Promoter in the lac operon

Promoter in the lac operon

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Here is the question:

Suppose an experimenter becomes proficient with a technique that allows her to move DNA sequences within a prokaryotic genome. If she moves the promoter for the lac operon to the region between the beta galactosidas gene and the permease gene which of the following would be likely?

Here is the answer:

Beta galactosidase will be produced.

My question to you is: why is this so? I have no idea where this conclusion came from. Thanks.

Since @biogirl has given an answer, I'll add my opinion:

β-galactosidase would be expressed but the permease and transacetylase would not.

The operator lies adjacent to/slightly overlaps the promoter, upstream of the lacZ gene. Binding of the repressor to the operator blocks the promoter, and induction of theoperon involves the repressor leaving the operator unoccupied. If you precisely excised the operator and transplanted it between the lacZ and lacY genes then I think the promoter would be constitutively expressed leading to β-galactosidase synthesis, but the operator would block the polymerase from transcribing the permease gene. In this view the operator would be acting more like a terminator.

Then, if an inducer was added it would relieve the effect of the transplanted operator and the downstream genes would be transcribed. It's even possible that this experiment has been done sometime in the last three decades.

added later:

The OP has now edited the question in such a way as to make my answer, and the answer from @biogirl, nonsensical. The original wording referred to moving "the lac operon" not "the lac promoter", hence my question in a comment asking "operon" or "operator"?

I'll leave my answer here, but this question is now so compromised that I advise everyone to just forget about it.

I believe the answer should be that permease is expressed.

As you can see from the image, if the operator is moved between Z gene and Y gene, Y gene should be expressed and permease should be made.

16.1: The lac Operon

  • Contributed by Todd Nickle and Isabelle Barrette-Ng
  • Professors (Biology) at Mount Royal University & University of Calgary

Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. In E. coli, and many other bacteria, genes encoding several different proteins may be located on a single transcription unit called an operon. The genes in an operon share the same transcriptional regulation, but are translated individually. Eukaryotes generally do not group genes together as operons (exception is C. elegans and a few other species).

Regulation of Arabinose Operon

The arabinose operon is regulated both positively and negatively, like a Lac-operon model.

Positive Regulation

Here, a term positive indicates that the synthesis of mRNA will occur. Therefore, the ara BAD mRNA is formed in positive regulation. Arabinose operon is positively regulated by the two conditions that are explained below:

Case-I (when both inducer and repressor protein is absent):

There will be no repression of the arabinose operon in the absence of both an inducer and a repressor protein. In this condition, the RNA polymerase will bind with the specific promoter region and transcribe the ara-BAD genes to form mRNA. But in this case, the rate of transcribing mRNA is much slower.

Case-II (when both inducer and repressor are present):

The inducer (arabinose) will bind with the repressor protein to regulate the mRNA transcription. The Ara-C protein plus arabinose forms a complex that will not allow the loop formation. The arabinose binds with the Ara-C dimer and changes its structural configuration. This change in structural configuration will allow the RNA polymerase to transcribe the ara-BAD genes to form mRNA. The mRNA will further translate into proteins.

Negative Regulation

Here, a term negative indicates that the mRNA transcription will not occur. To understand the process in a simple way, let us take condition III to compare it with the before two conditions.

Case-III (when only repressor protein is present):

The main role of arabinose operon involves the breakdown of arabinose. In the absence of arabinose, there will be no transcription and translation of the DNA molecule. Arabinose acts as an inducer, which binds and inactivates the repressor protein ARA-C. In the absence of inducer, the repressor protein will be produced by the ara-C gene. ARA-C repressor protein forms a dimer with the operator and the inducer gene by forming a loop. The loop formation will not allow the RNA polymerase to transcribe the ara-BAD genes to form mRNA.

Regulation of Gene Expression in Prokaryotes (With Diagram)

(ii) Those that are synthesized only after a specific stimulation. The first type was named constitutively synthesized and the latter the inducible enzymes.

Analyzing a variety of E. coli that were defective for the induction of the lactose utilizing enzymes, Jacob and Monod hit upon the possible molecular mechanism that controls the repression and de-repression of a set of genes. The E. coli requires a set of three genes to be able to metabolize lactose. When a little lactose is added to a glucose-free growth medium, it is seen that these three lactose utilizing genes (lac genes) named lac z, lac y and lac a are synthesized simultaneously.

The product of lac z is the enzyme β-gaIactosidase that catalyzes the conversion of lactose into galactose and glucose. These genes are note expressed in the absence of lactose. Jacob and Monod (1961) proposed the operon model to explain the genetic basis of induction and repression of lac genes in prokaryotes. They were awarded Nobel Prize for this work in 1965.

The Operon:

1. Operons are segments of genetic material (DNA) that function as regulated unit that can be switched on or off.

2. An operon consists of minimum four types of genes: regulator, operator, promoter and structural (Fig. 8.4.A).

3. Regulator gene is a gene which forms a biochemical for suppressing the activity of operator gene.

4. Operator gene is a gene which receives the product of regulator gene. It allows the functioning of the operon when it is not covered by the biochemical produced by regulator gene.

5. The functioning of operon is stopped when operator gene is covered.

6. Promoter gene is the gene which provides point of attachment to RNA polymerase required for transcription of structural genes.

7. Structural genes are genes which transcribe mRNA for polypeptide synthesis.

8. An operon may have one or more structural genes, e.g., 3 in lac operon, 5 in tryptophan operon, 9 in histidine operon.

9. The polypeptides may become component of structural proteins, enzymes, transport proteins, hormones, antibodies, etc. Some structural genes also form non-coding RNAs.

10. The mechanism of regulation of protein synthesis utilizing operon model can be illustrated using two examples (lac & tryptophan) in bacteria.”

Inducible Operon System (Induction of Operon):

1. Inducible operon system is (a) regulated operon system in which the structural genes remain switched off unless and until an inducer is present in the medium. (Fig. 8.4B)

2. It occurs in catabolic pathways.

3. Lac operon of Escherichia coli is an inducible operon system which was discovered by Jacob and Monod (1961).

4. Lac operon of Escherichia coli has three structural genes, z, y, and a.

5. In the induced operon the structural genes transcribe a polycistronic mRNA which produces three enzymes. These are β-galactosidase, galactoside permease and galactoside acetylase.

6. β-galactoside brings about hydrolysis of lactose or galactoside to form glucose and galactose.

7. Galactoside permease is required for entry of lactose or galactoside into the bacterium.

8. Galactoside acetylase is a transacetylase which can transfer acetyle group to β-galactoside.

9. The initiation codon of structural gene z is TAG (corresponding to AUG of mRNA) and is located 10 base pairs away from the end of the operator gene.

10. The substance whose addition induces the synthesis of enzyme is called inducer.

11. Inducer is a chemical which attaches to repressor and changes the shape of operator binding site so that repressor no more remains attached to operator.

12. In the lac operon allolactose is the actual inducer while lactose is the apparent (visible) inducer.

13. Inducers which induce enzyme synthesis without getting metabolized are called gratuitous inducers, e.g. IPTG (Isopropyl thiogalactoside).

14. Regulator gene (gene) produces mRNA that synthesises a biochemical repressor.

15. Repressor is a small protein formed by regulator gene which binds to operator gene and blocks structural enzyme thus checking mRNA synthesis.

16. The represseor of lac operon is a tetrameric protein having a molecular weight of 1, 60,000. It is made up of 4 subunits each having molecular weight of 40,000.

17. The repressor protein has two sites, a head for attaching to operator gene and a groove for attachment of inducer.

18. Promoter gene functions as a recognition point for RNA polymerase. RNA polymerase initially binds to this gene. It becomes functional only when it is able to pass over the operator gene and reach structural genes.

19. Operator gene controls the expressibility of the operon. It is normally switched off due to binding of repressor over it.

20. However, if the repressor is withdrawn by the inducer, the gene allows RNA polymerase to pass from promoter gene to structural gene.

21. In lac operon the operator gene is small, 27 base pairs long. The gene is made of palindromic or self-complementary sequences.

22. If lactose is added, the repressor is rendered inactive so that it cannot attach on operator gene and synthesis of mRNA takes place.

23. Transcription is under negative control when lac repressor is inactivated by inducer.

24. Transcription in lac operon is under positive control through cyclic AMP receptor protein (CAP).

25. The catabolite gene activator protein (Cga protein) or cyclic AMP receptor protein (CAP) binds to the Cga site.

26. When CAP is attached to the binding site the promoter becomes a stronger one.

27. CAP only attaches to the binding site when bound with cAMP.

28. When glucose level is high cAMP does not occur and so CAP does not bind and hence RNA polymerase do not bind, resulting in low transcription.

29. Lac operon will not however remain operative indefinitely despite presence of lactose in the external environment.

30. It will stop its activity with the accumulation of glucose & galactose in the cell beyond the capacity of the bacterium for their metabolism.

Repressible Operon System (Repression of Operon e.g. Tryptophan Operon of E.coli):

1. A repressible operon system is a regulated segment of genetic material which normally remains operational but can be switched off when its product is either not required or crosses a threshold value.

2. This system is commonly found in anabolic pathways.

3. Tryptophan operon of Escherichia coli is one such repressible operon system. (Fig. 8.5).

4. Tryptophan operon has 5 structural gene – E, D, C, and B A.

5. The gene E and D encodes for enzyme anthranilate synthetase, gene C for glycerol phosphate synthetase, gene B for β subunit of tryptophan synthetize and A for α subunit of tryptophan synthetize.

6. Regulator gene (trp-R) produces a biochemical, generally a proteinaceous substance, called aporepressor.

7. Aporepressor alone is unable to block the operator gene because of the absence of the binding head. Therefore, the operon system remains switched on.

8. A complete repressor is formed only when a non-proteinaceous corepressor joins the aporepressor,

9. Corepressor is a non-proteinaceous component or repressor which is also an end product of reaction catalysed by enzymes produced through the activity of structural genes.

10. It (corepressor) combines with aporepressor and forms repressor which then blocks the operator gene to switch off the operon.

11. The structural genes stop transcription and the phenomenon is known as feed-back repression.

12. Corepressor of tryptophan operon is amino acid tryptophan.

13. In tryptophan the repressor gene is not adjacent to promoter but located in another part of E. coli genome.

16. Promoter gene (trp-P) is the recognition as well as initiation point for RNA polymerase. RNA polymerase attaches to promoter gene. It can pass to structural genes provided the operator gene is in the functional state.

17. Operator gene (trp-O) lies in the passage-way between promoter and structural genes. Normally it remains switched on so that RNA polymerase can pass over from promoter gene to structural gene and bring about transcription.

18. The operator gene can be switched off when both aporepressor and corepressor join together to form repressor. The repressor binds to operator gene to interrupt movement of RNA polymerase.

19. In absence of tryptophan, the RNA polymerase binds to the operator site and thus structural genes are transcribed.

20. The transcription of structural gene leads to the production of enzyme (tryptophan synthetize) that synthesizes tryptophan.

21. When tryptophan becomes available, the enzymes for synthesizing tryptophan are not needed, co-repressor (tryptophan) – repressor complex blocks transcription.

22. One element of tryptophan operon is the leader sequence ‘L’ that is immediately 5′ end of trp. E gene.

23. This ‘L’ sequence controls expression of the operon through a process called attenuation.

24. Attenuation is the termination of the transcription prematurity at the leader region.

25. The tryptophan operon is a negative control.

The two operon models described above can be summarized as given below:

Active Repressor + Operator → System OFF

Active Repressor + Inducer = Inactive Repressor → System ON

(ii) Repressible System:

Apo-repressor and co-repressor complex = Active repressor → System OFF

Apo-repressor = Inactive Repressor → System ON

Importance of Gene Regulation:

1. There are two types of gene action – constitutive and regulated.

2. The constitutive gene action occurs in those systems which operate all the time and the cell cannot live without them, e.g., glycolysis. It does not require repression. Therefore, regulator and operator genes are not associated with it.

3. In regulated gene action all the genes required for a multistep reaction can be switched on or off simultaneously.

4. The genes are switched on or off in response to particular chemicals whether required for metabolism or are formed at the end of a metabolic pathway.

5. Gene regulation is required for growth, division and differentiation of cells. It brings about morphogenesis.

In prokaryote genomes, groups of structural genes with related functions are often linked together, with their expression being controlled by a single set of regulatory elements . These gene “bundles” are referred to as operons. Operons are an efficient way to streamline gene expression in prokaryotes. In this module we’ll be looking specifically at the Escherichia coli lac operon, which is often used as a model system in genetics and has real, practical applications in molecular biology.

1. The lac operon

1.1 Structure

The lac operon contains three enzyme-coding structural genes and three regulatory elements. The enzymes work together to allow E. coli to digest the disaccharide lactose , and the regulatory elements control the transcription of these enzymes.

These coding genes always come in a specific order within the operon, and during transcription, they are all transcribed together onto a single polycistronic mRNA strand. Please explore Figure 1 thoroughly by clicking on the “?” icons, to familiarize yourself with the key regulatory elements, structural genes, and protein products of the lac operon.

1.2 Regulatory Elements

  • Repressor (I): A coding sequence for the repressor protein. The repressor protein is a trans-regulatory element , and it’s transcription is regulated by an entirely separate set of regulatory sequences.
  • Promoter (P): A non-coding cis-regulatory element . RNA polymerase (RNApol) must bind to the promoter region to begin mRNA transcription.
  • Operator (O): A non-coding cis-regulatory element. Contains a binding site for the repressor protein I. When I is bound to the operator, RNA polymerase cannot bind to the promoter.

1.3 Structural Genes

  • Beta-galactosidase (lacZ): A coding sequence for beta-galactosidase, an enzyme that takes lactose as a substrate and cleaves it into the monosaccharides galactose and glucose. This is the first reactions necessary for the breakdown of lactose.
  • Permease (lacY): A coding sequence for permease, a membrane-bound protein that allows lactose to enter the cell.
  • Beta-galactoside transacetylase (lacA): A coding sequence for beta-galactoside transacetylase, an enzyme that adds acetyl groups to lactose and other galactose-containing sugars. The role of this enzyme in lactose digestion is not well defined, and we will mostly be leaving it out of our lac operon models.

Figure 1: The lac operon

Click on the “?” icons in this Figure to see more information about the component parts of the operon.

2. Function

We can see from Figure 1 that the lac operon coordinates the transcription of three enzymes with related functions. This is evidently very practical, but true beauty of this system lies in the fact that it ensures that these genes only get transcribed under specific environmental conditions.

Lactose is a relatively rare sugar, and most E. coli don’t need to be producing the beta-galactosidase and permease enzymes at a constant rate. Luckily for E. coli, the lac operon only activates in the presence of lactose! Watch this short video, courtesy of Virtual Cell, to see how this is accomplished:

In order to understand this video, you’ll need a good understanding of gene transcription and mRNA translation. If anything in this video seems unfamiliar, please take some time to brush up on these topics.

Video Notes:

  • In this video, Virtual Cell never specifically refers to the operator and promoter regions, choosing instead to lump them into a single regulatory element called the “Controlling region”. For this course, you’ll need to consider them as separate elements within the operon.
  • Remember, although it isn’t explicitly referenced in this video, lacA is always transcribed and translated along with lacZ and lacY. The function of this gene product is still unclear, so it’s left out of most educational resources.

Hopefully, this video has given you a basic idea of how the lac operon functions. In Section 3, we’ll take a deeper dive into how the individual components of the operon interact with each other by considering what happens if one or more of them is altered by a mutation.

3. Mutations

In molecular biology, one of the most common methods for figuring out a gene’s function is to mutate it and measure the resulting effects on its organism’s phenotype. In this section, we’ll be looking at a variety of mutations that can occur in lac operon genes, and discussing the effects of those mutations on E. coli. To do this, we’ll be using the following symbols to represent the individual components of the lac operon:

In this model, all the genetic elements in the operon are lined up in the same orientation as they are in an actual E. coli genome (see Figure 1). Since the function of lacA is not yet well defined, we’ll be leaving it out of this model more often than not. When all the sequences are wild type , the lac operon functions normally. We’ll represent this using the following notation:

If a given gene is mutated, we’ll change the superscript above that gene. Listed below are the specific mutations we are going to be looking at for this course:

  • Null mutation: Denoted by X – (where X can be any genetic element on the operon), DNA sequences with this mutation have completely lost their normal activity. In protein-coding genes, this means no protein is produced. In regulatory genes, this means that regular binding sites are non-functional (ie. the RNApol binding site in the promoter region, and the RNApol binding site in the operator region).
  • Constitutive activity: Denoted by O c , this mutation is specific to the operator region. Constitutively active operator regions always block the binding of repressor protein to the operator region. This results in transcription of the operon whether or not lactose is present, because the repressor is unable to block RNApol from binding to the promoter.
  • Super-repressor: Denoted by I s , this mutation is specific to the repressor-coding gene. Super-repressor genes produce special repressor proteins, which can still bind to the operator but not to lactose.

In these next exercises, we’ll consider what happens in a typical haploid E. coli when some of these mutations occur. As a hint, remember that all regulatory elements in the operon need to be functioning normally before any structural genes can be transcribed.

4. Merodiploids

Typically, we represent E. coli and other prokaryotes as being completely haploid, with only one circular chromosome and only one copy of each gene. You may remember, however, from our chapter on prokaryote genetics that this isn’t always the case. Bacteria, including E. coli, can acquire DNA from their environment (translation), from phages (transduction) or from other bacteria (conjugation). This may result in E. coli with two copies of certain genes! We call these partially diploid prokaryotes merodiploids (“mero-” comes from the Greek word for “part”, or “partial”). Merodiploids can be produced in a lab setting, using Hfr/F+ strains of E. coli.

Merodiploid E. coli are a fantastic research tool. They allow us to examine how wild-type and mutated alleles interact within a living organism, with all the added bonuses of working with E. coli (fast reproduction/growth, easy colony maintenance, etc.) In this module, we’ll be representing merodiploids using the following notation:

In this notation, we show a chromosomal lac operon and an Hfr plasmid lac operon side by side. Again, we’ve included the lacA gene here for completeness, but will be leaving it out of our exercises.

Because merodiploids have two copies of a given set of genes, mutations affect them differently. For example, if a single copy of a protein coding gene is inactivated, the second copy may still continue to produce viable protein, effectively masking the mutation.

Try out your understanding using this next set of exercises:

5. Regulators and Effectors

We’ve seen in Section 2 that the lac operon has a built-in lactose sensor: the repressor protein. When there is no lactose present, the repressor prevents lac operon products from being translated by binding to the operator region. When lactose is plentiful in the environment, it is taken up by the cell and binds to the repressor, removing its ability to bind to the operator region. In general, we call any molecule that modifies a protein’s function in this way an effector molecule. To be a true effector, a molecule must modify a protein’s activity by selectively binding at an allosteric site .

In molecular biology terms, we would say that the repressor protein is a negative regulator of the lac operon, because it’s binding to the operon decreases transcription. In contrast, a positive regulator would be a molecule that binds to the operon and increases transcription. The lac operon does indeed have a positive regulator: Catabolite Activator Protein, or CAP. Keeping pace with the repressor protein, CAP has its own effector molecule: cyclic AMP, or cAMP.

cAMP is produced by E. coli as a metabolic byproduct when glucose is scarce. It binds to the allosteric site on CAP, activating the protein and forming what we’ll call the cAMP-CAP complex. Thus activated, CAP binds to the lac operon promoter region, just upstream of the binding site for RNApol. This increases the affinity of the promoter region for RNApol, which leads to a huge increase in lac operon transcription (Figure 2). Without the cAMP-CAP complex, the lac operon is still transcribed in the presence of lactose, but at a much slower rate.

Figure 2: The cAMP-CAP complex

Now we might wonder, if the lac operon already has a negative regulator, why does it also need a positive regulator? Ultimately, it all comes down to efficiency. E. coli are more efficient at digesting glucose than lactose, so when glucose is plentiful, it’s wasteful to transcribe lac operon enzymes. The most efficient regulatory system would be one which activates not only in the presence of lactose, but also in the absence of glucose this is what the cAMP-CAP complex accomplishes.

Test your understanding using the next set of exercises:

A structural gene codes for a product that does not regulate gene expression. Examples include enzymes, structural proteins, siRNA, etc.

Regulatory elements are non-coding regions of DNA that function to regulate gene expression. They may contain binding sites for polymerase enzymes, transcription factors, repressor proteins, etc.

A disaccharide made up of the two monosaccharides glucose and galactose.

A single mRNA strand that contains coding sequences for multiple products. Separate ribosome binding-sites exist for each coding sequence, allowing for simultaneous translation of all sequences.

DNA sequences that modify or regulate the expression of distant genes.

DNA sequence that modifies or controls the expression of an adjacent gene.

An enzyme that transcribes mRNA using DNA as a template

The most common form of a gene or phenotype found in nature

A binding site other than the protein's active site. In an enzyme, the active site is the site of catalysis. In a DNA-binding protein, the active site is the binding site for DNA.

What is lac operon in biology?

The lac, or lactose, operon is found in E. coli and some other enteric bacteria. This operon contains genes coding for proteins in charge of transporting lactose into the cytosol and digesting it into glucose. This glucose is then used to make energy.

Also, what is operon in biology? Operon: A set of genes transcribed under the control of an operator gene. More specifically, an operon is a segment of DNA containing adjacent genes including structural genes, an operator gene, and a regulatory gene. An operon is thus a functional unit of transcription and genetic regulation.

Herein, what is meant by lac operon?

The lac operon (lactose operon) is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria. The gene product of lacZ is &beta-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose.

What is the function of Lac A?

These are referred to as lac z, lac y, and lac a. The lac z gene encodes beta-galactosidase, the lac y gene encodes a permease, and the lac a gene encodes the transacetylase enzyme. Together, these gene products act to import lactose into cells and break it down for use as a food source.

What is the t7 promoter?

T7 expression hosts, such as DE3 strains or T7 Express strains, carry a chromosomal copy of the phage T7 RNA Polymerase gene, which is controlled by a lac promoter. When inducer is added, T7 RNA Polymerase is expressed and becomes dedicated to transcription of the gene of interest.

Likewise, what is a promoter? In genetics, a promoter is a region of DNA that leads to initiation of transcription of a particular gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand).

Also, what is the t7 expression system?

The T7 expression system allows high-level expression from the strong bacteriophage T7 promoter. It's ideal for expressing soluble, nontoxic recombinant proteins in E. coli. The T7 expression vectors are designed to facilitate cloning using Gateway® technology, and easy protein purification and detection.

What does t7 RNA polymerase do?

T7 RNA polymerase. T7 RNA Polymerase (blue) producing mRNA (light-blue) from a double-stranded DNA template (orange). T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'&rarr 3' direction.

Regulation of Gene Expression in Prokaryotes | Gene Regulation

Gene transcription is regulated in bacteria through a complex of genes termed operon. These are transcriptional units in which several genes, with related functions, are regulated together. Other genes also occur in operons which encode regulatory proteins that control gene expression. Operons are classified as inducible or repressible.

Inducible and Repressible System:

The β galactosidase in E. coli is responsible for hydro­lysis of lactose into glucose and galactose.

If lactose is not supplied to E. coli cells, the presence of β galactosidase is hardly detectable. But as soon as lactose is added, the production of β galactosidase enzyme increases. The enzyme falls as quickly as the substrate (lactose) is removed.

Such enzymes whose synthesis can be induced by adding the substrate are known as inducible enzymes and the genetic system responsible for the synthesis of such an enzyme is called inducible system. The substrate whose addition induces the synthesis of an enzyme is inducer.

In some other cases, the situation is reverse. For instance, when no amino acids are supplied from outside, the E. coli cells can synthesize all the enzymes needed for the synthesis of different amino acids. However, if a particular amino acid, for instance, histidine, is added, the production of histidine synthesizing enzyme falls.

In such a system, the addition of the end product of biosyn­thesis checks the synthesis of the enzymes needed for the biosynthesis. Such enzymes whose synthesis can be checked by the addition of the end product are repressible enzymes and the genetic system is known as repressible sys­tem. The end product, the addition of which check the synthesis of the enzyme is co-repressor.

A class of molecules called repressors are found in cells and these repressors check the activity of genes. An active repressor can be made inactive by adding inducer, while an inac­tive repressor can be made active by adding a co-repressor.

Operon Model:

A hypothesis to explain the induction and repression of enzyme synthesis was first pro­posed by Jacob and Monod. The scheme pro­posed by them is called Operon Model.

This consists of the components:

These are directly con­cerned with the synthesis of cellular proteins. They produce the mRNAs through transcription and determine the sequence of amino acids in the synthesized proteins. All the structural genes under an operon may form one long poiycistronic or polygenic mRNA molecule.

This is located adjacent to the structural gene. It determines whether the structural genes are to be repressed by the repre­ssor protein, a product of regulator gene. The operator gene is the site of binding of the repre­ssor protein, the latter binds to the operator form­ing an operator-repressor complex. When the repressor binds to the operator, transcription of the structural genes cannot occur.

These genes synthesize repressor. Repressor may be either an active repressor or an inactive repressor. Repressor pro­tein has one active site for operator recognition and other active site for inducer. In absence of an inducer protein, the repressor binds to the ope­rator gene and blocks the path of RNA poly­merase. Thus the structural genes are unable to transcribe mRNA and consequently protein syn­thesis does not occur.

In presence of an inducer, the repressor protein binds to the inducer to form an inducer-repressor complex. The repressor when binds with inducer undergoes a change and becomes ineffective and as a result it cannot bind to the operator gene and the protein syn­thesis is possible.

The actual site of transcrip­tion initiation is known as promoter gene which lies to the left of the operator gene. It is believed that RNA polymerase binds to and moves from the promoter site.

Effector is a small molecule (sugar or amino acid) that can be linked to a regulator protein and will determine whether repressor will bind the operator or not. In the inducible operon, these effector molecules are called inducer. In repressible operon, these effector molecules are called co-repressor.

Inducible Operon:

The best known operon is the lac operon. The lac operon exercises both positive and nega­tive control. Negative control is in the sense that the operon is normally “on” but is kept “off” by the regulator gene, i.e., the genes are not allowed to express unless required.

The lac repressor exercises negative control. Positive control is that in which the regulator gene will stimulate the production of the enzyme. Catabolite activator protein (CAP) facilitates transcription, so it exer­cises positive control. Two unique proteins are thus involved in the regulation of the lac operon which are lac repressor and CAP.

Lactose is a disaccharide molecule. In order to utilize lactose as a carbon and energy source, the lactose molecules must be transported from the extracellular environment into the ceil, and then undergo hydrolysis into glucose and galac­tose. These reactions are catalysed by three enzymes. The lac operon consists of three struc­tural genes (lac Z, Y, A) which code for these three enzymes (Fig. 17.2).

lac Z gene — codes for enzyme β galactosidase which breaks lactose into galactose and glucose

lac Y gene — codes for permease which transports lactose into the cell

lac A gene — codes for transacetylase which transfer the acetyl group from acetyl CoA to galactose.

Negative Control of lac Operon:

lac repres­sor is synthesized through the activity of the lac I gene called the regulator gene. This repressor is an allosteric protein

(i) That can bind the lac DNA at the operator site, or

(ii) That can bind to inducer.

In the absence of inducer, DNA binding site of repressor is functional. The repressor protein binds to the DNA at the operator site of the lac locus and blocks the transcription of the lac genes by RNA polymerase. Thus lac enzyme syn­thesis is inhibited (Fig. 17.3A).

Lactose is not the real inducer of the lac operon. It binds to repressor to increase its affi­nity for operator. On the other hand, the bound protein of the inactive repressor is the allolactose. While β galactosidase breaks lactose into glucose and galactose, a side reaction changes galactose to allolactose and galactobiose.

This allolactose prevents the anti-inducing lac I lac lac effect of lactose. When the allolactose (inducer) binds to the repressor, it changes the form of DNA binding site making the repressor inactive and release from- the operator site. Thus trans­cription of lac genes are possible.

Positive Control of lac Operon:

It is an additional regulatory mechanism which allows the lac operon to sense the presence of glucose, an alternative and preferred energy source to lactose. If glucose and lactose are both present, cells will use up the glucose first and will not uti­lize energy splitting lactose into its component sugars.

The presence of glucose in the cell switches off the lac operon by a mechanism called catabolite repression which involves a regulatory protein called the catabolite activator protein (CAP). CAP binds to a DNA sequence upstream of the lac promoter and enhances bind­ing of the RNA polymerase and transcription of the operon is enhanced (Fig. 17.3B).

CAP only binds in the presence of a deri­vative of ATP called cyclic adenosine monophos­phate (cAMP) whose levels are influenced by glucose. The enzyme adenylate cyclase cata­lyzes the formation of cAMP and is inhibited by glucose. When glucose is available to the cell, adenylate cyclase is inhibited and cAMP levels are low.

Under these conditions CAP does not bind upstream of the promoter and the lac ope­ron is transcribed at a very low level. Conversely, when glucose is low, adenylate cyclase is not inhibited, cAMP is higher and CAP binds increasing the level of transcription from the operon.

If glucose and lactose are present together, the lac operon will only be transcribed at a low level. However when the glucose is used up, catabolite repression will end and trans­cription from the lac operon increases allowing the available lactose to be used up.

Repressible Operon:

The trp operon consists of the following components:

(i) Structural genes (trp E, D, C, B and A):

This operon contains five structural genes encoding enzymes involved in biosynthe­sis of the amino acid tryptophan. The genes are expressed as a single mRNA transcribed from an upstream promoter.

(ii) Promoter gene (trp P):

It is the promoter region which is the binding site for RNA polymerase.

(iii) Operator gene (trp O):

It is the operator region which binds with the repressor.

It is the leader region which is made of 162 nucleotides prior to the first structural gene trp E. It has four regions, region 1 has the codon for tryp­tophan, region 2, 3 and 4 regulate the mRNA synthesis of the structural genes.

Expression of the operon is regulated by the level of tryptophan in the cell (Fig. 17.4). A regu­latory gene upstream of the trp operon encodes a protein called the trp repressor. This protein binds a DNA sequence called the trp operator which lies just downstream of the trp promoter partly overlapping it.

When tryptophan is present in the cell it binds to the trp repressor protein enabling it to bind the trp operator sequence, obstructing binding of the RNA polymerase to the trp promoter and preventing transcription of the operon.

In the absence of tryptophan, the trp repressor is incapable of binding the trp operator and transcription of the operon proceeds. Tryptophan, the end product of the enzymes encoded by the trp operon, thus acts as a co-repressor with the trp repressor protein and inhibits its own synthesis by end product inhibition.

Attenuation is an alternative regulatory mechanism that allows fine adjust­ment of expression of the trp operon and other operons (phe, his, leu, thr operon). The trans­cribed mRNA sequence between the trp promo­ter and the first trp gene are capable of forming either a large stem-loop structure that does not influence transcription or a smaller stem loop which acts as transcription terminator (Fig. 17.5).

The relative position of the sequences does not allow the formation of both stem-loops at a time. Attenuation depends on the fact that transcrip­tion and translation are linked, i.e., ribosomes attach to mRNAs as they are being transcribed and begin translating them into protein.

Binding of ribosomes to the trp mRNA influences which of the two stem-loops can form and so deter­mines whether termination occurs or not (Fig. 17.5).

A short coding region upstream of the stem-loop region contains tryptophan codons which is translated before the structural genes. When tryptophan levels are adequate, RNA polymerase transcribes the leader region closely followed by a ribosome which prevents forma­tion of the larger stem-loop, allowing the termi­nator loop to form ending transcription.

If trypto­phan is lacking, transcription is initiated, but not subsequently terminated because the ribosome is stalled, the RNA polymerase moves ahead and the large stem-loop forms. Formation of the ter­minator loop is blocked and transcription of the operon proceeds. When tryptophan present at intermediate levels, some transcripts will termi­nate and others not.

Attenuation thus allows the cell to synthesize tryptophan according to its exact requirements. Overall, the trp repressor determines whether the operon is switched on or off and attenuation determines how efficiently it is transcribed.

The sequence of the mRNA suggests that ribosome stalling influences termination at the attenuator. The ability of the ribosome to pro­ceed through the leader region may control transition between these structures. The structure determines whether the mRNA can provide the features needed for termination or not.

When tryptophan is present, ribosomes are able to synthesize the leader peptide. They will continue along the leader section of the mRNA to the UGA codon, which lies between regions 1 and 2. By progressing to this point, the ribosomes extend over region 2 and prevent it from base pairing.

The result is that region 3 is available to base pair with region 4, generating the termi­nator hairpin. Under these conditions, therefore, RNA polymerase terminates at the attenuator.

However, when there is no tryptophan, ribo­somes initiate translation of the leader peptide but stall at the trp codons which is at the region 1. Thus the region 1 cannot base pair with region 2. If this happens, even while the mRNA itself is being synthesized, region 2 and 3 will be base- paired before region 4 has been transcribed.

This compels region 4 to remain in a single stranded form. In the absence of the terminator hairpin, RNA polymerase continues transcription past the attenuator.

The ara (arabinose) operon of F. coli con­tains:

(i) Three structural genes (ara A, ara B and ara D) – which encode three different enzymes (isomerase, kinase, epimerase) for metabolism of arabinose three sructuretural genes are co-transcribed on a single mRNA.

(ii) Promoter gene(PBAD)- which initiates transcription.

(iii) Regular gene (ara C)- the regulatory protein of this gene ara C.

(iv) Promoter gene (Pc)- This initiates transcription of are C.

Two promoters PBAD and Pc are situated 100 nucleotide pairs away in the same inducer region and they initiate transcription in opposite direc­tions.

The induction of ara operon depends on the positive regulatory effects of two proteins, the ara C protein and CAP (the cAMP binding catabolite activator protein), the binding sites of these two proteins are located in a region called ara I which is situated in between the three structural genes (ara B, ara A and ara D) and the regulator gene (ara C) (Fig. 17.6A).

The ara C protein acts as a negative regulator (a repressor) of transcription of the ara B, ara A and ara D structural genes from the PBAD promoter in absence of arabinose and cyclic AMP (cAMP). But it acts as a positive regulator (an activator) of tran­scription of these genes from the PBAD promoter when arabinose and cAMP are present.

Depending on the presence or absence of effector molecule like arabinose and cAMP, the ara C regulatory gene product may exert either a positive or negative effect on transcription of the ara B, ara A and ara D structural genes (Fig. 17.6B).

Post-Transcriptional Regulation of Gene Expression in Prokaryotes:

Gene regulation may also occur in prokaryotes at the time of translation.

Autogenous Regulation of Translation:

There are number of examples where a protein or RNA regulates its own production. Several proteins work as repressors, bind to the ribosome binding site (or SD-Shine-Dalgarno sequence) or initiation codon of mRNA. In these cases mRNA remains intact but cannot be translated. There are some other systems where mRNA may be degraded by the binding of protein on the short specific sequences of mRNA.

Regulation by Anti-sense RNA:

Translational control of protein synthesis can be exercised by using RNA which is complementary to mRNA, these complementary RNA will form RNA- mRNA hybrids and prevent mRNA from being translated. These kind of RNAs are called anti- sense RNA or micRNA (mic = mRNA interfering complementary RNA).

Repression of Translation:

Repression of translation occurs by the following ways:

(a) A repressor-effector molecule may recog­nise and bind to a specific sequence or to a specific secondary structure (involving SD region and AUG codon), thus blocking initiation of translation through blocking of the ribosomal bind­ing region.

(b) A repressor-effector molecule may bind to an operator (not involving SD region and AUG codon) thus stabilizing an inhibitory mRNA secondary structure.

(c) An effector molecule (an endonuclease) can inhibit initiation of translation by endonucleolytic cleavage of SD region.

Activation of Translation:

Some positive effectors or activators cause activation of trans­lation by destabilizing the inhibitory secondary structures in mRNA either through simple bind­ing or by endonucleolytic cleavage. Translation of certain genes may be influenced by certain other genes – the phenomenon is called trans­lational coupling.

In some cases, the end product of a particular biosynthetic pathway gets accumulated and this accumulation may stop further synthesis of this substance. The end product acts through allosteric transformation of the first enzyme of biosynthetic pathway (Fig. 17.7).

Art Connection

Regulation of the lac operon. Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source.

Question: In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think this is the case?

If glucose is present, then CAP fails to bind to the promoter sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these conditions is met, then transcription remains off. Only when glucose is absent and lactose is present is the lac operon transcribed (Table).

Signals that Induce or Repress Transcription of the lac Operon
GlucoseCAP bindsLactoseRepressor binds Transcription

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