Which of the following statements represent switch off condition for lac operon

Introduction

Gene expression is a very strictly regulated process and is altered in response to developmental cues, therapeutic drugs, environmental changes, or diseases. Gene regulation is critical for an organism to optimize their metabolic activity and respond to changes in a variety of extracellular and intracellular signals. The pioneering work by Francois Jacob and Jacques Monod, in 1961, depicted a classic example of how genetic mechanisms can be altered in response to changing environmental stimulants to regulate the bacterial metabolic activities. The operon model depicting this phenomenon was based on the metabolism of lactose in Escherichia coli.[1]

Genes involved in bacterial metabolic pathways are clustered together and coordinately transcribed under a common promoter. These structural genes, the promoter, and additional sequences that regulate their expression are called an operon. The clustering of genes allows coordinated regulation and expression of the genes and provides rapid adaptation to various environmental changes. An inducible operon is one whose expression increases quantitatively in response to an enhancer, an inducer, or a positive regulator. The lac operon is a classic example of an inducible operon and is induced by lactose and its structural analogs: isopropyl beta-D-1 thiogalactopyranoside(IPTG) and thiomethyl galactoside(TMG).[2][3]

Many other inducible operons have been identified since introducing the operon model that plays a key role in bacterial metabolism and survival against host defense mechanisms. Examples include the gal operon regulating the metabolism of galactose and induced in the presence of D-galactose; L-arabinose operon metabolizing arabinose and induced by it.[4][5]

Development

The operon model has been the paradigm for understanding gene regulation and has laid a foundation for the development of modern molecular biology. In 1941, experiments by Jacques Monod showed that bacterial colonies of Escherichia grown in the presence of glucose and lactose metabolized glucose until it is completely exhausted. The metabolism of lactose only began after a lag period, which resulted in a diauxic bacterial growth curve. It shows two distinct exponential growth phases separated by a brief period of lag phase, signifying the sequential utilization of different carbon sources. The first phase corresponds to the utilization of glucose, followed by the second phase for lactose. The lag phase arises due to the time taken by bacterial cells to synthesize enzymes required for lactose metabolism. Based on this behavior, Karstrom coined the term “enzyme adaptation” for the enzymes produced only in the presence of their particular substrate. This was later redefined and termed “enzyme induction.”[6][7]

These observations were followed by Monod’s experiments to study the kinetics of enzyme induction in Escherichia coli. He observed that the addition of a small metabolite to bacterial colonies increased the production of beta-galactosidase (an enzyme, then called lactase). The metabolite was recognized as allolactose, a natural inducer of beta-galactosidase, produced from a side reaction catalyzed by beta-galactosidase. Many other inducers were synthesized that could mimic the activity of allolactose and induce the synthesis of beta-galactosidase. One such effective molecule is isopropyl beta-d-1 thiogalactopyranoside (IPTG). Although capable of inducing the enzyme, it cannot be catabolized by beta-galactosidase, unlike allolactose, which can be hydrolyzed to glucose and galactose. Therefore, it is called a gratuitous inducer. Many mutant bacterial strains were produced that affected the synthesis of the enzyme, depending on the deleted genes. While some strains could produce the enzyme constitutively, others lost this ability even in the presence of an inducer.[8]

Andre Lwoff and Francois Jacob observed a similar induction phenomenon while exploring the development of a lambda bacteriophage. The bacteriophage could switch from a lysogenic phage to a lytic phage in response to noxious environmental stimulus. This raised the same fundamental question of how cellular events and genetic elements are switched on and off. The PaJaMa experiments in 1959 demonstrated that these inducers work by relieving the genetic synthetic machinery from a reversible inhibition. These observations led to the development of the operon model for which Francois Jacob, Jacques Monod, and Andre Michel Lwoff received the Nobel Prize in Physiology or Medicine in 1965.[8]

Cellular

The lac operon spans over 5300 base pairs and contains three structural genes: LacZ, LacY, and LacA. Their transcription is coordinately regulated by a single lac promoter, located between base pairs: -36 and -7, from the transcription start site. During each transcription cycle, the DNA-dependent RNA-polymerase binds to the promoter and initiates transcription. This produces a single polycistronic mRNA containing multiple independent Stop and Start codons such that each of the three proteins is translated separately and not cleaved from a single polypeptide precursor.[1][6][9]

Structural genes of the lac operon encode proteins that coordinately metabolize lactose. LacZ gene encodes for a tetrameric enzyme, beta-galactosidase. It contains four identical polypeptide subunits and plays a central role in the breakdown of lactose. It catalyzes the hydrolysis of lactose and allolactose to glucose and galactose and the transgalactosylation of lactose to its structural analog allolactose.[10]

LacY gene encodes for lactose permease (LacY), a transmembrane protein belonging to the oligosaccharide/H+ symporter subfamily. It is a secondary active transporter and drives the reversible transport of galactosides (such as lactose) with a proton molecule into the cell.[11]

LacA gene encodes for thiogalactoside transacetylase, also called galactoside acetyltransferase. It transfers an acetyl group from coenzyme A to the 6-O-methyl position of pyranose rings in beta-galactosides. Although the roles of the former two enzymes are well known, the biological function of galactoside acetyltransferase remains uncertain. Acetylation of these galactosides causes their efflux and reduces their re-entry into the cells. This prevents the toxic accumulation of these compounds and reduces cell stress by decreasing the intracellular concentration of the inducers.[3][12]

The lac operon also contains regulatory genes that switch on or off the operon depending on the nutritional environment of the bacterial cells. A primary operator locus(O1) located upstream of the structural genes plays a key role in regulating their transcription. Centered around +11 base pairs from the transcription start site, it binds to a repressor protein and negatively regulates the lac operon. In addition to the primary operator, two additional auxiliary operators, formerly called the “pseudo-operators,” have been recognized. O2 is located 401 base pairs downstream from O1, while O3 is positioned 82 base pairs upstream from the primary operator. They differ from O1 in a few nucleotide sequences and their binding affinity for lac repressor.[13]

The repressor protein is encoded by a regulatory gene, LacI, located on a different operon having its independent promoter. It encodes constitutively for a homo-tetrameric polypeptide: lac repressor. A protein made of 360 amino acids has a molecular mass of 154520 Dalton. It contains a separate DNA binding site, with which it associates with the operator, and an inducer binding site, with which it binds to lactose and its structural analogs. Each tetrameric unit of the protein is arranged in two functional dimers, allowing the repressor to simultaneously bind to two different operators. The lac repressor acts as a master switch for the lac operon and optimizes the synthesis of enzymes from the structural genes.[14]

Immediately upstream to the lac promoter and centered between base pairs -72 and -50 is the catabolite activator protein (CAP) binding site. The transcription factor, CAP, contains two functional dimers that interact with the alpha subunit C-terminal domain of RNA polymerase, increasing its recruitment and binding with the lac promoter.[6][15]

Molecular

The lac operon ensures the sequential utilization of different carbon sources depending on the bacterial cells' nutritional environment. Glucose is the preferred source of energy, following which lactose and other carbohydrates are metabolized. This occurs due to the positive and negative regulation of the lac operon.

In the absence of lactose, the genetic switch, lac repressor binds to the primary operator O1 and physically blocks RNA polymerase from forming the pre-initiation complex, inhibiting the synthesis of enzymes required for lactose metabolism. This repression is further enhanced by the two auxiliary operators, O2 and O3. The bidentate repressor binds to two operator sites simultaneously and leads to the formation of stable DNA loops. DNA looping enhances the effective concentration of the repressor at the primary regulatory operator, further inhibiting the lac operon. This cooperative binding between O1 and O2 or O1 and O3 can increase the repression intensity by as high as 1300 times. Inhibiting the synthesis of enzymes required for lactose metabolism allows the cell to conserve its metabolic energy and utilize the available carbon sources.[13][16][17]

However, in the presence of lactose and the absence of its preferred carbohydrate source, the cells switch their genetic machinery to metabolize lactose as its primary source of energy. Due to the inherent leakiness of the lac operon, basal levels of lac permease and beta-galactosidase are produced even in the absence of lactose. This allows lactose to enter the cell via lac permease and get isomerized to allolactose by beta-galactosidase. Allolactose binds to the lac repressor and produces a conformational change, decreasing its affinity for the operator locus. Thus, the repressor dissociates from the operator, allowing the synthesis of metabolic enzymes. This provides evidence that the lac repressor can exhibit two conformational states, one where it binds to the operator and inhibits the lac operon and the other when it allows induction of the operon by binding to an inducer. The phenomenon by which the lac repressor can exhibit two structural states with different biological properties is also called allostery.[18]

Extracellular glucose can also regulate the lac operon by carbon catabolite repression (CCR), a phenomenon formerly called the "glucose effect." The phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) couples the transport and phosphorylation of glucose as it moves into the cell and plays a central role in exerting the effects of glucose on the expression of the lac operon. The transfer of a phosphate group from phosphoenolpyruvate to glucose occurs in two-steps, each catalyzed by different proteins. The phosphoryl group is first accepted by enzyme I and HPr, which transfers it to a sugar specific Enzyme II. Enzyme II specific for the transport of glucose is EIIAGlc. As glucose levels increase in the environment, it is transported into the cells and phosphorylated by EIIAGlc. Dephosphorylated EIIAGlc binds to LacY and prevents the formation of lac permease. This blocks the entry of lactose into the cell, decreasing the intracellular concentration of the inducer, a phenomenon called inducer exclusion. This ensures that the cells use glucose, and other carbons sources such as lactose, are not metabolized.[19][20]

In the absence of glucose, phosphorylated EIIAGlc activates the enzyme adenylyl cyclase, which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). cAMP binds to and activates the cAMP receptor protein(CRP), also called the Catabolite Activator Protein (CAP). CAP interacts with the CAP binding site, increases the recruitment of RNA polymerase, and enhances its interaction with the lac promoter, positively regulating the transcription mechanism. Thus, the synthesis of enzymes required for lactose metabolism is increased in the absence of glucose.[15][21]

Another mechanism regulating the synthesis of enzymes is the presence of a positive feedback loop. Increased production of lac permease and beta-galactosidase increases the uptake of lactose and its conversion into allolactose, which further reduces the concentration of active repressor and induces the operon. As a consequence, more lac permease and beta-galactosidase are produced, and the cycle continues.[6]

Function

The clustering of genes involved in a metabolic pathway and their co-regulation under a single promoter helps in rapid adaptation to fluctuating levels of food supplies in the environment. The lac operon enables bacterial cells to take full advantage of the available carbon sources by assimilating and utilizing rapidly metabolizable substrates (such as glucose) and switching to other food supplies only after its supply is exhausted. Regulation at the level of transcription prevents metabolic energy from getting lost in the process of transcription and translation. The formation of a single polycistronic mRNA decreases gene expression noise and ensures better stoichiometry.[1][22][23]

Many other inducible operons have been identified that function to protect the bacterial cells from host defense mechanisms. Inducible operons also play an important role in rendering anti-microbial resistance to pathogenic organisms. The mef(E)/mel operon carried on the Macrolide Efflux Genetic Assembly(MEGA) confers resistance to macrolides in Streptococcus pneumoniae. This inducible operon encodes for a macrolide efflux pump and a ribosomal protection protein preventing the entry of macrolides and their action on ribosomes. The expression of anti-microbial resistance proteins is controlled at the level of transcription and induced in the presence of 14-and 15-membered macrolides, tilmicosin and rosamicin, and tetracyclines.[24][25] 

The marRAB operon in Escherichia coli belongs to the multidrug resistance (mdr) system and confers resistance to a wide range of functionally and structurally diverse compounds by decreasing the synthesis of porins and activating efflux pumps. It is induced in the presence of tetracycline, chloramphenicol, and salicylates.[26]

Mechanism

The mechanisms by which transcription regulation is determined can be positive or negative. If the regulatory protein is an activator, we can say that it is a positive regulation; if the protein is a repressor, the regulation will be negative. We can distinguish inducible operons with negative and positive regulation; repressible operons with positive and negative regulation.

Clinical Significance

The identification of inducible systems, playing an important role in mediating anti-microbial resistance, opens doors for the development of newer anti-microbial therapy.  Understanding the molecular basis of sensing and responding to antibiotics in these operons, including the molecular process of induction, can help develop potential antimicrobials that target these induction mechanisms and have non-inducing properties.[25]

In recent years, the concept of an inducible system has been extended to mammalian cells to develop tools that aid in studying gene expression and their regulation. Allolactose and IPTG have been exploited to induce the expression of target genes to study their function and roles in various disease processes. Gene regulation using these inducible systems can aid in developing cancer therapy and the treatment of various genetic disorders.[27]

Man's manipulation of operons is a major step in the process of enabling bacterial cells to produce medically important proteins such as human insulin.

Review Questions

Figure

Figure 1: Lac operon and its regulatory elements Figure 2-4: The expression of lac operon under different nutritional conditions. Contributed by Tanisha Sanganeria

References

Osbourn AE, Field B. Operons. Cell Mol Life Sci. 2009 Dec;66(23):3755-75. [PMC free article: PMC2776167] [PubMed: 19662496]

Sáenz-Lahoya S, Bitarte N, García B, Burgui S, Vergara-Irigaray M, Valle J, Solano C, Toledo-Arana A, Lasa I. Noncontiguous operon is a genetic organization for coordinating bacterial gene expression. Proc Natl Acad Sci U S A. 2019 Jan 29;116(5):1733-1738. [PMC free article: PMC6358700] [PubMed: 30635413]

Marbach A, Bettenbrock K. lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. J Biotechnol. 2012 Jan;157(1):82-8. [PubMed: 22079752]

Schleif R. Regulation of the L-arabinose operon of Escherichia coli. Trends Genet. 2000 Dec;16(12):559-65. [PubMed: 11102706]

Lewis DE, Adhya S. Molecular Mechanisms of Transcription Initiation at gal Promoters and their Multi-Level Regulation by GalR, CRP and DNA Loop. Biomolecules. 2015 Oct 16;5(4):2782-807. [PMC free article: PMC4693257] [PubMed: 26501343]

Santillán M, Mackey MC. Quantitative approaches to the study of bistability in the lac operon of Escherichia coli. J R Soc Interface. 2008 Aug 06;5 Suppl 1:S29-39. [PMC free article: PMC2504340] [PubMed: 18426771]

JACOB F, MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. [PubMed: 13718526]

Lewis M. A tale of two repressors. J Mol Biol. 2011 May 27;409(1):14-27. [PMC free article: PMC3104267] [PubMed: 21392509]

Hediger MA, Johnson DF, Nierlich DP, Zabin I. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6414-8. [PMC free article: PMC390726] [PubMed: 3901000]

Juers DH, Matthews BW, Huber RE. LacZ β-galactosidase: structure and function of an enzyme of historical and molecular biological importance. Protein Sci. 2012 Dec;21(12):1792-807. [PMC free article: PMC3575911] [PubMed: 23011886]

Kaback HR, Guan L. It takes two to tango: The dance of the permease. J Gen Physiol. 2019 Jul 01;151(7):878-886. [PMC free article: PMC6605686] [PubMed: 31147449]

Roderick SL. The lac operon galactoside acetyltransferase. C R Biol. 2005 Jun;328(6):568-75. [PubMed: 15950163]

Oehler S, Eismann ER, Krämer H, Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990 Apr;9(4):973-9. [PMC free article: PMC551766] [PubMed: 2182324]

Lewis M. The lac repressor. C R Biol. 2005 Jun;328(6):521-48. [PubMed: 15950160]

Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol. 2004 Feb;14(1):10-20. [PMC free article: PMC2765107] [PubMed: 15102444]

Deuschle U, Gentz R, Bujard H. lac Repressor blocks transcribing RNA polymerase and terminates transcription. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4134-7. [PMC free article: PMC323685] [PubMed: 3520567]

Becker NA, Peters JP, Maher LJ, Lionberger TA. Mechanism of promoter repression by Lac repressor-DNA loops. Nucleic Acids Res. 2013 Jan 07;41(1):156-66. [PMC free article: PMC3592455] [PubMed: 23143103]

Lewis M. Allostery and the lac Operon. J Mol Biol. 2013 Jul 10;425(13):2309-16. [PubMed: 23500493]

Hogema BM, Arents JC, Bader R, Eijkemans K, Yoshida H, Takahashi H, Aiba H, Postma PW. Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc. Mol Microbiol. 1998 Nov;30(3):487-98. [PubMed: 9822815]

Vinuselvi P, Kim MK, Lee SK, Ghim CM. Rewiring carbon catabolite repression for microbial cell factory. BMB Rep. 2012 Feb;45(2):59-70. [PubMed: 22360882]

Ammar EM, Wang X, Rao CV. Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose. Sci Rep. 2018 Jan 12;8(1):609. [PMC free article: PMC5766520] [PubMed: 29330542]

Deutscher J. The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol. 2008 Apr;11(2):87-93. [PubMed: 18359269]

Stoebel DM, Dean AM, Dykhuizen DE. The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products. Genetics. 2008 Mar;178(3):1653-60. [PMC free article: PMC2278061] [PubMed: 18245823]

Schroeder MR, Lohsen S, Chancey ST, Stephens DS. High-Level Macrolide Resistance Due to the Mega Element [mef(E)/mel] in Streptococcus pneumoniae. Front Microbiol. 2019;10:868. [PMC free article: PMC6491947] [PubMed: 31105666]

Chancey ST, Zähner D, Stephens DS. Acquired inducible antimicrobial resistance in Gram-positive bacteria. Future Microbiol. 2012 Aug;7(8):959-78. [PMC free article: PMC3464494] [PubMed: 22913355]

Seoane AS, Levy SB. Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli. J Bacteriol. 1995 Jun;177(12):3414-9. [PMC free article: PMC177043] [PubMed: 7768850]

Myung SH, Park J, Han JH, Kim TH. Development of the Mammalian Expression Vector System that can be Induced by IPTG and/or Lactose. J Microbiol Biotechnol. 2020 Aug 28;30(8):1124-1131. [PMC free article: PMC9728253] [PubMed: 32423185]

In which condition lac operon is switch off?

Which of the following statement represents which of condition of lac operon?

Which of the following is responsible for switching on and off of the lac operon?

How does switch off mechanism occur in lac operon?