The lactose operon (also known as the lac operon) is a set of genes that are specific for uptake and metabolism of lactose and is found in E. coli and other bacteria.
From: Brenner's Encyclopedia of Genetics (Second Edition), 2013
A repressor protein regulates transcription of thelac operon inE. coli
Escherichia coli can use the disaccharide lactose (milk sugar) as a source of metabolic energy. Lactose is first transported across the plasma membrane by the membrane carrierlactose permease, then it is cleaved to free glucose and galactose by the enzymeβ-galactosidase (Fig. 6.31). A third protein,β-galactoside transacetylase, is not required for lactose catabolism, but it acetylates several other β-galactosides. It probably is involved in the removal of nonmetabolizable β-galactosides from the cell.
As an intestinal bacterium,E. coli needs these three proteins only when its host drinks milk. In the absence of lactose, the cell contains only about 10 molecules of β-galactosidase, but several thousand molecules are present when lactose is the only carbon source. The levels of the permease and the transacetylase parallel those of β-galactosidase.
The genes for these three proteins are lined up head to tail in the bacterial chromosome. They are regulated in concert becausethey are transcribed from a single promoter. The product of transcription is apolycistronic mRNA (fromcistron meaning “gene”). The ribosome can synthesize three different polypeptides from this large mRNA because the stop codons of the first two genes are followed by a Shine-Dalgarno sequence and a start codon at which the synthesis of the next polypeptide is initiated.
The array of protein-coding genes, shared promoter, and associated regulatory sites is called anoperon, and the protein-coding genes of the operon are calledstructural genes.
Wedged between the promoter and the first structural gene is anoperator (Fig. 6.32), a short regulatory DNA sequence that binds thelac repressor. The repressor binding site (operator) overlaps with the binding site for RNA polymerase (promoter). Thereforethe RNA polymerase cannot bind to the promoter when the lacrepressor is bound to the operator.
Thelac repressor is a tetrameric (from Greek τετρα meaning “four” and μερoσ meaning “part”) protein with four identical subunits, encoded by a regulatory gene that is constitutively transcribed at a low rate. This gene is located immediately upstream of thelac operon.
In the absence of lactose, thelac repressor binds tightly to the operator and prevents transcription of the structural genes. In the presence of lactose, however, a small amount of 1,6-allolactose is formed. This minor side product of the β-galactosidase reaction (seeFig. 6.31) binds tightly to thelac repressor, changing its conformation by an allosteric mechanism. The repressor- allolactose complex no longer binds to the operator, and the structural genes can be transcribed. Thus 1,6-allolactose functions as aninducer of thelac operon.
In addition to the primary operator, LacO, shown in Figure 1, two ‘pseudo-operator’ sequences are present within the lac operon sequence and contribute to repression. The DNA sequences of the pseudo-operators are very similar, but not identical, to LacO and are bound by LacI more weakly. The presence of two DNA-binding sites in LacI protein tetramer suggested a mechanism by which pseudo-operators could enhance repression – one LacI tetramer could bind two separate operators and generate a looped DNA structure. Experimental evidence for DNA looping has been obtained from a variety of laboratories. Recent evidence indicates that the angle between the two dimers must open for loop formation to occur, as illustrated in Figure 4. These looped structures are highly stabilized, accounting for the significant repression of lacZYA expression observed in bacterial cells. Indeed, DNA containing multiple operator sequences and with the supercoiling density characteristic of E. coli exhibits a half-life for the complex that exceeds 2 days. However, even these looped complexes respond rapidly (in less than 30 s) to the presence of inducer sugars, allowing quick adaptation to an external lactose source that may be transient.
Figure 4. Looped DNA structure. The teal blue curved line depicts the lac operon DNA (with shading to indicate nearness to observer), which contains three possible LacI-binding sites (two of which, O1 and O2, are shown bound to LacI). The pseudo-operator sequence O2 is located within the lacZ gene, and the primary operator sequence O1 overlaps the promoter sequence for the lacZYA metabolic genes (Figure 1). Tetrameric LacI is shown at the bottom of the figure as simultaneously interacting with O1 and O2. This structure loops the DNA and generates a complex with very high stability. Note that the dimers within a LacI tetramer separate and adopt a larger angle between them when looping between O1 and O2 than in the absence of looping (i.e., the structure shown in Figure 3(a)). The need for flexibility between the dimers for looping to occur is supported by experimental evidence.
Patrick R. Murray PhD, F(AAM), F(IDSA), in Medical Microbiology, 2021
Control of Gene Expression
Bacteria have developed mechanisms to adapt quickly and efficiently to changes and triggers from the environment. This allows them to coordinate and regulate the expression of genes for multicomponent structures or the enzymes of one or more metabolic pathways. For example, temperature change could signify entry into the human host and indicate the need for a global change in metabolism and upregulation of genes important for parasitism or virulence. Many bacterial genes are controlled at multiple levels and by multiple methods.
Promoters and operators are DNA sequences at the beginning of a gene or operon that are recognized by sigma factors, which are activator and repressor proteins that control expression of a gene or an operon. Thus all the genes coding for the enzymes of a particular pathway can be coordinately regulated.
Coordination of a large number of processes on a global level can also be mediated by small molecular activators, such as cyclic adenosine monophosphate (cAMP). Increased cAMP levels indicate low glucose levels and the need to use alternative metabolic pathways. Similarly, in a process calledquorum sensing, each bacterium produces a specific small molecule, and when a sufficient number of bacteria are present, the concentration of the molecule will be sufficient to coordinate the expression of genes to support the colony rather than the individual bacterium. The trigger for biofilm production byPseudomonas spp. is triggered by a critical concentration ofN-acyl homoserine lactone (AHL) produced when sufficient numbers of bacteria (quorum) are present. Activation of biofilm, toxin production, and more virulent behavior byStaphylococcus aureus accompanies the increase in concentration of a cyclic peptide.
The genes for some virulence mechanisms are organized into apathogenicity island under the control of a single promoter to coordinate their expression and ensure that all the proteins necessary for a structure or process are produced when needed. The many components of the type III secretion devices ofE. coli, Salmonella, orYersinia are grouped together within pathogenicity islands.
Transcription also can be regulated by the translation process. Unlike eukaryotes, the absence of a nuclear membrane in prokaryotes allows the ribosome to bind to the mRNA as it is being transcribed from the DNA. The position and speed of ribosomal movement along the mRNA can determine whether loops form in the mRNA, influencing the ability of the polymerase to continue transcription of new mRNA. This allows control of gene expression at both the transcriptional and translational levels.
Initiation of transcription may be under positive or negative control. Genes undernegative control are expressed unless they are switched off by arepressor protein. This repressor protein prevents gene expression by binding to a specific DNA sequence within the operator, blocking the RNA polymerase from initiating transcription at the promoter sequence. Conversely, genes whose expression is underpositive control are not transcribed unless an active regulator protein, called anapoinducer, is present. The apoinducer binds to a specific DNA sequence and assists the RNA polymerase in the initiation steps by an unknown mechanism.
Liskin Swint-Kruse, Kathleen S. Matthews, in Encyclopedia of Biological Chemistry, 2004
The role of LacI is to inhibit mRNA production for proteins encoded by the lac operon. Transcription is not completely eliminated, but lacZYA mRNA is transcribed only at very low levels. This function is accomplished by specific binding of LacI protein to the lac operator DNA sequence to inhibit transcription via a variety of mechanisms. Since the lac operator (LacO) overlaps the promoter, LacI binding directly competes with RNA polymerase for binding this region. LacI can also impede transcription initiation and/or block elongation of mRNA. The LacI·LacO association and consequent transcription repression occur when no lactose is available to serve as the substrate of the lac metabolic proteins.
When lactose is available as a carbon source, the low levels of metabolic enzymes allow a small amount of this sugar to be transported into the bacterium by LacY. Next, residual LacZ metabolizes lactose to glucose and galactose, which produces energy for the bacterium. Notably, this catalytic process also generates low levels of allolactose (a rearrangement of the β-1,4 linkage between glucose and galactose to a β-1,6 linkage; Figure 2). The by-product allolactose binds to LacI and elicits a conformational change in the protein that results in release of the operator DNA sequence (induction). Consequently, RNA polymerase is freed to generate numerous copies of mRNA encoding the lac enzymes. When translated into proteins, these enzymes allow the bacterium to transport and metabolize large quantities of lactose as its carbon energy source, taking advantage of environmental opportunity. One result of the studies by Jacob and Monod was the discovery that a variety of non-natural galactoside sugars (e.g., IPTG; Figure 2) can induce LacI and relieve transcription repression of lacZYA.
Richard V. Goering BA MSc PhD, in Mims' Medical Microbiology and Immunology, 2019
The principles of gene regulation in bacteria can be illustrated by the regulation of genes involved in sugar metabolism
Bacteria use sugars as a carbon source for growth and prefer to use glucose rather than other less well-metabolized sugars. When growing in an environment containing both glucose and lactose, bacteria such asE. coli preferentially metabolize glucose and at the same time prevent the expression of the lac operon, the products of which transport and metabolize lactose (Fig. 2.9). This is known as catabolite repression. It occurs because the transcriptional initiation of the lac operon is dependent upon a positive regulator: the cyclic adenosine monophosphate (cAMP)-dependent catabolite activator protein (CAP), which is activated only when cAMP is bound. When bacteria grow on glucose the cytoplasmic levels of cAMP are low and so CAP is not activated. CAP is therefore unable to bind to its DNA binding site adjacent to the lac promoter and facilitate transcription initiation by RNA polymerase. When the glucose is depleted, the cAMP concentration rises resulting in the formation of activated cAMP–CAP complexes, which bind the appropriate site on the DNA, increasing RNA polymerase binding and lac operon transcription.
CAP is an example of a global regulatory protein that controls the expression of multiple genes; it controls the expression of over 100 genes inE. coli. All genes controlled by the same regulator are considered to constitute a regulon (seeFig. 2.8). In addition to the influence of CAP on the lac operon, the operon is also subject to negative regulation by the lactose repressor protein (LacI, seeFig. 2.9). LacI is encoded by thelacI gene, which is located immediately upstream of the lactose operon and transcribed by a separate promoter. In the absence of lactose, LacI binds specifically to the operator region of the lac promoter and blocks transcription. An inducer molecule, allolactose (or its non-metabolizable homologue, isopropyl-thiogalactoside – IPTG) is able to bind to LacI, causing an allosteric change in its structure. This releases it from the DNA, thereby alleviating the repression. The lac operon therefore illustrates the fine tuning of gene regulation in bacteria – the operon is switched on only if lactose is available as a carbon source for cell growth, but remains unexpressed if glucose, the cell's preferred carbon source, is also present.
John W. Pelley, in Elsevier's Integrated Review Biochemistry (Second Edition), 2012
Regulation of the Lac Operon
The lack of a nuclear membrane in prokaryotes gives ribosomes direct access to mRNA transcripts, allowing their immediate translation into polypeptides. This makes transcription the rate-limiting step in prokaryotic gene expression and, therefore, a major point of regulation. The classic example of prokaryotic gene regulation is that of the lac operon. This operon is a genetic unit that produces the enzymes necessary for the digestion of lactose (Fig. 16-13).
The lac operon consists of three contiguous structural genes that are transcribed as continuous mRNA by RNA polymerase. An operator sequence located at the 5′ end serves as a binding site for a repressor protein that blocks RNA polymerase. The repressor protein is produced constitutively (continuously) by the i gene, which is not under regulatory control. The repressor itself is formed from subunits that self-assemble to form the active tetramer. When present, the inducer, allolactose, binds to the repressor subunits, preventing their assembly into an active tetramer. Allolactose is produced from lactose by β-galactosidase at a steady low rate and thus serves as a lactose signal. Another regulatory component is the catabolite activator protein (CAP). CAP forms an active complex with intracellular cyclic adenosine monophosphate (cAMP), which accumulates in the absence of glucose (cAMP is a starvation signal). RNA polymerase binds to the lac promoter effectively only when the CAP-cAMP complex is also bound. This ensures that the lac operon will be expressed only when glucose is absent.
The lac operon exhibits both negative control and positive control. Under negative control, a regulatory factor is needed to prevent expression of the lac operon, whereas under positive control, a regulatory factor is needed to permit expression of the lac operon.
Negative control (conditions: glucose only; prevent expression of lac operon). If lactose is absent and glucose is present (see Fig. 16-13A), the gene products from the lac operon are not needed. Thus a regulatory factor, the repressor protein, prevents lac operon expression. Since the repressor is produced constitutively and spontaneously assembles as its active tetrameric form, it is available to bind to the operon and prevent transcription.
Positive control (conditions: lactose only; permit expression of lac operon). If no glucose is present and lactose is present (see Fig. 16-13B), the gene products from the lac operon are needed to use the lactose for energy. Thus a regulatory factor, the CAP-cAMP complex is needed to permit expression of the operon. Because cAMP is a starvation signal indicating an absence of glucose, it is available to form the CAP-cAMP complex and permit transcription.
Positive control (conditions: lactose and glucose; do not permit expression of the lac operon even if not prevented by repressor). If both lactose and glucose are present (see Fig. 16-13C), the regulatory mechanisms act to avoid wasteful expression of the lac operon. Even though the repressor is inactivated by the presence of lactose, RNA polymerase cannot bind to the promoter, since the CAP-cAMP complex is absent owing to the presence of glucose.
The lactose operon (also known as the lac operon) is a set of genes that are specific for uptake and metabolism of lactose and is found in E. coli and other bacteria. The lac operon consists of three structural genes: lacZ, which codes for β-galactosidase, which acts to cleave lactose into galactose and glucose; lacY, which codes for lac permease, which is a transmembrane protein necessary for lactose uptake; and lacA, which codes for a transacetylase that transfers an acetyl group from coenzyme A (CoA) to the hydroxyl group of galactosides. In the 5′ end with respect to lacZ is the lacI gene, which encodes a repressor of the lac operon, which is transcribed independently from the structural genes (Figure 1).
In front of the lacZ gene is the promoter whose expression is modulated by the LacI repressor and the catabolite activator protein (CAP, also known as cAMP receptor protein (CRP)). Expression of this operon is activated only when lactose levels outside the cell are high and glucose levels are low. E. coli utilizes preferentially glucose and, as a result, will not activate the genes to metabolize lactose until there is a sufficiently high level of external lactose, that acts as an effector and a sufficiently low level of glucose.
The lac operon in the bacterium Escherichia coli functions by a repression mechanism in which an inhibitor protein (lacI) binds to regulatory sites (lacO) in the promoter and turns off transcription (Fig. 59-2). On the addition of lactose, the lacI protein undergoes a conformational change, which changes its binding affinity for the lacO sequences. The lacI protein thereby comes off the lacO sites, and transcription can occur. E. coli uses this system to tightly control the genes required for the use of lactose, and it is completely reversible.
Figure 59-2. Bacterial lac operon. The lac operon functions by a repression mechanism. (A) An inhibitor protein, lacI, binds to regulatory sites lacO in the promoter (P) and turns off transcription of the genes required for lactose metabolism. (B) On the addition of lactose, the lacI protein undergoes a conformational change, which changes its binding affinity for the lacO sequences. The lacI protein thereby comes off the lacO sites and transcription of the lac genes can occur. (A, transacetylase; Y, permease; and Z, β-galactosidase.)
The lactose operon of E. coli is the classic example of an operon and is often used when discussing prokaryotic regulation. The lac operon consists of three coding regions in tandem, lacZ, lacY, and lacA. The lacZ gene encodes β-galactosidase, which degrades lactose. The lacY gene product, lactose permease, transports lactose into the cell, and the lacA gene product, lactose acetylase, has an unknown and not usually necessary function. The lac operon is repressed by LacI, encoded by lacI. The lacI gene is upstream of lacZYA and faces in the opposite direction. The repressor, LacI, binds to the operator sequence upstream of lacZYA and prevents transcription of those genes unless the inducer molecule is present. The inducer molecule, allo-lactose, signifies the presence of lactose. Allo-lactose binds to LacI and prevents it from repressing transcription of lacZYA, thus the operon is derepressed. In addition to allo-lactose, LacI may bind other chemicals such as IPTG (see Figures 16.11 and 16.12Figure 16.11Figure 16.12).
Activator and repressor proteins take part in positive and negative transcriptional regulation.
Some regulatory proteins may function as either a repressor or an activator depending on the situation.
Activators typically bind upstream of a promoter sequence on the DNA and help RNA polymerase bind to the promoter to initiation transcription of the genes. Repressors bind to operator sequences downstream of the promoters to prevent RNA polymerase from either binding or moving forward, which prevents transcription.
Some regulatory proteins behave as both activators and repressors, depending upon the conditions. The AraC regulatory protein is involved in regulation of the utilization of arabinose, a sugar source for cells. When arabinose binds to AraC, the regulatory protein is converted from a repressor to an activator. The araBAD and araFG operons are repressed in the absence of arabinose and activated in the presence of arabinose.
Regulatory proteins are often controlled in turn by binding small signal molecules.
Small inducer molecules often bind to repressors and change the shape of the protein such that it is no longer able to bind DNA and repress transcription of its target genes. An example includes allo-lactose for the lac operon of E. coli. Additionally, some repressors are not able to bind DNA unless they have a small signal molecule present, called a co-repressor. This type of regulation is often seen for biosynthetic pathways where if the product of the pathway is present in the cell, then the pathway is repressed.
David P. Clark, .. Michelle R. McGehee, in Molecular Biology (Third Edition), 2019
Although the lactose operon genes were the first whose regulation was characterized in detail, and although they are often cited as a typical example, they are aberrant in several ways. Curiously, lactose itself is not the inducer. Lactose, which consists of glucose linked to galactose, is converted to allo-lactose, an isomer in which the same two sugars are linked differently. This transformation is carried out by β-galactosidase, which normally splits lactose, but makes a small amount of allo-lactose as a side reaction. It is allo-lactose that actually binds to the LacI protein and acts as an inducer.