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23.1: Gene regulation: Bacterial - Biology

23.1: Gene regulation: Bacterial - Biology


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Examples of Bacterial Gene Regulation

This section describes two examples of transcriptional regulation in bacteria. Be on the lookout in class, in discussion, and in the study-guides for extensions of these ideas and use these to explain the regulatory mechanisms used for regulating other genes.

Gene Regulation Examples in E. coli

The DNA of bacteria and archaea are usually organized into one or more circular chromosomes in the cytoplasm. The dense aggregate of DNA that can be seen in electron micrographs is called the nucleoid. In bacteria and archaea, genes, whose expression needs to be tightly coordinated (e.g. genes encoding proteins that are involved in the same biochemical pathway) are often grouped closely together in the genome. When the expression of multiple genes is controlled by the same promoter and a single transcript is produced these expression units are called operons. For example, in the bacterium Escherschia coli all of the genes needed to utilize lactose are encoded next to one another in the genome. This arrangement is called the lactose (or lac) operon. It is often the case in bacteria and archaea that nearly 50% of all genes are encoded into operons of two or more genes.

The Role of the Promoter

The first level of control of gene expression is at the promoter itself. Some promoters recruit RNA polymerase and turn those DNA-protein binding events into transcripts more efficiently than other promoters. This intrinsic property of a promoter, it's ability to produce transcript at a particular rate, is referred to as promoter strength. The stronger the promoter, the more RNA is made in any given time period. Promoter strength can be "tuned" by Nature in very small or very large steps by changing the nucleotide sequence the promoter (e.g. mutating the promoter). This results in families of promoters with different strengths that can be used to control the maximum rate of gene expression for certain genes.

UC Davis Undergraduate Connection:

A group of UC Davis students interested in synthetic biology used this idea to create synthetic promoter libraries for engineering microbes as part of their design project for the 2011 iGEM competition.

Example #1: Trp Operon

Logic for regulating tryptophan biosynthesis

E. coli, like all organisms, needs to either synthesize or consume amino acids to survive. The amino acid tryptophan is one such amino acid. E. colican either import tryptophan from the environment (eating what it can scavenge from the world around it) or synthesize tryptophan de novo using enzymes that are encoded by five genes. These five genes are encoded next to each other in the E. coli genome into what is called the tryptophan (trp) operon (Figure below). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when environmental tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized. See the figure and paragraphs below for a mechanistic explanation.

Organization of the trp operon

Five genomic regions encoding tryptophan biosynthesis enzymes are arranged sequentially on the chromosome and are under the control of a single promoter - they are organized into an operon. Just before the coding region is the transcriptional start site. This is, as the name implies, the location where the RNA polymerase starts a new transcript. The promoter sequence is further upstream of the transcriptional start site.

A DNA sequence called an "operator" is also encoded between the promoter and the first trp coding gene. This operator is the DNA sequence to which the transcription factor protein will bind.

A few more details regarding TF binding sites

It should be noted that the use of the term "operator" is limited to just a few regulatory systems and almost always refers to the binding site for a negatively acting transcription factor. Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory proteins allowing them to perform their appropriate function (e.g. repress or activate transcription). This theme will be repeated universally across biology whether the "operator" term is used or not.

Moreover, while the specific examples you will be show depict TF binding sites in their known locations, these locations are not universal to all systems. Transcription factor binding sites can vary in location relative to the promoter. There are some patterns (e.g. positive regulators are often upstream of the promoter and negative regulators bind downstream), but these generalizations are not true for all cases. Again, the key thing to remember is that transcription factors (both positive and negatively acting) have binding sites with which they interact to help regulate the initiation of transcription by RNA polymerase.

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 to the transcription factor and allow the TF-tryptophan complex to bind at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes. When tryptophan is absent, the transcription factor does not bind to the operator and the genes are transcribed.
Attribution: Marc T. Facciotti (own work)

Regulation of the trp operon

When tryptophan is present in the cell: two tryptophan molecules bind to the trp repressor protein. When tryptophan binds to the transcription factor it causes a conformational change in the protein which now allows the TF-tryptophan complex to bind to the trp operator sequence. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes. When tryptophan is not present in the cell, the transcription factor does not bind to the operator; therefore, the transcription proceeds, the tryptophan utilization genes are transcribed and translated, and tryptophan is thus synthesized.

Since the transcription factor 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.

Suggested discussion

Do you think that the constitutive expression levels of the trp operon are high or low? Why?

Suggestion discussion

Suppose nature took a different approach to regulating the trp operon. Design a method for regulating the expression of the trp operon with a positive regulator instead of a negative regulator. (hint: we ask this kind of question all of the time on exams)

External link

Watch this video to learn more about the trp operon.

Example #2: The lac operon

Rationale for studying the lac operon

In this example, we examine the regulation of genes encoding proteins whose physiological role is to import and assimilate the disaccharide lactose, the lac operon. The story of the regulation of lac operon is a common example used in many introductory biology classes to illustrate basic principles of inducible gene regulation. We choose to describe this example second because it is, in our estimation, more complicated than the previous example involving the activity of a single negatively acting transcription factor. By contrast, the regulation of the lac operon is, in our opinion, a wonderful example of how the coordinated activity of both positive and negative regulators around the same promoter can be used to integrate multiple different sources of cellular information to regulate the expression of genes.

As you go through this example, keep in mind the last point. For many Bis2a instructors it is more important for you to learn the lac operon story and guiding principles than it is for you to memorize the logic table presented below. When this is the case, the instructor will usually make a point to let you know. These instructors often deliberately do NOT include exam questions about the lac operon. Rather they will test you on whether you understood the fundamental principles underlying the regulatory mechanisms that you study using the lac operon example. If it's not clear what the instructor wants you should ask.

The utilization of lactose

Lactose is a disaccharide composed of the hexoses glucose and galactose. It is commonly found in high abundance in milk and some milk products. As one can imagine, the disaccharide can be an important food-stuff for microbes that are able to utilize its two hexoses. coli is able to use multiple different sugars as energy and carbon sources, including lactose and the lac operon is a structure that encodes the genes necessary to acquire and process lactose from the local environment. Lactose, however, has not been frequently encountered by E. coli during its evolution and therefore the genes of the lac operon must typically be repressed (i.e. "turned off") when lactose is absent. Driving transcription of these genes when lactose is absent would waste precious cellular energy. By contrast, when lactose is present, it would make logical sense for the genes responsible for the utilization of the sugar to be expressed (i.e. "turned on"). So far the story is very similar to that of the tryptophan operon described above.

However, there is a catch. Experiments conducted in the 1950's by Jacob and Monod clearly demonstrated that E. coli prefers to utilize all the glucose present in the environment before it begins to utilize lactose. This means that the mechanism used to decide whether or not to express the lactose utilization genes must be able to integrate two types of information (1) the concentration of glucose and (2) the concentration of lactose. While this could theoretically be accomplished in multiple ways, we will examine how the lac operon accomplishes this by using multiple transcription factors.

The transcriptional regulators of the lac operon

The lac repressor - a direct sensor of lactose

As noted, the lac operon normally has very low to no transcriptional output in the absence of lactose. This is due to two factors: (1) the constitutive promoter strength for the operon is relatively low and (2) the constant presence of the LacI repressor protein negatively influences transcription. This protein binds to the operator site near the promoter and blocks RNA polymerase from transcribing the lac operon genes. By contrast, if lactose is present, lactose will bind to the LacI protein, inducing a conformational change that prevents LacI-lactose complex from binding to its binding sites. Therefore, when lactose is present the negative regulatory LacI is not bound to the its binding site and transcription of lactose utilizing genes can proceed.

CAP protein - an indirect sensor of glucose

In E. coli, when glucose levels drop, the small molecule cyclic AMP (cAMP) begins to accumulate in the cell. cAMP is a common signaling molecule that is involved in glucose and energy metabolism in many organisms. When glucose levels decline in the cell, the increasing concentrations of cAMP allow this compound to bind to the positive transcriptional regulator called catabolite activator protein (CAP) - also referred to as CRP. cAMP-CAP complex has many sites located throughout the E. coli genome and many of these sites are located near the promoters of many operons that control the processing of various sugars.

In the lac operon, the cAMP-CAP binding site is located upstream of the promoter. Binding of cAMP-CAP to the DNA helps to recruit and retain RNA polymerase to the promoter. The increased occupancy of RNA polymerase to its promoter, in turn, results in increased transcriptional output. In this case the CAP protein is acting as a positive regulator.

Note that the CAP-cAMP complex can, in other operons, also act as a negative regulator depending upon where the binding site for CAP-cAMP complex is located relative to the RNA polymerase binding site.

Putting it all together: Inducing expression of the lac operon

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. When this condition is achieved the LacI-lactose complex dissociates the negative regulator from near the promoter, freeing the RNA polymerase to transcribe the operon's genes. Moreover, high cAMP (indirectly indicative of low glucose) levels trigger the formation of the CAP-cAMP complex. This TF-inducer pair now bind near the promoter and act to positively recruit the RNA polymerase. This added positive influence boosts transcriptional output and lactose can be efficiently utilized. The mechanistic output of other combinations of binary glucose and lactose conditions are descried in the table below and in the figure that follows.

Truth Table for 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.
Attribution: Marc T. Facciotti (own work)
Signals that Induce or Repress Transcription of the lac Operon
GlucoseCAP bindsLactoseRepressor bindsTranscription
+--+No
+-+-Some
-+-+No
-++-Yes

A more nuanced view of lac repressor function

The description of the lac repressor's function correctly describes the logic of the control mechanism used around the lac promoter. However, the molecular description of binding sites is a bit overly simplified. In reality the lac repressor has three similar, but not identical, binding sites called Operator 1, Operator 2, and Operator 3. Operator 1 is very close to the transcript start site (denoted +1). Operator 2 is located about +400nt into the coding region of the LacZ protein. Operator 3 is located about -80nt before the transcript start site (just "outside" of the CAP binding site).

The lac operon regulatory region depicting the promoter, three lac operators, and CAP binding site. The coding region for the Lac Z protein is also shown relative to the operator sequences. Note that two of the operators are in the protein coding region - there are multiple different types of information simultaneously encoded in the DNA.
Attribution: Marc T. Facciotti (own work)

The lac repressor tetramer (blue) depicted binding two operators on a strand of looped DNA (orange).
Attribution: Marc T. Facciotti (own work) - Adapted from Goodsell (https://pdb101.rcsb.org/motm/39)


Gene Regulation in Prokaryotes | Genetics

Gene regulation refers to the control of the rate or manner in which a gene is expressed. In other words, gene regulation is the process by which the cell determines (through interactions among DNA, RNA, proteins, and other substances) when and where genes will be activated and how much gene product will be produced.

Thus, the gene expression is controlled by a complex of numerous regulatory genes and regulatory proteins. The gene regulation has been studied in both prokaryotes and eukaryotes.In prokaryotes, the operon model of gene regulation is widely accepted.

This model of gene regulation was proposed by Jacob and Monod in 1961 for which they were awarded Nobel Prize in 1965. The operon refers to a group of closely linked, genes which together code for various enzymes of a particular biochemical pathway.

In other words, operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in-DNA recognized by regulator gene product(s). Thus operon is a model which explains the on-off mechanism of protein synthesis in a systematic manner. The main points of operon model of gene regulation are presented below.

(i) Developed By:

In prokaryotes, the operon model of gene regulation was developed by Jacob and Monod in 1961 for which they were awarded Nobel prize in 1965. Now this model of gene regulation is widely accepted.

(ii) Organism Used:

The operon model was developed working with lactose region [lac region] of human intestine bacteria E. coli. The gene regulation was studied for degradation of the sugar lactose.

(iii) Genes Involved:

In the operon model of gene regulation, four types of genes viz:

(iv) Regulator gene are involved.

In addition, repressor, co-repressor, and inducer molecules are also involved.

(iv) Enzymes Involved:

Four types of enzymes are involved in gene regulation of prokaryotes. These are beta-galactosidase, galactosidase permease, transacetylase and RNA polymerase. The beta-galactosidase catalyses the breakdown of lactose into glucose and galactose.

The galactosidase permease permits entry of lactose from the medium into the bacterial cell. The enzyme transacetylase transfers an acetyl group from acetyl co-enzyme A to beta galactosidase. The enzyme mRNA polymerase controls on-off of the transcription.

Types of Operon in Gene Regulation:

In prokaryotes, operons are of two types, viz., inducible and repressible. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism. An example of repressible operon is the Trp operon, which encodes enzymes responsible for the synthesis of the amino acid tryptophan (trp for short).

A. Inducible Operon:

An enzyme whose production is enhanced by adding the substrate in the culture medium is called inducible enzyme, and such system is called inducible system. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism.

In bacteria, operon refers to a group of closely linked genes which act together and code for the various enzymes of a particular biochemical pathway.

The model of lac operon of E. coli looks like this:

There are three structural genes of the lac operon i.e. lac Z, lac Y and lac A. The main function of structural genes is to control of protein synthesis through messenger RNA. Function of these genes is as follows.

It encodes the enzyme beta-galactosidase, which catalyses the breakdown of lactose into glucose and galactose.

It encodes the enzyme galactosidase permease, which permits entry of lactose from the medium into the bacterial cell.

It encodes the enzyme transacetylase, which transfers an acetyl group from acetyl co-enzyme A to beta galactosidase.

The above three structural genes genes are under the control of the promoter gene [designated P]. In the promoter, RNA polymerase binds to the DNA and prepares to initiate transcription. The main function of promoter gene is to initiate mRNS transcription.

The other regulatory element in an operon is the operator (designated O). This is the element that determines whether or not the genes of the operon are transcribed. The main function of operator gene is to control function of structural genes.

This is designated as I. It is expressed all the time, or constitutively and plays an important role in operon function. This is the lac I gene, which encodes a protein called the lac repressor. The lac repressor has two functional domains or regions: one that binds to the DNA of the operator region, and one that binds to lactose.

When the repressor binds to the operator, it prevents RNA polymerase advancing along the operon, and transcription does not occur. The regulation of the operon depends on regulating whether or not the repressor binds to the operator. The function of regulator gene is to direct synthesis of repressor, a protein molecule. Its function differs in the presence and absence of lactose as discussed below.

When Lactose is absent:

When the lactose is absent in the environment, events take place in this way. The lac I gene is transcribed [constitutively i.e. continuously] and the mRNA is translated, producing the lac repressor. The repressor binds to the operator, and blocks RNA polymerase.

When RNA polymerase is blocked, there is no transcription. Thus the enzymes for lactose metabolism are not synthesized, because there is no lactose to metabolize. Thus when lactose is absent, lactose-metabolizing enzymes are not produced.

When Lactose is Present:

When the lactose is present in the environment, the events occur in a different way. A small amount of the lactose enters into the cell and affects regulation of the operon. The lac repressor is still synthesized. The repressor can bind to lactose.

After binding to lactose, the repressor undergoes a conformational change (change of shape). Molecules that change shape when they bind to another molecule are called allosteric molecules. With this change, the lac repressor is unable to bind to the operator region. Hence RNA polymerase is not blocked, and is able to transcribe the genes of the operon.

The enzymes encoded by those genes are produced. The lac permease transports more lactose into the cell and beta-galactosidase cleaves the lactose into glucose and galactose. This can be further metabolized by other enzymes, producing energy for the cell.

Lactose, therefore, is able to induce the synthesis of the enzymes necessary for its metabolism (by preventing the action of the repressor). As such, lactose is the inducer of the lac operon. Thus when lactose is absent, lactose-metabolizing enzymes are not produced, and when lactose is present, those enzymes are produced.

Mutations of the Lac Operon:

Mutations can affect the regulation of the lac operon in different ways as given below:

(i) Mutation of the lac I gene in such a way that the repressor encoded no longer binds to lactose. In this case, the repressor would bind to the operator regardless of the presence or absence of lactose, and the operon would never be transcribed at high levels.

(ii) Mutation of the lac I gene in such a way that the repressor no longer binds to the operator. In this case, the operon would never be repressed, and transcription would be carried out continuously. This is known as constitutive transcription.

(iii) Mutation in the operator region in such a way that the wild-type repressor does not recognize it (the repressor recognizes the specific DNA sequence of the operator legion): In this case, there will be no binding of the repressor to the operator, and transcription will go on continuously.

Catabolite Repression:

Expression of the lac operon can also be regulated in another way. Glucose is preferable to lactose as an energy source. Hence if glucose is present in the environment, the transcription is reduced or lac operon is down-regulated.

Transcription of the lac operon requires another protein, called catabolite activator protein (CAP for short). This CAP protein binds to the lac promoter and enhances transcription. But it occurs only after CAP binds to a small molecule called cyclic AMP (cAMP).

Without cAMP, CAP will not bind to the promoter, and no transcription will occur. In the previous examples involving the lac operon, we can assume that cAMP was present, and the CAP-cAMP complex was bound to the promoter.

The cAMP is produced by an enzyme called adenyl-cyclase. In the presence of glucose in the environment, adenyl-cyclase is inhibited, and cAMP production drops. Thus there is no cAMP to bind to CAP. In this situation, the CAP will not bind to the lac promoter, and no lac transcription takes place.

In this way, the bacterium does not produce enzymes for lactose metabolism when they are not necessary because of the presence of glucose. Beta-galactosidase breaks lactose in to glucose and galactose. When enough lactose has been metabolized, glucose (one of the products) accumulates and causes repression of the lac operon.

Merits of Operon Model in Gene Regulation:

1. It is a very simple yet informative model of gene regulation in prokaryotes.

2. It is a very well understood model of gene regulation in prokaryotes.

3. This model is based on empirical results and has been studied on different prokaryotes.

4. This model is of two types, viz:

B. Repressible Operon:

A protein molecule which prevents transcription is called repressor and the process of inhibition of transcription is called repression. Repressible operons are regulated by the end product of the metabolic pathway and not by a reactant in the metabolic pathway (such as lactose in lac operon).

An example of repressible operon is the Trp operon. This encodes enzymes which are responsible for the synthesis of the amino acid tryptophan (trp for short). The trp operon is regulated by trp, which is the product of the metabolic pathway.

In trp operon, the trp repressor only binds to the operator when trp is present, (opposite to the lac repressor). The repressor binds to trp, and undergoes a conformational change [change of shape]. This change in shape allows it to bind to the operator, blocking transcription. Because trp is needed for repression, it is referred to as a co-repressor in this system (as opposed to lactose being an inducer).

When trp is absent, the repressor will not bind to the operator, and transcription occurs. Thus, if there is plenty of trp around [and no more is needed], the transcription is blocked. If there is no trp around [it needs to be synthesized], transcription occurs. In other words, it allows production of the enzymes for trp synthesis.

Repressible operons are organized in much the same way as inducible operons: there are structural genes under the control of a promoter and operator, and there is a gene encoding a repressor.

The mutation will affect the gene regulation as follows:

(i) mutation in the repressor gene in such a way that it no longer binds trp When repressor does not bind trp, there will be no change in its structure and it will not bind with operator and transcription will occur.

(ii) mutation in the repressor gene in such a way that it no longer binds the repressor: In such situation transcription will take place.

(iii) mutation in the operator in such a way that it no longer binds to the repressor: In such situation also transcription will occur.

Mechanism of Gene Regulation:

The mechanism of gene regulation is of two types, viz:

(1) Negative regulation, and

The mechanism of gene regulation in E. coli operon and tryptophan operon are discussed below:

1. Negative Control:

The first switch in the lac operon of E. coli, is the repressor protein. In negative control, the transcription is controlled by repressor protein, which is an allosteric protein. The repressor protein binds to operator region and prevents transcription. It prevents transcription by blocking RNA polymerase. Thus, when repressor is bound to operator, the transcription is switched off.

Thus the on-off switch of protein synthesis is governed by free or occupied position of the operator gene. When the operator is free, transcription will take place and when the operator gene is blocked, the transcription is prevented. If an isomer of lactose [allolaptose] is present, it will bind to repressor protein and change its shape. The changed repressor does not bind to operator and thus allows transcription.

2. Positive Control:

The second switch in the lac operon of E. coli, is the catabolite activator protein [CAP].The CAP is an allosteric protein. The CAP binds to DNA and small molecule called cyclic adenosine mono phosphate [cAMP], The CAP only binds to promoter region and stimulates transcription when cAMP binds to allosteric site.

The concentration of cAMP is controlled by ATP concentrations. The low ATP leads to high cAMP and high ATP leads to low cAMP. If E. coli is growing on glucose, there will be high [ATP] & low [cAMP], If no glucose is present, there will be a low [ATP] & high [cAMP]

In the absence of glucose, [cAMP] is high, binds to CAP which binds to promoter region and stimulates transcription. If glucose is present, [cAMP] is low. doesn’t bind to CAP which cannot bind to promoter and doesn’t allow transcription.

Tryptophan Operon:

The tryptophan operon [in short trp operon] is regulated by trp, which is the product of .the metabolic pathway. The trp operon contains genes that make 5 enzymes in the biosynthetic pathway for the production of amino acid tryptophan.

In trp operon, the negative control is associated with a repressor protein. However, the repressor protein only binds with operator gene when an allosteric effector is bound to it. The tryptophan is an allosteric effector, which is called a co-repressor in trp operon also, the transcription is controlled by the free or occupied position of repressor.

If the repressor protein doesn’t bind with operator gene, transcription will take place. If tryptophan is present, there is no need to synthesize enzymes. In such situation tryptophan binds to repressor protein and both these [trp and repressor] bind to operator gene preventing transcription. When trp is absent, the repressor will not bind to the operator, and transcription will take place.

In the negative control, repressor protein binds DNA and stops transcription. In positive control, activator protein binds DNA and stimulates transcription. In the inducible system, allosteric effector binds and releases repressor protein from DNA resulting in transcription. In the repressible system, allosteric effector binds and causes repressor protein to bind to DNA preventing transcription.


Regulation of bacterial RecA protein function

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.


23.1: Gene regulation: Bacterial - Biology

Unlike other more complex organisms, bacteria are in direct contact with their environment. Sudden variations in temperature, nutrient availability or pH can be fatal and bacteria have evolved mechanisms of adaptation that allow them to survive these ever-changing conditions. What allows bacteria to adapt to the selection pressures is their ability to very rapidly express proteins and enzymes that can quickly provide protection against the harsh external environment. Proteins are encoded by genes (DNA) in the chromosome. Genes are transcribed into messenger RNAs (mRNAs), which are then read and translated by the ribosomes, which are ribonucleoproteins decoding the RNA messages. Along with this multi-step process there are some genes that do not actually code for a protein. They rather produce functional non-coding RNA molecules that fulfil various essential roles in the cell. Some non-coding RNAs, for example, act as powerful regulators of gene expression.

Small RNAs (sRNAs) have the unique role of reprogramming gene expression and reroute metabolisms in response to the environment. The main mechanism by which sRNA regulate gene expression is by directly interacting with an mRNA, interfering with, or – in some instances – facilitating the process of protein synthesis. Negative regulation occurs when a sRNA induces degradation of the mRNA and/or interferes with the translational machinery. On the other hand, positive regulation occurs when an sRNA stabilises an mRNA and/or facilitates translation initiation. In doing so, sRNAs reprogram gene expression and reroute metabolisms in response to the environment.

Although sRNAs were previously believed to present common general characteristics such as their small length or their non-coding nature, new evidence suggest that sRNAs are more versatile than anticipated. Some sRNAs even encode short regulatory peptides and, contrary to what was previously thought, sRNAs do not always originate from independent genes.

Prof Eric Massé and PhD student Marie-Claude Carrier from the University of Sherbrooke, Canada, aim to decipher the complex regulatory networks that exist between bacterial sRNAs and their targets. A better understanding of these networks will allow scientists to shed light on how bacteria survive the most challenging extracellular conditions.

When the predator becomes the prey: the role of sRNA ‘sponges’

A single sRNA can regulate a considerable number of target mRNAs, acting as a bridge between various cellular metabolisms. Almost a decade ago, in an effort to identify mRNA targets rapidly and reliably, the Massé Lab combined RNA affinity purification and RNA sequencing in a single technique called MAPS, an acronym for MS2 Affinity Purification (coupled to RNA) Sequencing. This allows the genome-wide identification of a sRNA interactome in bacterial cells.

The gut microbiome is an important risk factor for colon cancer. crystal light/Shutterstock.com

A recurring theme in the Massé Lab is the understanding of the role of a sRNA, called RyhB, in the response to iron starvation. MAPS was performed on the RyhB sRNA and lead to one of the most interesting discoveries of the Massé Lab: a new type of regulatory RNAs in Escherichia coli. The team was able to identify the role of a fragment deriving from a transfer RNA molecule. The tRNA fragment (tRF) acts as a sRNA sponge: it interacts with the RyhB sRNA and sequesters it to prevent its action. In a surprising twist, the predator becomes the prey as RyhB is degraded following its interaction with the tRF. In this case, the tRF sets a concentration threshold that RyhB needs to overcome before regulatory events can be detected. The necessity of the tRF is explained by the fact that even if RyhB is essential during iron starvation, its expression is never totally shut down, even when plenty of iron is available. If the resulting RyhB molecules were not sponged, they would regulate their targets even when iron is available, decreasing bacterial fitness. For example, unwanted RyhB increases bacterial sensitivity to a type of natural antibiotics called colicines.

One of the most interesting discoveries of the Massé Lab is the identification of a new type of regulatory RNAs in E. coli.

Navigating the sRNA web: ongoing studies

Early research on sRNA showed that most interactions with their targets led to very strong regulatory effects. However, sRNA regulation is not exclusively of the ‘all-or-nothing’ type. The Massé Lab team members are currently working on identifying subtle regulatory events that are at the base of bacterial adaptation.

As mentioned, a group of people is directing their efforts in understanding the extended network surrounding RyhB, expressed in the response to iron starvation. Other lab members are studying how sRNAs are involved in the survival to oxidative stress and how sRNAs coordinate cellular division. PhD student Marie-Claude Carrier is specifically interested in how E. coli adapts to different growth phases, particularly by studying novel regulatory mechanisms by which sRNAs modulate expression of membrane transporters.

The researchers aim to decipher the complex regulatory networks that exist between bacterial sRNAs and their targets.

Future directions in the study of sRNAs

Prof Massé and the other members of the lab are currently carrying out studies on the model non-pathogenic bacterium Escherichia coli K-12. In the future, and with the help of Prof Massé’s collaborators, the team aims to investigate the importance of sRNA subtle regulatory events in pathogenic bacteria. The team hopes to unravel the intricate molecular mechanisms behind sRNA-dependent modulation of pathogenicity and to understand how sRNAs interact with metabolic processes to coordinate the cellular responses to changes in the environmental conditions.

Towards new frontiers in cancer screening: researching the microbiome

Colorectal cancer (CRC) is the third most common cause of cancer mortality in the world. While CRC is largely preventable by removal of intestinal polyps, there is a dramatic decline in survival following tumour establishment. This is why early detection and removal of polyps at the precancerous stage is critical for patient survival. A common screening test for polyps and tumours is the immunochemical-based faecal occult blood test (iFOBT), which consists in the detection of blood in patient’s stool. However, this test is nonspecific and leads to many invasive nonessential procedures. Recent studies demonstrated that the microbiome, bacteria living in the human gut, has emerged as an important risk factor for colon cancer. Bacteria can directly foster tumorigenesis by interacting with the immune system.

The team at the Massé Lab observed significant differences in the intestinal bacterial composition that could be solely caused by the presence of blood in stools. More precisely, they identified 4 bacterial species whose abundance increased in the presence of blood and 8 species that showed decreased abundance in patients with blood in their stools.

The team published these findings in a recent paper (2020) where it is concluded that in the absence of disease, blood in the stools has a major influence on the composition of the microbiome. This suggests that blood itself should be taken into consideration when investigating the microbiome signatures of intestinal diseases.

Taking this into consideration, the team will conduct further studies with the aim of establishing the microbiome as a biomarker for colorectal cancer, including the precancerous polyp stage. The study will involve the use of hospital screenings for CRC combined with bioinformatic tools to improve prediction of most at risk patients. The Massé Lab hopes to offer a robust methodology for the diagnosis of CRC and aims to dramatically increase the quality of CRC prevention for patients participating in the early stage pilot studies.

In eukaryotic organisms, RNA-based regulation is a major determinant of gene expression, just as it is in bacterial cells. The close relatives of bacterial sRNAs are termed microRNAs (miRNAs). The sponging mechanisms observed in bacteria is also observed in eukaryotes. Indeed, in these organisms of higher complexity, sponge RNAs, sometimes called competitive inhibitors, are usually circular RNA molecules and harbour multiple binding sites for a miRNA, efficiently sequestering them from their mRNA targets. Currently, synthetic RNA sponges are designed for loss-of-function miRNA studies. MicroRNAs are known to be involved in a plethora of human pathologies, such as cancer, viral diseases, immune-related diseases and neurodegenerative diseases. Accumulating evidence suggest that synthetic miRNA sponges could hold vital clues in the fight against these serious diseases.

  • Chénard, T., Malick, M., Dubé, J., and Massé, E. (2020). The influence of blood on the human gut microbiome. BMC microbiology, 20(1), 44.
  • Carrier, M.C., Lalaouna, D., and Massé, E. (2018). Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action. Annual review of microbiology, 72, 141–161.
  • Carrier, M.C., Laliberté, G., and Massé, E. (2018). Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification Coupled to RNA Sequencing. Methods in molecular biology (Clifton, N.J.), 1737, 77–88.
  • Lalaouna, D., Carrier, M.C., Semsey, S., Brouard, J.S., Wang, J., Wade, J.T., and Massé, E. (2015). A 3’ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise. Molecular cell, 58(3), 393–405.

Research Objectives
The Eric Massé laboratory is deciphering RNA-based regulations in bacteria.

  • Natural Sciences and Engineering Research Council (NSERC)
  • Canadian Institutes of Health Research (CIHR)
  • Fonds de Recherche du Québec – Nature et Technologies (FRQNT)
  • National Institutes of Health (NIH)

Collaborators

  • Jingyi Fei (Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA)
  • Charles Dozois (Institut national de recherche scientifique (INRS)-Institut Armand Frappier, Laval, Québec, Canada)
  • Cari Vanderpool (Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA)
  • Daniel Lafontaine (Department of Biology, Faculty of Science, RNA Group, University of Sherbrooke, Sherbrooke, Québec, Canada)
  • Emanuele Biondi (Aix Marseille University, CNRS, IMM, LCB, 13009 Marseille, France)
  • Isabelle Laforest-Lapointe (Department of Biology, Faculty of Science, University of Sherbrooke, Sherbrooke, Québec, Canada

Bio
Eric Massé received his PhD from the University of Montreal in 2000. He then completed his post-doctoral training at the National Institutes of Health (Bethesda, MD, USA). In 2004, Prof Massé opened his lab at University of Sherbrooke as an Associate Professor, focusing his research on small regulatory RNAs in bacteria. Since then, he supervised multiple postdocs, PhD, and MSc students and welcomed more than 30 undergraduate trainees.

Marie-Claude Carrier received her BSc in Microbiology from the University of Sherbrooke in 2013. During her BSc studies, she completed more than 12 months of training in Prof Eric Massé’s lab. In 2014, Marie-Claude started her MSc-PhD cursus under Prof Massé’s supervision, during which she participated in multiples studies on bacterial small regulatory RNAs. She plans to obtain her PhD degree in the spring of 2021.

Contact
Prof Eric Massé
Faculté de Médecine et des Sciences de la santé – Université de Sherbrooke
Département de Biochimie et de Génomique fonctionnelle
Pavillon de Recherche Appliquée sur le Cancer (PRAC)
3201, rue Jean-Mignault
Sherbrooke, Québec, Canada
J1E 4K8

Marie-Claude Carrier
E: [email protected]
T: +1 819 821 8000 ext. 72197
W: researchgate.net/profile/Marie_Claude_Carrier


Cytokine Release Affect

The binding of PRRs with PAMPs triggers the release of cytokines, which signal that a pathogen is present and needs to be destroyed along with any infected cells. A cytokine is a chemical messenger that regulates cell differentiation (form and function), proliferation (production), and gene expression to affect immune responses. At least 40 types of cytokines exist in humans that differ in terms of the cell type that produces them, the cell type that responds to them, and the changes they produce. One type cytokine, interferon, is illustrated in Figure 23.4.

One subclass of cytokines is the interleukin (IL), so named because they mediate interactions between leukocytes (white blood cells). Interleukins are involved in bridging the innate and adaptive immune responses. In addition to being released from cells after PAMP recognition, cytokines are released by the infected cells which bind to nearby uninfected cells and induce those cells to release cytokines, which results in a cytokine burst.

A second class of early-acting cytokines is interferons, which are released by infected cells as a warning to nearby uninfected cells. One of the functions of an interferon is to inhibit viral replication. They also have other important functions, such as tumor surveillance. Interferons work by signaling neighboring uninfected cells to destroy RNA and reduce protein synthesis, signaling neighboring infected cells to undergo apoptosis (programmed cell death), and activating immune cells.

In response to interferons, uninfected cells alter their gene expression, which increases the cells’ resistance to infection. One effect of interferon-induced gene expression is a sharply reduced cellular protein synthesis. Virally infected cells produce more viruses by synthesizing large quantities of viral proteins. Thus, by reducing protein synthesis, a cell becomes resistant to viral infection.

Figure 23.4. Interferons are cytokines that are released by a cell infected with a virus. Response of neighboring cells to interferon helps stem the infection.


Gene regulation

Cells express (transcribe and translate) only a subset of their genes. Cells respond and adapt to environmental signals by turning on or off expression of appropriate genes. In multicellular organisms, cells in different tissues and organs differentiate, or become specialized by making different sets of proteins, even though all cells in the body (with a couple of exceptions) have the same genome. Such changes in gene expression, or differential gene expression among cells, are most often regulated at the level of transcription.
There are three broad levels of regulating gene expression:

  • transcriptional control (whether and how much a gene is transcribed into mRNA)
  • translational control (whether and how much an mRNA is translated into protein)
  • post-translational control (whether the protein is in an active or inactive form, and whether the protein is stable or degraded)

Based on our shared evolutionary origin, there are many similarities in the ways that prokaryotes and eukaryotes regulate gene expression however, there are also many differences. All three domains of life use positive regulation (turning on gene expression), negative regulation (turning off gene expression), and co-regulation (turning multiple genes on or off together) to control gene expression, but there are some differences in the specifics of how these jobs are carried out between prokaryotes and eukaryotes.

Similarities between prokaryotes and eukaryotes: promoters and regulatory elements

Promoters are sites in the DNA where RNA polymerase binds to initiate transcription. Promoters also contain, or have near them, binding sites for transcription factors, which are DNA-binding proteins that can either help recruit, or repel, RNA polymerase. A regulatory element is a DNA sequence that certain transcription factors recognize and bind to in order to recruit or repel RNA polymerase. The promoter along with nearby transcription factor binding elements regulate gene transcription.
Regulatory elements can be used for either positive and negative transcriptional control. When a gene is subject to positive transcriptional control, the binding of a specific transcription factor to the regulatory element promotes transcription. When a gene is subject to negative transcriptional control, the binding of a specific transcription factor to a regulator elements represses transcription. A single gene can be subject to both positive and negative transcriptional control by different transcription factors, creating multiple layers of regulation.

Some genes are not subject to regulation: they are constitutively expressed, meaning they are always transcribed. What sorts of genes would you imagine a cell would always need to have on, regardless of the environment or situation?

Differences between prokaryotes and eukaryotes: mechanisms of co-regulation

Often a set of proteins are needed together to respond to a certain stimulus or carry out a certain function (for example, many metabolic pathways). There are often mechanisms to co-regulate such genes such that they are all transcribed in response to the same stimulus. Both prokaryotic and eukaryotic cells have ways of co-regulating genes, but they use very different mechanisms to accomplish this goal.
In prokaryotes, co-regulated genes are often organized into an operon, where two or more functionally related genes are transcribed together from a single promoter into one long mRNA. This mRNA is translated to make all of the proteins encoded by the genes in the operon. Ribosomes start at the 5′ end, begin translating at the first AUG codon, terminate when they run into a stop codon, and then re-initiate at the next AUG codon.

A generic operon in prokaryotes. R = a regulatory protein (transcription factor) P = promoter Pol = RNA polymerase

With a few exceptions (C. elegans and related nematodes), eukaryotic genomes do not have genes arranged in operons. Instead, eukaryotic genes that are co-regulated tend to have the same DNA regulatory element sequence associated with each gene, even if those genes are located on completely different chromosomes. This means that the same transcriptional activator or repressor can regulate transcription of every single gene that has that particular DNA regulatory element associated with it. For example, eukaryotic HSP (heat shock protein) genes are located on different chromosomes. HSPs help cells survive and recover from heat shock (a type of cellular stress). All HSP genes are transcribed simultaneously in response to heat stress, because they all have a DNA sequence element that binds a heat shock response transcription factor.

Additional complexities specific to eukaryotic gene regulation: chromatin and alternative splicing

Another major difference between prokaryotic gene regulation and eukaryotic gene regulation is that the eukaryotic (but not prokaryotic) DNA double helix is organized around proteins called histones which organize the DNA into nucleosomes. This combination of DNA + histones is called chromatin.
Chromatin can be condensed in a 30-nm fiber formation (tightly compacted nucleosomes) or loosely arranged as “beads-on-a-string,” where the DNA between and around nucleosomes is more accessible. This compaction is controlled by post-translational modifications which are added to the histones in the nucleosomes. When histones have acetyl groups added to them by enzymes called histone acetyl transferases (HATs), the acetyl groups physically obstruct the nucleosomes from packing too densely and help to recruit other enzymes that further open the chromatin structure. Conversely, when the acetyl groups are removed by histone deacetylases (HDACs), the chromatin assumes a condensed formation that prevents transcription factors from being able to access the DNA. In the image below, you can clearly see how much more compact and inaccessible the 30-nm fiber is (top) compared to the beads-on-a-string formation (bottom).

Chromatin plays a fundamental role in positive and negative gene regulation, because transcriptional activators and RNA polymerase cannot physically access the DNA regulatory elements when chromatin is in a compact form.
Prokaryotic DNA does have some associated proteins that help to organize the genomes, but it is fundamentally different from chromatin prokaryotic DNA can essentially be thought of as ‘naked’ compared to eukaryotic chromatin, so prokaryotic cells lack this layer of gene regulation.
Another difference between prokaryotic and eukaryotic gene regulation is that eukaryotic mRNAs must be properly processed with addition of the 5′ cap, splicing out of introns, and addition of the 3′ poly(A) tail (discussed in more detail here). Each of these processing steps is also subject to regulation, and the mRNA will be degraded if any of them are not properly completed. The export of mRNAs from the nucleus to the cytoplasm is also regulated, as is stability of the properly processed mRNA in the cytoplasm.
Finally, eukaryotic genes often have different splice variants, where different exons can be included in different mRNAs that are transcribed from the same gene. Here you can see a cartoon of a gene with color-coded exons, and two different mRNA molecules transcribed from this gene. The different mRNAs encode for different proteins because they contain different exons. This process is called alternative splicing and we will discuss it more here.


Often different types of cells in different tissues express different splice variants of the same gene, such that there is a heart-specific transcript and a kidney-specific transcript of a particular gene.
In general, eukaryotic gene regulation is more complex than prokaryotic gene regulation. The upstream regulatory regions of eukaryotic genes have binding sites for multiple transcription factors, both positive regulators and negative regulators, that work in combination to determine the level of transcription. Some transcription factor binding sites, called enhancers and silencers, work at quite a distance, thousands of base pairs away from the promoter. Activators are examples of positive regulation and repressors are examples of negative regulation.

Eukaryotic transcription initiation, from biology.kenyon.edu (after Tjian)

Overall differences and similarities

If you understand the similarities and differences in eukaryotic and prokaryotic gene regulation, then you know which of the following process are exclusive to eukaryotes, which are exclusive to prokaryotes, which occur in both, and how each is accomplished:

  • coupled transcription and translation
  • 5′ cap and 3′ poly(A) tail
  • AUG as the translation initiation codon
  • regulation of gene expression by proteins binding to DNA regulatory elements
  • alternative mRNA splicing
  • regulation of gene expression through chromatin accessibility

Putting it all together: the lac operon in E. coli

The lac operon is a good model gene for understanding gene regulation. You should use the information below to make sure you can apply all of the details of gene regulation described above to a specific gene model.
E. coli lac operon: dual positive and negative regulation

lacI is the gene that encodes the lac Repressor protein CAP = catabolite activator protein O = Operator P = promoter lacZ = gene that encodes beta-galactosidase lacY encodes permease lacA encodes transacetylase. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Lac_operon-2010-21-01.png)

The lac operon of E. coli has 3 structural genes required for metabolism of lactose, a disaccharide found at high levels in milk:

  • lacZ encodes the enzyme beta-galactosidase, which cleaves lactose into glucose and galactose
  • lacY encodes permease, a membrane protein for facilitated diffusion of lactose into the cell
  • lacA encodes transacetylase, an enzyme that modifies lactose

An mRNA encoding all 3 proteins is transcribed at high levels only when lactose is present, and glucose is absent.
Negative regulation by the Repressor – In the absence of lactose, the lac Repressor protein, encoded by the lacI gene with a separate promoter that is always active, binds to the Operator sequence in the DNA. The Operator sequence is a type of DNA regulatory element as described above. Repressor protein bound to the Operator prevents RNA polymerase from initiating transcription.
When lactose is present, an inducer molecule derived from lactose binds allosterically to the Repressor, and causes the Repressor to leave the Operator site. RNA polymerase is then free to initiate transcription, if it successfully binds to the lac promoter.
Positive regulation by CAP – Glucose is the preferred substrate for energy metabolism. When glucose is present, cells transcribe the lac operon only at very low levels, so the cells obtain most of their energy from glucose metabolism. RNA polymerase by itself binds rather poorly to the lac promoter.
Glucose starvation causes a rise in the level of cyclic adenosine monophosphate (cAMP), an intracellular alarm signal. Cyclic AMP binds to the catabolite activator protein (CAP). The CAP+cAMP complex binds to the CAP binding site near the lac promoter and recruits RNA polymerase to the promoter.
High level transcription of the lac operon requires both that CAP+cAMP be bound to the CAP binding site, and that Repressor is absent from the Operator. These conditions normally occur only in the absence of glucose and presence of lactose.

The lac operon in E. coli is a classic example of a prokaryotic operon which is subject to both positive and negative regulation. Positive regulation and negative regulation are universal themes for gene regulation in both prokaryotes and eukaryotes.


5 Conclusion

Mathematical and computational modeling is often viewed as a specialized task. To facilitate modeling, we automated the development of RBMs, as these types of models show simulation flexibility, a reasonable degree of readability, modularity for integrative modeling and good simulation scalability.

Atlas produces sub-models from genome graphs, and protein–protein, protein–metabolites and protein–DNA interaction and metabolic networks. We developed, in this work, a divide-and-conquer strategy supported by the modularity of RBMs, as it is the pathway for the development of whole-cell models ( Szigeti et al., 2018). The software produces RBMs for the PySB framework ( Lopez et al., 2013) and rules can be added in any order while PySB checks on whether new rules are compatible with the current model. In addition, PySB could export to kappa language and we employed the KaSA software ( Boutillier et al., 2018) to further assess the coherence of the developed RBMs. Simulation of RBMs could be done within PySB and calibration of exported models could be performed with pyBioNetFit ( Mitra et al., 2019, only BNGL models) or Pleione ( Santibáñez et al., 2019, BNGL and kappa models) to compare the reconstructed models with experimental data or available models.

Atlas contrasts with available software because it lacks a graphical interface [e.g. RuleBender ( Smith et al., 2012) and VirtualCell ( Blinov et al., 2017)], although the user could employ Atlas within a Jupyter notebook and use pyViPR ( Ortega and Lopez, 2020) to visualize the model structure. Also, Atlas relies on the user to obtain formatted data to model interactions, in contrast to INDRA ( Gyori et al., 2017), which can use natural language processing to read information and reconstruct models. In turn, Atlas can model metabolism, transcription and translation, as well as widespread protein–protein interactions found in signaling pathways that INDRA ( Gyori et al., 2017) and KAMI ( Harmer et al., 2019) can model.

Finally, the models and the Atlas software are extensible, for instance, to model cooperative behavior not currently supported. The utilization of the law of mass action for the metabolic network (and other reactions) limits the utility of the resulting RBMs in the current form, but exporting to BNGL or kappa leverages this imposition, as they support mathematical expressions as reaction rates. However, we expect to extend Atlas to consider enzyme–metabolite interactions and describe the detailed mechanisms of enzyme reactions ( Saa and Nielsen, 2017) and allosteric regulations of metabolic activity, as well as to model the assembly of ribosomes ( Davis et al., 2016 Gupta and Culver, 2014 Shajani et al., 2011). Notably, Atlas is already compatible with metabolic and interaction data from eukaryotes and we obtained a model from data for 1991 metabolic reactions of the yeast Saccharomyces cerevisiae from BioCyc. In addition, we expect further interoperability with INDRA models of signaling pathways to model protein modifications, such as phosphorylation and the collaboration from researchers. With collaboration in mind, we shared the developed models in this work at https://github.com/networkbiolab/atlas/tree/master/examples.


23.1: Gene regulation: Bacterial - Biology

The DNA of prokaryotes is organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function 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.

In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors are proteins that suppress transcription of a gene in response to an external stimulus, whereas activators are proteins that increase the transcription of a gene in response to an external stimulus. Finally, inducers are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate.

Learning Outcomes

Understand the basic steps in gene regulation in prokaryotic cells

In bacteria and archaea, structural proteins with related functions—such as the genes that encode the enzymes that catalyze the many steps in a single biochemical pathway—are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter. This forms a polycistronic transcript (Figure 1). The promoter then has simultaneous control over the regulation of the transcription of these structural genes because they will either all be needed at the same time, or none will be needed.

Figure 1. In prokaryotes, structural genes of related function are often organized together on the genome and transcribed together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing transcription.

French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the lac operon of E. coli. They found that in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. For this work, they won the Nobel Prize in Physiology or Medicine in 1965.

Each operon includes DNA sequences that influence its own transcription these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.

Other genes in prokaryotic cells are needed all the time. These gene products will be constitutively expressed, or turned on continually. Most consitutively expressed genes are “housekeeping” genes responsible for overall maintenance of a cell.


Art Connection

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.

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 absent, then CAP can bind to the operator sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is met, then transcription remains off. Only when both conditions are satisfied is the lac operon transcribed (Table).

Signals that Induce or Repress Transcription of the lac Operon
GlucoseCAP bindsLactoseRepressor binds Transcription
+--+No
+-+-Some
-+-+No
-++-Yes


The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are three ways to control the transcription of an operon: repressive control, activator control, and inducible control. Repressive control, typified by the trp operon, uses proteins bound to the operator sequence to physically prevent the binding of RNA polymerase and the activation of transcription. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains off. Activator control, typified by the action of CAP, increases the binding ability of RNA polymerase to the promoter when CAP is bound. In this case, low levels of glucose result in the binding of cAMP to CAP. CAP then binds the promoter, which allows RNA polymerase to bind to the promoter better. In the last example—the lac operon—two conditions must be met to initiate transcription. Glucose must not be present, and lactose must be available for the lac operon to be transcribed. If glucose is absent, CAP binds to the operator. If lactose is present, the repressor protein does not bind to its operator. Only when both conditions are met will RNA polymerase bind to the promoter to induce transcription.

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.


Watch the video: Gene Regulation and the Order of the Operon (October 2022).