* Catabolite repression

References

External links

*
* ''lac'' operon in NCBI Bookshel

* Virtual Cell Animation Collectio
Introducing: The ''Lac'' Operon

The ''lac'' Operon: Bozeman Science

Staining Whole Mouse Embryos for β-Galactosidase (lacZ) Activity

{{DEFAULTSORT:Lac Operon
Gene expression
Bacterial genetics
Operons