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How can a continuous RNA be transcribed in the lac operon?

How can a continuous RNA be transcribed in the lac operon?


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The lac operon has 3 genes: lacZ , lacY and lacA. I have seen that the m-RNA transcript of these genes has stop codons in between. So, how can the RNA be made continuously? Won't the RNA Pol detach if there is a stop codon in between?


RNA Pol doesn't worry about stop codons. Transcription termination can occur through the formation of a hairpin in the new RNA sequence, or through the action of Rho proteins. A lot of the time prokaryotes have polycistronic mRNAs, that is, mRNAs with multiple protein coding regions. The stop codons are detected during translation, so you will often have all three lac proteins being translated at once off of the same mRNA.

Here's a nice section on transcription termination, or look up Rho-independent or Rho-dependent transcription termination for more info.


How can a continuous RNA be transcribed in the lac operon? - Biology

By the end of this section, you will be able to do the following:

  • Describe the steps involved in prokaryotic gene regulation
  • Explain the roles of activators, inducers, and repressors in gene regulation

The DNA of prokaryotes is organized into a circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA.

In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors and activators are proteins produced in the cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent to the genes they control. In general, activators bind to the promoter site, while repressors bind to operator regions. Repressors prevent transcription of a gene in response to an external stimulus, whereas activators increase the transcription of a gene in response to an external stimulus. Inducers are small molecules that may be produced by the cell or that are in the cell’s environment. Inducers either activate or repress transcription depending on the needs of the cell and the availability of substrate.

The trp Operon: A Repressible Operon

Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesize using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon ((Figure)). The genes are transcribed into a single mRNA, which is then translated to produce all five enzymes. If tryptophan is present in the environment, then E. coli does not need to synthesize it and the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are translated, and tryptophan is synthesized.

Figure 1. The tryptophan operon. The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site. The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.

The trp operator contains the DNA code to which the trp repressor protein can bind. However, the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes the shape of the repressor protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding to the promoter and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.

Link to Learning

Watch this video to learn more about the trp operon.

Catabolite Activator Protein (CAP): A Transcriptional Activator

Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the promoter sequences that act as positive regulators to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate sugars must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. Accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons which control the processing of alternative sugars. When cAMP binds to CAP, the complex then binds to the promoter region of the genes that are needed to use the alternate sugar sources ((Figure)). In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. CAP binding stabilizes the binding of RNA polymerase to the promoter region and increases transcription of the associated protein-coding genes.

Figure 2. Transcriptional activation by the CAP protein. When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the CAP protein, a positive regulator that binds to a promoter region upstream of the genes required to use other sugar sources.

The lac Operon: An Inducible Operon

The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. The Z gene of the lac operon encodes beta-galactosidase, which breaks lactose down to glucose and galactose.

However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed ((Figure)). In the absence of glucose, the binding of the CAP protein makes transcription of the lac operon more effective. When lactose is present, it binds to the lac repressor and changes its shape so that it cannot bind to the lac operator to prevent transcription. This combination of conditions makes sense for the cell, because it would be energetically wasteful to synthesize the enzymes to process lactose if glucose was plentiful or lactose was not available.

Art Connection

Figure 3. 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?

Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present.

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 ((Figure)).

Signals that Induce or Repress Transcription of the lac Operon
Glucose CAP binds Lactose Repressor binds Transcription
+ + No
+ + Some
+ + No
+ + Yes

Link to Learning

Watch an animated tutorial about the workings of lac operon here.

Section Summary

The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are two majors kinds of proteins that control prokaryotic transcription: repressors and activators. Repressors bind to an operator region to block the action of RNA polymerase. Activators bind to the promoter to enhance the binding of RNA polymerase. Inducer molecules can increase transcription either by inactivating repressors or by activating activator proteins. In the trp operon, the trp repressor is itself activated by binding to tryptophan. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains off. The lac operon is activated by the CAP (catabolite activator protein), which binds to the promoter to stabilize RNA polymerase binding. CAP is itself activated by cAMP, whose concentration rises as the concentration of glucose falls. However, the lac operon also requires the presence of lactose for transcription to occur. Lactose inactivates the lac repressor, and prevents the repressor protein from binding to the lac operator. With the repressor inactivated, transcription may proceed. Therefore glucose must be absent and lactose must be present for effective transcription of the lac operon.

Art Connections

(Figure) In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think that this is the case?

(Figure) Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present.

Review Questions

If glucose is absent, but so is lactose, the lac operon will be ________.

Prokaryotic cells lack a nucleus. Therefore, the genes in prokaryotic cells are:

  1. all expressed, all of the time
  2. transcribed and translated almost simultaneously
  3. transcriptionally controlled because translation begins before transcription ends
  4. b and c are both true

The ara operon is an inducible operon that controls the production of the sugar arabinose. When arabinose is present in a bacterium it binds to the protein AraC, and the complex binds to the initiator site to promote transcription. In this scenario, AraC is a(n) ________.

Free Response

Describe how transcription in prokaryotic cells can be altered by external stimulation such as excess lactose in the environment.

Environmental stimuli can increase or induce transcription in prokaryotic cells. In this example, lactose in the environment will induce the transcription of the lac operon, but only if glucose is not available in the environment.

What is the difference between a repressible and an inducible operon?

A repressible operon uses a protein bound to the promoter region of a gene to keep the gene repressed or silent. This repressor must be actively removed in order to transcribe the gene. An inducible operon is either activated or repressed depending on the needs of the cell and what is available in the local environment.


The trp Operon: A Repressible Operon

Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesize using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure). The genes are transcribed into a single mRNA, which is then translated to produce all five enzymes. If tryptophan is present in the environment, then E. coli does not need to synthesize it and the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are translated, and tryptophan is synthesized.

The tryptophan operon. The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site . The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.


pUC19 contains LacZ DNA as a reporter gene to illustrate the presence of the functioning gene. Transcription of this gene is driven by the binding site for the RNA Polymerase subunit called σ factor . The σ factor binding site determines the directionality of the RNA polymerase, since there is an option of transcribing in 2 directions. The standard σ factor binding site is often denoted as -35 TTGACA…TATAAT -10 from the transcription initiation.

The multiple cloning site within the plasmid provides a convenient location to shuttle a foreign piece of DNA. When no foreign DNA is inserted to this space, the LacZ gene product β-galactosidase is functional. Disruption of the reading frame for this gene likewise disables the functional product from being translated. By using chemical reporters, the integrity of this gene can be confirmed through enzymatic activity.

Hydrolysis of lactose to galactose and glucose Two chemical reporters used to reveal the presence of a functioning LacZ are X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside5-bromo-4-chloro-3-indolyl-β-D-galactoside) and ONPG (orthonitrophenol).

X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) yields a blue color when cleaved by β-galactosidase ONPG (orthonitrophenol) yields a yellow color upon cleavage by β-galactosidase
As in the case of the Lac operon, the LacI (repressor protein) will occupy the operator. This operator happens to be overlapping the -35 & -10 sequences. In order to fully activate these genes, the Lac repressor must be removed by binding to a lactose analog. In this case, the chemical IPTG (Isopropyl β-D-1-thiogalactopyranoside) is used since it is a non-cleavable analog that will perpetually bind to the Lac repressor.


LacI is an allosterically regulated repressor

One of the major trans-regulators of the lac operon is encoded by lacI. Four identical molecules of lacI proteins assemble together to form a homotetramer called a repressor (Figure (PageIndex<2>)). This repressor binds to two operator sequences adjacent to the promoter of the lac operon. Binding of the repressor prevents RNA polymerase from binding to the promoter (Figure (PageIndex<3>)). Therefore, the operon will not be transcribed when the operator is occupied by a repressor.

Figure (PageIndex<2>): Structure of lacI homotetramer bound to DNA (Origianl-Deyholos-CC:AN)

Besides its ability to bind to specific DNA sequences at the operator, another important property of the lacI protein is its ability to bind to lactose. When lactose is bound to lacI, the shape of the protein changes in a way that prevents it from binding to the operator. Therefore, in the presence of lactose, RNA polymerase is able to bind to the promoter and transcribe the lac operon, leading to a moderate level of expression of the lacZ, lacY, and lacA genes. Proteins such as lacI that change their shape and functional properties after binding to a ligand are said to be regulated through an allosteric mechanism. The role of lacI in regulating the lac operon is summarized in Figure (PageIndex<4>).

Figure (PageIndex<3>): When the concentration of lactose [Lac] is low, lacI tetramers bind to operator sequences (O), thereby blocking binding of RNApol (green) to the promoter (P). Alternatively, when [Lac] is high, lactose binds to lacI, preventing the repressor from binding to O, and allowing transcription by RNApol. (Origianl-Deyholos-CC:AN)


Contents

The term "operon" was first proposed in a short paper in the Proceedings of the French Academy of Science in 1960. [9] From this paper, the so-called general theory of the operon was developed. This theory suggested that in all cases, genes within an operon are negatively controlled by a repressor acting at a single operator located before the first gene. Later, it was discovered that genes could be positively regulated and also regulated at steps that follow transcription initiation. Therefore, it is not possible to talk of a general regulatory mechanism, because different operons have different mechanisms. Today, the operon is simply defined as a cluster of genes transcribed into a single mRNA molecule. Nevertheless, the development of the concept is considered a landmark event in the history of molecular biology. The first operon to be described was the lac operon in E. coli. [9] The 1965 Nobel Prize in Physiology and Medicine was awarded to François Jacob, André Michel Lwoff and Jacques Monod for their discoveries concerning the operon and virus synthesis.

Operons occur primarily in prokaryotes but also in some eukaryotes, including nematodes such as C. elegans and the fruit fly, Drosophila melanogaster. rRNA genes often exist in operons that have been found in a range of eukaryotes including chordates. An operon is made up of several structural genes arranged under a common promoter and regulated by a common operator. It is defined as a set of adjacent structural genes, plus the adjacent regulatory signals that affect transcription of the structural genes. 5 [11] The regulators of a given operon, including repressors, corepressors, and activators, are not necessarily coded for by that operon. The location and condition of the regulators, promoter, operator and structural DNA sequences can determine the effects of common mutations.

Operons are related to regulons, stimulons and modulons whereas operons contain a set of genes regulated by the same operator, regulons contain a set of genes under regulation by a single regulatory protein, and stimulons contain a set of genes under regulation by a single cell stimulus. According to its authors, the term "operon" is derived from the verb "to operate". [12]

An operon contains one or more structural genes which are generally transcribed into one polycistronic mRNA (a single mRNA molecule that codes for more than one protein). However, the definition of an operon does not require the mRNA to be polycistronic, though in practice, it usually is. [5] Upstream of the structural genes lies a promoter sequence which provides a site for RNA polymerase to bind and initiate transcription. Close to the promoter lies a section of DNA called an operator.

All the structural genes of an operon are turned ON or OFF together, due to a single promoter and operator upstream to them, but sometimes more control over the gene expression is needed. To achieve this aspect, some bacterial genes are located near together, but there is a specific promoter for each of them this is called gene clustering. Usually these genes encode proteins which will work together in the same pathway, such as a metabolic pathway. Gene clustering helps a prokaryotic cell to produce metabolic enzymes in a correct order. [13]

An operon is made up of 3 basic DNA components:

    – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for messenger RNA creation – and, by extension, control which proteins the cell produces.
  • Operator – a segment of DNA to which a repressor binds. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. [14] The main operator (O1) in the lac operon is located slightly downstream of the promoter two additional operators, O2 and O3 are located at -82 and +412, respectively. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes. – the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed gene which codes for repressor proteins. The regulatory gene does not need to be in, adjacent to, or even near the operon to control it. [15]

An inducer (small molecule) can displace a repressor (protein) from the operator site (DNA), resulting in an uninhibited operon.

Alternatively, a corepressor can bind to the repressor to allow its binding to the operator site. A good example of this type of regulation is seen for the trp operon.

Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various genes depending on environmental conditions. Operon regulation can be either negative or positive by induction or repression. [14]

Negative control involves the binding of a repressor to the operator to prevent transcription.

  • In negative inducible operons, a regulatory repressor protein is normally bound to the operator, which prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon. The lac operon is a negatively controlled inducible operon, where the inducer molecule is allolactose.
  • In negative repressible operons, transcription of the operon normally takes place. Repressor proteins are produced by a regulator gene, but they are unable to bind to the operator in their normal conformation. However, certain molecules called corepressors are bound by the repressor protein, causing a conformational change to the active site. The activated repressor protein binds to the operator and prevents transcription. The trp operon, involved in the synthesis of tryptophan (which itself acts as the corepressor), is a negatively controlled repressible operon.

Operons can also be positively controlled. With positive control, an activator protein stimulates transcription by binding to DNA (usually at a site other than the operator).

  • In positive inducible operons, activator proteins are normally unable to bind to the pertinent DNA. When an inducer is bound by the activator protein, it undergoes a change in conformation so that it can bind to the DNA and activate transcription.
  • In positive repressible operons, the activator proteins are normally bound to the pertinent DNA segment. However, when an inhibitor is bound by the activator, it is prevented from binding the DNA. This stops activation and transcription of the system.

The lac operon of the model bacterium Escherichia coli was the first operon to be discovered and provides a typical example of operon function. It consists of three adjacent structural genes, a promoter, a terminator, and an operator. The lac operon is regulated by several factors including the availability of glucose and lactose. It can be activated by allolactose. Lactose binds to the repressor protein and prevents it from repressing gene transcription. This is an example of the derepressible (from above: negative inducible) model. So it is a negative inducible operon induced by presence of lactose or allolactose.

Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the first repressible operon to be discovered. While the lac operon can be activated by a chemical (allolactose), the tryptophan (Trp) operon is inhibited by a chemical (tryptophan). This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which encodes tryptophan synthetase. It also contains a promoter which binds to RNA polymerase and an operator which blocks transcription when bound to the protein synthesized by the repressor gene (trp R) that binds to the operator. In the lac operon, lactose binds to the repressor protein and prevents it from repressing gene transcription, while in the trp operon, tryptophan binds to the repressor protein and enables it to repress gene transcription. Also unlike the lac operon, the trp operon contains a leader peptide and an attenuator sequence which allows for graded regulation. [16] This is an example of the corepressible model.

The number and organization of operons has been studied most critically in E. coli. As a result, predictions can be made based on an organism's genomic sequence.

One prediction method uses the intergenic distance between reading frames as a primary predictor of the number of operons in the genome. The separation merely changes the frame and guarantees that the read through is efficient. Longer stretches exist where operons start and stop, often up to 40–50 bases. [17]

An alternative method to predict operons is based on finding gene clusters where gene order and orientation is conserved in two or more genomes. [18]

Operon prediction is even more accurate if the functional class of the molecules is considered. Bacteria have clustered their reading frames into units, sequestered by co-involvement in protein complexes, common pathways, or shared substrates and transporters. Thus, accurate prediction would involve all of these data, a difficult task indeed.

Pascale Cossart's laboratory was the first to experimentally identify all operons of a microorganism, Listeria monocytogenes. The 517 polycistronic operons are listed in a 2009 study describing the global changes in transcription that occur in L. monocytogenes under different conditions. [19]


A. Mechanisms of Control of the Lac Operon

In the animal digestive tract (including ours), genes of the E. coli lac operon regulate the use of lactose as an alternative nutrient to glucose. Think cheese instead of chocolate! The operon consists of lacZ, lacY, and lacA genes that were called structural genes. By definition, structural genes encode proteins that participate in cell structure and metabolic function. As already noted, the lac operon is transcribed into an mRNA encoding the Z, Y and A proteins.

Let&rsquos take a closer look at the structure of the lac operon and the function of the Y, Z and A proteins (below).

The lacZ gene encodes &beta-galactosidase, the enzyme that breaks lactose (a disaccharide) into galactose and glucose. The lacY gene encodes lactose permease, a membrane protein that facilitates lactose entry into the cells. The role of the lacA gene (a transacetylase) in lactose energy metabolism is not well understood. The I gene to the left of the lac Z gene is a regulatory gene (to distinguish it from structural genes). Regulatory genes encode proteins that interact with regulatory DNA sequences associated with a gene to control transcription. The operator sequence separating the I and Z genes is a transcription regulatory DNA sequence.

The E. coli lac operon is usually silent (repressed) because these cells prefer glucose as an energy and carbon source. In the presence of sufficient glucose, a repressor protein (the I gene product) is bound to the operator, blocking transcription of the lac operon. Even if lactose is available, cells will not be use it as an alternative energy and carbon source when glucose levels adequate. However, when glucose levels drop, the lac operon is active and the three enzyme products are translated. We will see how limiting glucose levels induce maximal lac operon transcription by both derepression and direct induction, leading to maximal transcription of the lac genes only when necessary (i.e., in the presence of lactose and absence of glucose). Let&rsquos look at some of the classic experiments that led to our understanding of E. coli gene regulation in general, and of the lac operon in particular.

In the late 1950s and early 1960s, Francois Jacob and Jacques Monod were studying the use of different sugars as carbon sources by E. coli. They knew that wild type E. coli would not make the (eta )-galactosidase, (eta )-galactoside permease or (eta )-galactoside transacetylase proteins when grown on glucose. Of course, they also knew that the cells would switch to lactose for growth and reproduction if they were deprived of glucose! They then searched for and isolated different E. coli mutants that could not grow on lactose, even when there was no glucose in the growth medium. Here are some of the mutants they studied:

  1. One mutant failed to make active (eta )-galactosidase enzyme but made permease.
  2. One mutant failed to make active permease but made normal amounts of (eta )-galactosidase.
  3. Another mutant failed to make transacetylase. but could still metabolize lactose in the absence of glucose. Hence the uncertainty of its role in lactose metabolism.
  4. Curiosly, one mutant strain failed to make any of the three enzymes!

Since double mutants are very rare and triple mutants even rarer, Jacob and Monod inferred that the activation of all three genes in the presence of lactose were controlled together in some way. In fact, it was this discovery that defined the operon as a set of genes transcribed as a single mRNA, whose expression could therefore be effectively coordinated. They later characterized the repressor protein produced by the lacI gene. Jacob, Monod and Andre Lwoff shared the Nobel Prize in Medicine in 1965 for their work on bacterial gene regulation. We now know that negative and positive regulation of the lac operon (described below) depend on two regulatory proteins that together, control the rate of lactose metabolism.

1. Negative Regulation of the Lac Operon by Lactose

Refer to the illustration below to identify the players in lac operon derepression.

The repressor protein product of the I gene is always made and present in E. coli cells. I gene expression is not regulated! In the absence of lactose in the growth medium, the repressor protein binds tightly to the operator DNA. While RNA polymerase is bound to the promoter and ready to transcribe the operon, the presence of the repressor bound to the operator sequence close to the Z gene physically blocks its forward movement. Under these conditions, little or no transcript is made. If cells are grown in the presence of lactose, the lactose entering the cells is converted to allolactose. Allolactose binds to the repressor sitting on the operator DNA to form a 2-part complex, as shown below.

The allosterically altered repressor dissociates from the operator and RNA polymerase can transcribe the lac operon genes as illustrated below

2. Positive Regulation of the Lac Operon Induction by Catabolite Activation

The second control mechanism regulating lac operon expression is mediated by CAP (cAMP-bound catabolite activator protein or cAMP receptor protein). When glucose is available, cellular levels of cAMP are low in the cells and CAP is in an inactive conformation. On the other hand, if glucose levels are low, cAMP levels rise and bind to the CAP, activating it. If lactose levels are also low, the cAMP-bound CAP will have no effect. If lactose is present and glucose levels are low, then allolactose binds the lac repressor causing it to dissociate from the operator region. Under these conditions, the cAMP-bound CAP can bind to the operator in lieu of the repressor protein. In this case, rather than blocking the RNA polymerase, the activated Camp-bound CAP induces even more efficient lac operon transcription. The result is synthesis of higher levels of lac enzymes that facilitate efficient cellular use of lactose as an alternative to glucose as an energy source. Maximal activation of the lac operon in high lactose and low glucose is shown below.

cAMP-bound CAP is an inducer of transcription. It does this by forcing the DNA in the promoter-operator region to bend. And since bending the double helix loosens H-bonds, it becomes easier for RNA polymerase to find and bind the promoter on the DNA strand to be transcribed&hellip, and for transcription to begin. cAMP-CAPinduced bending of DNA is illustrated below.

3. Lac Operon Regulation by Inducer Exclusion and Multiple Operators

In recent years, additional layers of lac operon regulation have been uncovered. In one case, the ability of lac permease to transport lactose across the cell membrane is regulated. In another, additional operator sequences have been discovered to interact with a multimeric repressor to control lac gene expression.

A) Regulation of Lactose use by Inducer Exclusion

When glucose levels are high (even in the presence of lactose), phosphate is consumed to phosphorylate glycolytic intermediates, keeping cytoplasmic phosphate levels low. Under these conditions, unphosphorylated EIIAGlc binds to the lactose permease enzyme in the cell membrane, preventing it from bringing lactose into the cell.

The role of phosphorylated and unphosphorylated EIIA Glc in regulating the lac operon are shown below.

High glucose levels block lactose entry into the cells, effectively preventing allolactose formation and the derepression of the lac operon. Inducer exclusion is thus a logical way for the cells to handle an abundance of glucose, whether or not lactose is present. On the other hand, if glucose levels are low in the growth medium, phosphate concentrations in the cells rise sufficiently for a specific kinase to phosphorylate the EIIAGlc. Phosphorylated EIIAGlc then undergoes an allosteric change and dissociates from the lactose permease, making it active so that more lactose can enter the cell. In other words, the inducer is not &ldquoexcluded&rdquo under these conditions!

The kinase that phosphorylates EIIA Glc is part of a phosphoenolpyruvate (PEP)- dependent phosphotransferase system (PTS) cascade. When extracellular glucose levels are low, the cell activates the PTS system in an effort to bring whatever glucose is around into the cell. But the last enzyme in the PTS phosphorylation cascade is the kinase that phosphorylates EIIA Glc . Phosphorylated EIIA Glc dissociates from the lactose permease, re-activating it, bringing available lactose into the cell from the medium.

B) Repressor Protein Structure and Additional Operator Sequences

The lac repressor is a tetramer of identical subunits (below).

Each subunit contains a helix-turn-helix motif capable of binding to DNA. However, the operator DNA sequence downstream of the promoter in the operon consists of a pair of inverted repeats spaced apart in such a way that they can only interact two of the repressor subunits, leaving the function of the other two subunits unknown&hellip that is, until recently!

Two more operator regions were recently characterized in the lac operon. One, called O2, is within the lac z gene itself and the other, called O3, lies near the end of, but within the lac I gene. Apart from their unusual location within actual genes, these operators, which interact with the remaining two repressor subunits, went undetected at first because mutations in the O2 or the O3 region individually do not contribute substantially to the effect of lactose in derepressing the lac operon. Only mutating both regions at the same time results in a substantial reduction in binding of the repressor to the operon.

B. Mechanism of Control of the Tryptophan Operon

If ample tryptophan (trp) is available, the tryptophan synthesis pathway can be inhibited in two ways. First, recall how feedback inhibition by excess trp can allosterically inhibit the trp synthesis pathway. A rapid response occurs when tryptophan is present in excess, resulting in rapid feedback inhibition by blocking the first of five enzymes in the trp synthesis pathway. The trp operon encodes polypeptides that make up two of these enzymes.

Enzyme 1 is a multimeric protein, made from polypeptides encoded by the trp5 and trp4 genes. The trp1 and trp2 gene products make up Enzyme 3. If cellular tryptophan levels drop because the amino acid is rapidly consumed (e.g., due to demands for proteins during rapid growth), E. coli cells will continue to synthesize the amino acid, as illustrated below.

On the other hand, if tryptophan consumption slows down, tryptophan accumulates in the cytoplasm. Excess tryptophan will bind to the trp repressor. The trp-bound repressor then binds to the trp operator, blocking RNA polymerase from transcribing the operon. The repression of the trp operon by trp is shown below.

In this scenario, tryptophan is a co-repressor. The function of a co-repressor is to bind to a repressor protein and change its conformation so that it can bind to the operator.


Components of Lac-Operon

An operon primarily consists of two elements or genes:

Regulatory Elements

It includes the following regions:

  • Promotor Region: It codes the Lac-P gene. It lies between the regulator and the operator. RNA-polymerase binds to this site, as a promoter region initiates transcription. It is 100 base pairs long. It consists of palindromic sequences. This site promotes and controls the transcription of structural genes or m-RNA. The regulatory genes of the repressor regulate the functioning of the promoter region.
  • Operator Region: It codes the Lac-O gene. It lies between a promoter and the structural gene (Lac-Z). It contains an operator switch, which decides whether transcription should take place or not. The regulatory gene binds to the operator.
  • Regulator Region: It codes for regulator gene (Lac-I) that controls the activity of promotor and an operator gene. This regulatory gene produces regulatory proteins known as “Repressor proteins” that can bind to the promoter and operator.

Structural Elements

These are the regions of DNA, which contain genes for the protein synthesis, and they are of three kinds:

  • Lac-Z: Encodes for the enzyme beta-galactosidase.
    Function: Beta-galactosidase brings about the hydrolysis of lactose into galactose and glucose subunits.
  • Lac-Y: Encodes for the enzyme lactose permease.
    Function: Lactose permease brings lactose into the cell.
  • Lac-A: Encodes for the enzyme thiogalactoside transacetylase.
    Function: Thiogalactoside transacetylase function is not very clear, but it assists the activity of an enzyme beta-galactosidase.

These three, i.e. Lac Z, Y and A genes, are present adjacent to each other. Therefore, all the elements like promotor, operator, repressor and structural genes together form a unit called Operon.

Control of Gene Expression in Prokaryotes

In prokaryotes, the Lac-operon system is controlled by two ways:

Positive Control of Lac-Operon

It is also called Positive inducible system and includes the following steps:

  1. Firstly, a regulatory gene expresses the repressor protein.
  2. After that, repressor proteins are produced by the expression of a regulatory gene.
  3. A repressor protein has binding sites for the operator and the inducer (lactose).
  4. When lactose is present as an inducer, it binds with the repressor protein and forms R+I complex.
  5. After the binding of inducer to the repressor, the complex blocks the binding of the repressor to the operator.
  6. As the repressor protein does not block the operator, the RNA polymerase binds to the promotor and moves further to transcribe mRNA.

This concept is known as switch on of Lac-operon (by the presence of an inducer).

Negative Control of Lac-Operon

It is also called Negative control of repressor system. It includes the following steps:

  1. First, the regulatory gene is expressed by the repressor.
  2. A repressor protein is produced after the expression of a regulatory gene.
  3. In the absence of inducer or lactose, the repressor protein directly binds to an operator.
  4. This blocks the movement of RNA polymerase and its attachment to the promoter.
  5. At last, mRNA transcription will not occur.

This concept is known as switch off of Lac-operon (by the absence of an inducer).


How can a continuous RNA be transcribed in the lac operon? - Biology

58 notecards = 15 pages ( 4 cards per page)

Campbell Biology Chapter 18: Regulation of Gene Expression

1) Which of the following is a protein produced by a regulatory gene?
A) operon
B) inducer
C) promoter
D) repressor

2) A lack of which molecule would result in a cell's inability to "turn off" genes?
A) operon
B) inducer
C) promoter
D) corepressor

3) Which of the following, when taken up by a cell, binds to a repressor so that the repressor no longer binds to the operator?

4) Most repressor proteins are allosteric. Which of the following binds with the repressor to alter its conformation?

5) A mutation that inactivates a regulatory gene of a repressible operon in an E. coli cell would result in _____.

A) continuous transcription of the structural gene controlled by that regulator

B) complete inhibition of transcription of the structural gene controlled by that regulator

C) irreversible binding of the repressor to the operator

D) continuous translation of the mRNA because of alteration of its structure

A) continuous transcription of the structural gene controlled by that regulator

6) The lactose operon is likely to be transcribed when _____.

A) there is more glucose in the cell than lactose

B) there is glucose but no lactose in the cell

C) the cyclic AMP and lactose levels are both high within the cell

D) the cAMP level is high and the lactose level is low

C) the cyclic AMP and lactose levels are both high within the cell

7) Transcription of structural genes in an inducible operon _____.

A) occurs continuously in the cell

B) starts when the pathway's substrate is present

C) starts when the pathway's product is present

D) stops when the pathway's product is present

B) starts when the pathway's substrate is present

8) For a repressible operon to be transcribed, which of the following must occur?

A) A corepressor must be present.

B) RNA polymerase and the active repressor must be present.

C) RNA polymerase must bind to the promoter, and the repressor must be inactive.

D) RNA polymerase must not occupy the promoter, and the repressor must be inactive.

C) RNA polymerase must bind to the promoter, and the repressor must be inactive.

9) Altering patterns of gene expression in prokaryotes would most likely serve an organism's survival by _____.

A) organizing gene expression, so that genes are expressed in a given order

B) allowing each gene to be expressed an equal number of times

C) allowing an organism to adjust to changes in environmental conditions

D) allowing environmental changes to alter a prokaryote's genome

C) allowing an organism to adjust to changes in environmental conditions

10) In positive control of several sugar-metabolism-related operons, the catabolite activator protein (CAP) binds to DNA to stimulate transcription. What causes an increase in CAP activity in stimulating transcription?

A) an increase in glucose and an increase in cAMP

B) a decrease in glucose and an increase in cAMP

C) an increase in glucose and a decrease in cAMP

D) a decrease in glucose and a decrease in the repressor

B) a decrease in glucose and an increase in cAMP

11) There is a mutation in the repressor that results in a molecule known as a super-repressor because it represses the lac operon permanently. Which of these would characterize such a mutant?

A) It cannot bind to the operator.

B) It cannot make a functional repressor.

C) It cannot bind to the inducer.

D) It makes a repressor that binds CAP

C) It cannot bind to the inducer.

12) If she moves the promoter for the lac operon to the region between the beta galactosidase (lacZ) gene and the permease (lacY) gene, which of the following would be likely?

A) The three structural genes will be expressed normally.

B) RNA polymerase will no longer transcribe permease.

C) The operon will still transcribe the lacZ and lacY genes, but the mRNA will not be translated.

D) Beta galactosidase will not be produced.

D) Beta galactosidase will not be produced.

13) If she moves the operator to the far end of the operon, past the transacetylase (lacA) gene, which of the following would likely occur when the cell is exposed to lactose?

A) The inducer will no longer bind to the repressor.

B) The repressor will no longer bind to the operator.

C) The operon will never be transcribed.

D) The structural genes will be transcribed continuously.

D) The structural genes will be transcribed continuously.

14) If she moves the repressor gene (lacI), along with its promoter, to a position at some several thousand base pairs away from its normal position, we would expect the _____.

A) repressor will no longer bind to the operator

B) repressor will no longer bind to the inducer

C) lac operon will be expressed continuously

D) lac operon will function normally

D) lac operon will function normally

15) What would occur if the repressor of an inducible operon were mutated so that it could not bind the operator?

A) irreversible binding of the repressor to the promoter

B) reduced transcription of the operon's genes

C) continuous transcription of the operon's genes

D) overproduction of catabolite activator protein (CAP)

C) continuous transcription of the operon's genes

16) According to the lac operon model proposed by Jacob and Monod, what is predicted to occur if the operator is removed from the operon?

A) The lac operon would be transcribed continuously.

B) Only lacZ would be transcribed.

C) Only lacY would be transcribed.

D) Galactosidase permease would be produced, but would be incapable of transporting lactose.

A) The lac operon would be transcribed continuously.

17) The trp repressor blocks transcription of the trp operon when the repressor _____.

C) is not bound to tryptophan

D) is not bound to the operator

18) Extracellular glucose inhibits transcription of the lac operon by _____.

A) strengthening the binding of the repressor to the operator

B) weakening the binding of the repressor to the operator

C) inhibiting RNA polymerase from opening the strands of DNA to initiate transcription

D) reducing the levels of intracellular cAMP

D) reducing the levels of intracellular cAMP

19) CAP is said to be responsible for positive regulation of the lac operon because _____.

B) CAP binds to the CAP-binding site

C) CAP prevents binding of the repressor to the operator

D) CAP bound to the CAP-binding site increases the frequency of transcription initiation

D) CAP bound to the CAP-binding site increases the frequency of transcription initiation

20) Imagine that you've isolated a yeast mutant that contains histones resistant to acetylation. What phenotype do you predict for this mutant?

A) The mutant will grow rapidly.

B) The mutant will require galactose for growth.

C) The mutant will show low levels of gene expression.

D) The mutant will show high levels of gene expression

C) The mutant will show low levels of gene expression.

21) The primary difference between enhancers and promoter-proximal elements is that enhancers _____.
A) are transcription factors promoter-proximal elements are DNA sequences
B) enhance transcription promoter-proximal elements inhibit transcription
C) are at considerable distances from the promoter promoter-proximal elements are close to the promoter
D) are DNA sequences promoter-proximal elements are proteins

C) are at considerable distances from the promoter promoter-proximal elements are close to the promoter

22) The reason for differences in the sets of proteins expressed in a nerve and a pancreatic cell of the same individual is that nerve and pancreatic cells contain different _____.

C) sets of regulatory proteins

C) sets of regulatory proteins

23) Gene expression is often assayed by measuring the level of mRNA produced from a gene. If one is interested in knowing the amount of a final active gene product, a potential problem of this method is that it ignores the possibility of _____.
A) chromatin condensation control
B) transcriptional control
C) alternative splicing
D) translational control

24) Not long ago, it was believed that a count of the number of protein-coding genes would provide a count of the number of proteins produced in any given eukaryotic species. This is incorrect, largely due to the discovery of widespread _____.

A) chromatin condensation control

B) transcriptional control

25) One way to detect alternative splicing of transcripts from a given gene is to _____.

A) compare the DNA sequence of the given gene to that of a similar gene in a related organism

B) measure the relative rates of transcription of the given gene compared to that of a gene known to be constitutively spliced

C) compare the sequences of different primary transcripts made from the given gene

D) compare the sequences of different mRNAs made from the given gene

D) compare the sequences of different mRNAs made from the given gene

26) Which of the following mechanisms is (are) used to coordinate the expression of multiple, related genes in eukaryotic cells?

A) Environmental signals enter the cell and bind directly to promoters.

B) The genes share a single common enhancer, which allows appropriate activators to turn on their transcription at the same time.

C) The genes are organized into a large operon, allowing them to be coordinately controlled as a single unit.

D) A single repressor is able to turn off several related genes.

B) The genes share a single common enhancer, which allows appropriate activators to turn on their transcription at the same time.

27) DNA methylation and histone acetylation are examples of _____.

B) chromosomal rearrangements

28) In eukaryotes, general transcription factors _____

A) bind to other proteins or to the TATA box

B) inhibit RNA polymerase binding to the promoter and begin transcribing

C) usually lead to a high level of transcription even without additional specific transcription factors

D) bind to sequences just after the start site of transcription

A) bind to other proteins or to the TATA box

29) Steroid hormones produce their effects in cells by _____.

A) activating key enzymes in metabolic pathways

B) activating translation of certain mRNAs

C) promoting the degradation of specific mRNAs

D) binding to intracellular receptors and promoting transcription of specific genes

D) binding to intracellular receptors and promoting transcription of specific genes

30) Which of the following is most likely to have a small protein called ubiquitin attached to it?

A) a cyclin protein, that usually acts in G1, in a cell that is in G2

B) a cell surface protein that requires transport from the ER

C) an mRNA leaving the nucleus to be translated

D) an mRNA produced by an egg cell that will be retained until after fertilization

A) a cyclin protein, that usually acts in G1, in a cell that is in G2

Use this information to answer the question(s) below.

A researcher found a method she could use to manipulate and quantify phosphorylation and methylation in embryonic cells in culture.

31) In one set of experiments she succeeded in increasing acetlylation of histone tails. Which of the following results would she most likely see?

A) increased chromatin condensation

B) decreased chromatin condensation

C) decreased binding of transcription factors

D) inactivation of the selected genes

B) decreased chromatin condensation

Use this information to answer the question(s) below.

A researcher found a method she could use to manipulate and quantify phosphorylation and methylation in embryonic cells in culture.

32) One of her colleagues suggested she try increased methylation of C nucleotides in the DNA of promoters of a mammalian system. Which of the following results would she most likely see?

A) decreased chromatin condensation

B) activation of histone tails for enzymatic function

C) higher levels of transcription of certain genes

D) inactivation of the selected genes

D) inactivation of the selected genes

33) Which method is utilized by eukaryotes to control their gene expression that is NOT used in bacteria?

A) control of chromatin remodeling

B) control of RNA splicing

C) transcriptional control

D) control of both RNA splicing and chromatin remodeling

D) control of both RNA splicing and chromatin remodeling

34) The phenomenon in which RNA molecules in a cell are destroyed if they have a sequence complementary to an introduced double-stranded RNA is called _____.

35) At the beginning of this century there was a general announcement regarding the sequencing of the human genome and the genomes of many other multicellular eukaryotes. Many people were surprised that the number of protein-coding sequences was much smaller than they had expected. Which of the following could account for much of the DNA that is not coding for proteins?

A) DNA that consists of histone coding sequences

B) DNA that is translated directly without being transcribed

C) non-protein-coding DNA that is transcribed into several kinds of small RNAs with biological function

D) non-protein-coding DNA that serves as binding sites for reverse transcriptase

C) non-protein-coding DNA that is transcribed into several kinds of small RNAs with biological function

36) Among the newly discovered small noncoding RNAs, one type reestablishes methylation patterns during gamete formation and blocks expression of some transposons. These are known as _____.

37) Which of the following best describes siRNA?

A) a double-stranded RNA, one of whose strands can complement and inactivate a sequence of mRNA

B) a single-stranded RNA that can, where it has internal complementary base pairs, fold into cloverleaf patterns

C) a double-stranded RNA that is formed by cleavage of hairpin loops in a larger precursor

D) a portion of rRNA that allows it to bind to several ribosomal proteins in forming large or small subunits

A) a double-stranded RNA, one of whose strands can complement and inactivate a sequence of mRNA

Use this information to answer the question(s) below.

A researcher introduces double-stranded RNA into a culture of mammalian cells and can identify its location or that of its smaller subsections experimentally, using a fluorescent probe.

38) Some time later, she finds that the introduced strand separates into single-stranded RNAs, one of which is degraded. What does this enable the remaining strand to do?

A) attach to histones in the chromatin

B) bind to complementary regions of target mRNAs

C) activate other siRNAs in the cell

D) bind to noncomplementary RNA sequences

B) bind to complementary regions of target mRNAs

Use this information to answer the question(s) below.

A researcher introduces double-stranded RNA into a culture of mammalian cells and can identify its location or that of its smaller subsections experimentally, using a fluorescent probe.

39) When she finds that the introduced strand separates into single-stranded RNAs, what other evidence of this single-stranded RNA piece's activity can she find?

A) She can measure the degradation rate of the remaining single strand.

B) The rate of accumulation of the polypeptide encoded by the target mRNA is reduced.

C) The amount of miRNA is multiplied by its replication.

D) The cell's translation ability is entirely shut down.

B) The rate of accumulation of the polypeptide encoded by the target mRNA is reduced.

40) The fact that plants can be cloned from somatic cells demonstrates that _____.

A) differentiated cells retain all the genes of the zygote

B) genes are lost during differentiation

C) the differentiated state is normally very unstable

D) differentiation does not occur in plants

A) differentiated cells retain all the genes of the zygote

41) Your brother has just purchased a new plastic model airplane. He places all the parts on the table in approximately the positions in which they will be located when the model is complete. His actions are analogous to which process in development?


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.


Watch the video: F215 OCR A2 Biology - Lac Operon (May 2022).