ZOOLOGY Molecular Cell Biology Regulation of Gene Expression in Prokaryotes: Lac Operon

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Paper : 15 Molecular Cell Biology Module : 23 Regulation of gene expression in Prokaryotes: Lac Operon

Development Team

Principal Investigator : Prof. Neeta Sehgal Department of Zoology, University of Delhi

Co-Principal Investigator : Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator : Prof. Kuldeep K. Sharma Department of Zoology, University of Jammu

Content Writer : Dr. SudhirVerma Deen Dayal Upadhyaya College, University of Delhi

Content Reviewer : Prof. Rup Lal Department of Zoology, University of Delhi

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

Description of Module

Subject Name ZOOLOGY

Paper Name Zool 015: Molecular Cell Biology

Module Name/Title Regulation of gene expression in Prokaryotes

Module ID M23: Lac Operon

Keywords Operon, Inducible Operon, Repressible Operon, Catabolite repression, β-galactosidase, Permease, Transacetylase, Operator, Promoter, Regulator, Structural genes

Contents

1. Learning Outcomes 2. Introduction 3. Regulation of Gene expression in Prokaryotes 4. Types of Gene Expression: Constitutive, Inducible and Repressible 5. The Positive or Negative Control of Gene Expression 6. Concept of Operon 7. The Lac Operon of E.coli 8. Regulation of Lac Operon 9. Substitutes of allolactose, used for Lac Operon 10. Lac Mutations 10.1. Mutations in structural genes 10.2. Mutations in regulator gene 10.3. Mutations in operator 10.4. Mutations in promoter 11. Positive Control: Catabolite Repression 12. Summary

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

1. Learning Outcomes

After studying this module, you shall be able to

 Know: What is an Operon system in prokaryotes? What are the various types of Operon that exist in Bacteria? How lactose metabolism take place in bacteria?

 Learn: About various mechanisms of gene expression regulation in prokaryotes and how an Operon system contributes at transcriptional level gene regulation.

 Identify: The components that constitute an Operon and regulate the prokaryotic gene expression

2. Introduction

The genetic composition of all the cells within an organism is same yet they do differ from each other in terms of structure and function due to differential expression of genes which leads to difference in their protein composition. How this differential expression (which might be temporal or spatial) is achieved? The answer lies in the ‘mechanisms of transcriptional regulation’, also called as gene regulation. We will understand the gene regulation in prokaryotes with an example of lactose metabolizing enzymes. But before that, we shall briefly learn about the gene expression regulatory checkpoints in prokaryotes, the Operon concept and general regulatory principles of transcription in bacteria.

3. Regulation of Gene expression in Prokaryotes

The gene expression can be broadly regulated at five different levels in prokaryotes:

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

Fig. 1: Levels of gene regulation in prokaryotes. Source: https://en.m.wikipedia.org/wiki/Regulation_of_gene_expression

For the current module, we will restrict our discussion to transcriptional regulation only.

4. Types of Gene Expression: Constitutive, Inducible and Repressible

Types of Gene Expression:

Gene expression can be:

 Constitutive Gene Expression  Inducible Gene Expression  Repressible Gene Expression

Constitutive Gene Expression refers to the genes which are constitutively expressed in most cells e.g. genes which express housekeeping function proteins like ribosomal proteins, tRNA, rRNA etc. There are other genes whose products are required for cell growth only under certain environmental conditions only. Thus, constitutive expression of such genes is a waste of energy. The expression of such genes is switched ON only when their product is needed. This process of turning on the expression of genes in response to a substance in the environment is called the Inducible gene expression and these genes are

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

called as inducible genes. The catabolic pathway enzymes such as in lactose, galactose etc. are example of inducible expression. In contrast, the continued synthesis of some biosynthetic enzyme is wastage if that molecule is present in the environment where bacteria are growing. Thus, in case of externally available nutrient molecule, the enzymes required for biosynthesis of that nutrient needs to be turned off. Such an expression is called as repressible gene expression. 5. The Positive or Negative Control of Gene Expression

If the product of regulator gene is involved in turning on the expression of one or more structural genes, the control mechanism is termed as Positive Control Mechanism. And, if the product of regulator gene is involved in turning off the expression of structural genes, the control mechanism is termed as Negative Control Mechanism.

Fig. 2: Different types of gene expression control mechanisms i.e. negative inducible, negative repressible, positive inducible and positive repressible. (Source: genes.atspace.org/10.8.html)

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

6. Concept of Operon

The significant difference between bacterial and eukaryotic gene control lies in the organization of functionally related genes. The bacterial genes which have related functions are clustered and under the control of a single promoter. These genes are transcribed together in a single mRNA. Thus, in prokaryotes, the genes with related functions are coordinately regulated genetic units called the ‘Operons’. The Operon model was developed by Francois Jacob and Jacques Monod in 1961 (For which they were awarded Nobel Prize in physiology and medicine in year 1965) to explain the regulation of genes required for utilization of lactose in E.coli. The organization of a typical Operon is as follows:

Fig. 3: Diagrammatic representation of a typical Operon Source: http://courses.lumenlearning.com/microbiology/chapter/gene-regulation-Operon;theory/

Source: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1965/

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

At one end of Operon is present a set of structural genes e.g. gene A, B and C. These genes are coordinately expressed in a single mRNA which is further translated to form protein A, B and C respectively. Upstream of these structural genes is present a promoter which controls the transcription of all these structural genes. The RNA polymerase binds to this promoter and then goes downstream to transcribe the structural genes. A regulator gene also helps in controlling the transcription of structural genes. The regulator gene has its own promoter and not actually a part of Operon. The regulatory protein can bind to a region on Operon, called as Operator. The operator usually overlaps the 3’ end of promoter and 5’ end of first structural gene. Some Operons may have multiple operators as well.

The Operons can be inducible Operon (in which transcription is normally OFF and something must happen to switch it ON) and repressible Operon (in which transcription is normally ON and something must happen to turn it OFF).

Fig. 4: Some common Operons in prokaryotes Source: http://www.biologydiscussion.com/wp-content/uploads/2014/09/clip_image00832.jpg

7. The Lac Operon of E. coli

Francois Jacob and Jacques Monod explained the Operon model for genetic control of lactose metabolism in E. coli in in 1961. Their work and the subsequent research established ‘Operon’ as basic unit of transcriptional regulation in bacteria. Though no sequencing methods were available to determine the nucleotide sequence at that time, but Jacob and Monod deduced the Operon structure genetically by analyzing the interactions in mutations that interfered the lactose metabolism regulation. We’ll discuss these

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

mutations in subsequent sections but before that let’s first understand how lactose metabolism is regulated by lac Operon in bacteria.

Lactose metabolism in E. coli: The E. coli bacteria present in our gut helps in metabolizing the sugar Lactose which is the major carbohydrate found in milk. The lactose is not internalized passively by E. coli but actively, by a protein called permease. Once lactose is transported inside the bacterial cell, it is broken up into its constituent monosaccharide i.e. glucose and galactose by enzyme, β-galactosidase. The same enzyme can also convert the lactose into allolactose also which is involved in regulation of lactose metabolism. Once converted into monosaccharides, it can be used as energy source by bacteria. Yet another enzymes which is produced by lac Operon is thiogalactoside transacetylase whose function is not yet clearly understood.

Fig. 5: Catalytic action of β-galactosidase cleaving lactose into glucose and galactose. (Source: Author)

Fig. 6: Catalytic action of lac Operon genes in transport and metabolism of lactose in bacteria. Source: Modified from Lehninger Principles of Biochemistry, 4th Ed. (2006) 8

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

8. Regulation of Lac Operon

Lac Operon is an example of negative inducible Operon. In a negative inducible Operon, an active repressor is encoded by a regulator gene which readily binds to the operator. Binding of repressor protein to operator blocks the binding of RNA polymerase to promoter as operator site overlaps with promoter. Thus, no transcription takes place. For transcription to occur promoter should be free or indirectly, the repressor should not bind to operator site. The transcription in such a case is normally inhibited (or in OFF state) and needs to be induced (or turned ON). In order to switch on the transcription, an inducer binds to repressor. The repressor proteins are usually allosteric in nature. They usually have two binding sites: one for DNA and another for some other molecule such as inducer. Thus, when inducer binds to repressor, it alters its shape which leads to dissociation of repressor from DNA. Simple, presence of inducer switches ON the transcription leading to expression of structural genes whereas absence of inducer keeps the transcription OFF because of repressor leading to no expression of structural genes.

Structurally, lac Operon consists of a common promoter (lacP) and three adjacently located structural genes: lacZ, lacY and lacAwhich encode for β-galactosidase, permease and transacetylase respectively. Between the operator and promoter lies the operator (lacO). Upstream of promoter is present the regulator gene lacI, which has its own

promoter (PI). lacI encodes for a repressor which has two binding sites: one for DNA and another for allolactose. Allolactose acts as an inducer for lac Operon and is produced by action of β-galactosidase on lactose.

Fig. 7: Structural arrangement of lac Operon in E.coli Source: http://1.bp.blogspot.com/_NHHTvTVxCmc/SwtVnJU_dPI/AAAAAAAAAfc/1qzbX8spyDw/s1600/lac- Operon.gif

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

Fig. 8: Size of gene, gene products and their molecular weights involved in lac Operon Source: https://www.google.co.in/search?q=lac+Operon&espv=2&biw=1024&bih=623&source=lnms&tbm=isch&sa=X &ved=0ahUKEwj96aC51PTKAhWPG44KHewuBngQ_AUIBygC#tbm=isch&q=lac+Operon+creative+commo ns&imgrc=xi9tdINNkOq5_M%3A

Table 1: Function of structural genes of lactose Operon (Source: Author)

Structural genes of the Lac Operon Gene Protein (Enzyme) Coded Function lacZ β-galactosidase Catabolism of lactose to glucose and galactose lacy Permease Transport of lactose in the cell lacA Transacetylase Largely unknown but probably it rids the cell of toxic thiogalactosides.

When lactose is absent; no allolactose is formed and hence no inducer is present. The repressor binds to operator and blocks the promoter site. The RNA polymerase is unable to bind to promoter and thus, no transcription takes place causing no expression of structural genes required for lactose metabolism. In fact, it makes the physiological sense. If no lactose is present in the growth medium, there is no requirement to form the enzymes/ proteins to catabolise it and energy is saved.

When lactose is present; initially some of the lactose is converted to allolactose which then binds to repressor and causes a structural change in it leading to its release from DNA. Now, the promoter is free and RNA polymerase can bind to it and transcription of lac Operon genes takes place. It also makes physiological sense to synthesize the lactose metabolizing enzymes only when lactose is present in the growth medium to metabolize.

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

In what we have discussed above, you must have realized that:

1. Permease is required to transport lactose into the cell. But, if lac Operon is repressed how lactose does get into the cell to inactive the repression and turn on transcription?

Secondly,

2. The inducer for lac Operon is allolactose, which is formed from lactose. But, if β- galactosidase production is repressed, how can lactose metabolism be induced?

The answer for these questions is that repression doesn’t completely shuts down the transcription of lac Operon. There is always a low level of transcription, even if the repressor is actively bound to operator. Thus, a few molecules of β-galactosidase, transacetylase and permease are always present in the cell. When lactose appears in the medium, permease transports it inside the cell and β-galactosidaseconverts it into allolactose which then induces the transcription.

The lac Operon is ‘Leaky”.

9. Substitutes of allolactose, used for Lac Operon

Besides allolactose, many similar compounds are used as inducer for lac Operon. Isopropylthiogalactoside (IPTG) is one such molecule which is extensively used in research to observe the effects of induction, independent of metabolism as it’s not metabolized by β-galactosidase.

Fig. 9: Structures of ‘inducers’ of Lac Operon (Source: Author)

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

10. Lac Mutations

The structure and function of lac Operon was worked out by Jacob and Monod by analyzing the mutations that affected lactose metabolism. They used partial diploid strains of E. coli. These strains possess two different DNA molecules: one complete bacterial chromosome and an extra piece of DNA. Jacob and Monod created such strains by conjugation process. As we know, in conjugation, small circular piece of DNA i.e. F plasmid is transferred from one bacterium to another. Jacob and Monod used the F plasmid of lac Operon. So, the recipient bacterium became partially diploid as it possessed two copies of lac Operon. They used different combination of mutations on F plasmid and bacterial DNA and deduced that some of the lac Operon components are cis-acting (controlling the gene expression only when present on same chromosome) and others are trans-acting (controlling the gene expression on other DNA molecules). The experiments in which genes or gene clusters are tested pairwise are called as complementation tests.

Let us discuss the mutations in all the components of lac Operon i.e. in structural gene, regulator gene, operator and promoter.

10.1. Mutations in structural genes

Using partial diploids, Jacob and Monod established that mutations at lacZ and lacY genes were independent and usually affected the product of the gene in which they occurred. The partial diploid with lacZ+lacY- on bacterial chromosome and lacZ-lacY+ on plasmid functioned normally, producing permease and β-galactosidase in presence of lactose. The single functional β-galactosidase gene (lacZ+) in this partial diploid is sufficient to form β- galactosidase irrespective of its coupling with defective (lacY-) or functional (lacY+) permease. The same is applicable for lacY+ gene.

10.2. Mutations in regulator gene

Mutations in lacI gene affect both β-galactosidase and permease as both the genes are in same Operon and regulated in co-ordinate manner. The mutations which were constitutive in nature i.e. causing lac proteins to be produced all the times irrespective of whether lactose was present or not were designated as lacI-. The lacI+ gene is dominant over lacI- gene; means a single copy of lacI+ gene is sufficient to bring normal regulation. Further, 12

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

lacI+ is trans-acting in nature; means it can regulate the Operon normally even if Operon is located on some other DNA molecule.

Some lacI mutations did not allow transcription to take place even in the presence of lactose and other inducers e.g. IPTG. Such mutations are referred as super repressor mutations (lacIs). The repressor produced by such mutations is defective in a way that inducer could not inactivate it. The lacIs mutations are dominant over lacI+. The partial diploids with genotype lacIslacZ+/ lacI+lacZ+ were unable to synthesize permease and β- galactosidase irrespective of presence or absence of lactose.

10.3. Mutations in operator

The mutations in operator region are referred as lacOc (denoting constitutive mutations on operator region). These mutations alter the operator DNA sequence such that repressor is unable to bind it. A partial diploid with genotype lacI+lacOclacZ+/ lacI+lacO+lacZ+ showed constitutive expression of β-galactosidase indicating that lacOc is dominant over lacO+. Additionally, lacO is cis-acting in nature; means it affects the genes present on same DNA molecule only. The partial diploid with genotype lacI+lacO+lacZ-/ lacI+lacOclacZ+ was constitutive in β-galactosidase production with or without lactose. But, a partial diploid genotype lacI+ lacO+lacZ+/ lacI+lacOclacZ- produced β-galactosidase only in the presence of lactose. In lacI+lacO+lacZ-/ lacI+lacOclacZ+ genotype, both lacOc and functional lacZ+ are present on same DNA but in lacI+ lacO+lacZ+/ lacI+lacOclacZ-, the lacOcand functional lacZ+ are present on different DNA. The lacOmutations regulate genes present on same DNA only by preventing binding of repressor to operator sequence but they cannot prevent a repressor from binding to a normal operator sequence present on some other DNA. Hence they are cis-acting.

10.4. Mutations in promoter

Mutations at promoter region are denoted as lacP-. These mutations interfere with binding of RNA polymerase to promoter region hence no transcription. Thus, lacP- mutation leads to no transcription of structural genes irrespective of whether lactose is present or not. Like operator mutations (lacOc), promoter mutations (lacP-) are also cis-acting.

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

Fig. 10: Lac regulatory mutants (lac A is omitted for simplicity). (a)- (d) show haploid states i.e. cell carries a single copy of lac genes. (a) shows repression, (b) shows IPTG mediated induction, (c) and (d) show effect of mutation to lacI gene or operator respectively.(e) shows complementation test for repressor is shown. (f) and (g) show complementation test for operator mutation which is cis-dominant. Source:https://en.m.wikipedia.org/wiki/Lac_Operon

11. Positive Control: Catabolite Repression

Glucose is always the preferred source of energy over other sugars (such as, lactose, arabinose, maltose etc.) and thus, is always the first one to be metabolized in presence of other sugars. If E. coli is grown in a medium which has both glucose and lactose, it always prefers to consume glucose first and then the lactose. Hence in such a growth medium, E. coli shows diauxic growth as depicted in biphasic growth curve below. During the first phase (I) of exponential growth the bacteria will utilize glucose as source of energy until all the glucose is exhausted. Then it will utilize lactose and so, after a secondary lag phase it will show a second phase (II) of exponential growth.

Fig. 11: Biphasic growth curve of E.coli, grown in presence of glucose and lactose Source: http://en.wikipedia.org/wiki/Lac_operon 14

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

The lac promoter contains two binding sites: one for RNA polymerase and another one for protein, called as ‘catabolite activator protein (CAP)’. The binding of CAP to promoter prevents the Operon from being induced in presence of glucose. Simply, this mechanism ensures the preferential utilization of glucose as energy source if its available in growth medium, which makes the physiological sense to avoid waste of energy in making lactose catabolising enzymes when ready source of energy i.e. glucose is available. This phenomenon is called as ‘Catabolite Repression”.

If the glucose is present in growth medium, it prevents the induction of lac Operon. Glucose, which is a catabolite of lactose metabolism is a repressor of lac Operon, hence this phenomenon is termed as “Catabolite Repression’ (also called as glucose effect or positive regulation). The regulatory protein CAP and cAMP molecules mediate this type of regulation. CAP is also called as CRP i.e. cAMP receptor protein because CAP binds to cAMP when cAMP is present in sufficient concentrations. The Cap/cAMP complex must be present at its binding site on lac promoter in order to lac Operon being induced normally. Since it exerts a positive control on lac Operon, it is called as positive regulator and phenomenon is called as positive control of lac Operon induction. The CAP/ cAMP complex bends the DNA by 90° when it is bound to it. Since CAP/ cAMP complex binding site and RNA polymerase binding sites are located adjacently, this bending makes the RNA polymerase binding site more accessible and thus positively regulate the induction of lac Operon.

Fig. 12: Structure of CAP protein and its binding sequence. (A) Two identical CAP dimer subunits bind to the activator binding sequence and induce a bend in the DNA (by ~80°). A molecule of cAMP (coactivator) is

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

bound to each monomer. (B) Symmetric half sites of the CAP binding sequence possessing dyad axis. CAP binding site is centered at position −61.5. Source:http://en.wikipedia.org/wiki/Cyclic_nucleotide- binding_domainhttp://biowiki.ucdavis.edu/Genetics/Unit_IV%3A_Regulation_of_Gene_Expression/Chapter_1 5._Positive_and_negative_control_of_gene_expression

It is the CAP/ cAMP complex that binds to lac promoter and not the CAP alone which means cAMP is major effector molecule. The intracellular concentration of cAMP is affected by presence or absence of glucose. Low concentration of glucose causes higher concentration of cAMP and vice-versa showing an inverse relationship. The glucose prevents the activation of adenylatecycalse, the enzyme which catalyzes the formation of cAMP from ATP. The connection between glucose and cAMP involves the mechanisms by which sugars such as glucose are transported into the bacterial cell and fact that adenylatecyclise is a membrane bound enzyme.

Many sugars are transported in E. coli by phosphoenolpyruvate-dependent phosphotransferase systems (PTS). They are phosphorylated as soon as they are transported into the cells. The phosphate group is transferred from phosphoenol pyruvate (PEP) through a series of intermediary proteins. The pathway is summarized below:

(A)

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

(B)

Fig. 13: A) The link between adenylatecyclase activation and glucose transport. B) The phosphoenolpyruvate-dependent phosphotransferase system (PTS) Source: Author

The connection between sugar transport and cAMP involves the interaction of EIIA~P and adenylatecyclase. EIIA~P activates adenylatecyclase. Thus,

If Glucose is scarce, the EIIA~P will be plentiful, adenylatecyclase will be activated and cAMP will increase and vice-versa.

TABLE 2: Signals that Induce or Repress Transcription of the Lac Operon Source: Author Glucose CAP binds Lactose Repressor binds Transcription + - - + No + - + - Some - + - + No - + + - Yes

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

Also, when glucose is absent, the EIIA will be less and thus, lactose permease transport will not be inhibited by EIIA.

12. Summary

 Though the genetic composition of all the cells within an organism is same, the difference in cells in terms of structure and function exists. This is because of differential expression of genes which leads to difference in their protein composition.

 This differential expression (which might be temporal or spatial) is achieved by ‘mechanisms of transcriptional regulation’, also called as gene regulation.

 The gene expression can be broadly regulated at five different levels in prokaryotes i.e. at transcription level, RNA processing level, RNA transport level, Translational level and Post-translational modification level.

 Gene regulation at transcription level in prokaryotes can be constitutive, inducible or repressible.

 It can be positive regulation as well as negative regulation.

 The significant difference between bacterial and eukaryotic gene control lies in the organization of functionally related genes. The bacterial genes which have related functions are clustered and under the control of a single promoter. These genes are transcribed together in a single mRNA. Thus, in prokaryotes, the genes with related functions are co-ordinately regulated genetic units called the ‘Operons’.

 The Operon model was developed by Francois Jacob and Jacques Monod in 1961 to explain the regulation of genes required for utilization of lactose in E.coli.

 An Operon consists of some structural genes, operator, promoter and regulator genes.

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon

 In lactose Operon, the structural genes are β-galactosidase, permease and transacetylase. These genes synthesize the lactose metabolizing enzymes, if lactose is present in the growth medium.

 The lac Operon is tightly regulated in a way that these genes are transcribed only when lactose is present otherwise transcription remains largely OFF. However, some transcription is also on, making lac Operon ‘leaky’.

 Lac Operon is a negative inducible Operon.

 Besides allolactose, IPTG also acts as substitute for lac induction.

 The structure and function of lac Operon was worked out by Jacob and Monod by analyzing the mutations that affected lactose metabolism. They used partial diploid strains of E.coli. The experiments in which genes or gene clusters are tested pairwise are called as complementation tests.

 E.coli shows diauxic growth if grown in medium consisting both glucose and lactose as glucose is preferential source of energy

 Glucose acts as catabolite repressor of lac Operon.

 Lac Operon is positively regulated by CAP-cAMP complex.

 There is a connection between glucose transport and cAMP formation as both the mechanism share common system i.e. phosphoenolpyruvate-dependent phosphotransferase systems (PTS).

 Thus, lac Operon is an example of negative inducible Operon in prokaryotes which is also regulated by CAP-cAMP complex and repressed by presence of glucose in the medium.

ZOOLOGY Molecular Cell Biology Regulation of gene expression in Prokaryotes: Lac Operon