80 Prokaryotic Gene Regulation
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.
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 trpoperator 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.
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.
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.
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.
In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think this is the case?
<!–<para> 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|
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.
Visual Connection Questions
(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.
If glucose is absent, but so is lactose, the lac operon will be ________.
- activated, but only partially
Prokaryotic cells lack a nucleus. Therefore, the genes in prokaryotic cells are:
- all expressed, all of the time
- transcribed and translated almost simultaneously
- transcriptionally controlled because translation begins before transcription ends
- 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) ________.
Critical Thinking Questions
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.
- protein that binds to prokaryotic operators to increase transcription
- catabolite activator protein (CAP)
- protein that complexes with cAMP to bind to the promoter sequences of operons which control sugar processing when glucose is not available
- inducible operon
- operon that can be activated or repressed depending on cellular needs and the surrounding environment
- lac operon
- operon in prokaryotic cells that encodes genes required for processing and intake of lactose
- negative regulator
- protein that prevents transcription
- region of DNA outside of the promoter region that binds activators or repressors that control gene expression in prokaryotic cells
- collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells
- positive regulator
- protein that increases transcription
- protein that binds to the operator of prokaryotic genes to prevent transcription
- transcriptional start site
- site at which transcription begins
- trp operon
- series of genes necessary to synthesize tryptophan in prokaryotic cells
- amino acid that can be synthesized by prokaryotic cells when necessary
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16.1 Regulation of Gene Expression
In this section, you will explore the following question:
- How does prokaryotic gene regulation differ from eukaryotic gene regulation?
Connection for AP® Courses
Structure and function in biology result from the presence of genetic information and the correct expression of this information. In the chapter on DNA structure and function, we explored how genes are translated into proteins, which in turn determine the nature of the cell. But how does a cell know when to “turn on” its DNA? With few exceptions, each cell in your body contains identical genetic information. If each cell has the same exact DNA make up, how is it that a liver cell differs from a nerve or muscle cell?
As we will discover, although each cell shares the same genome and DNA sequence, each cell does not express exactly the same genes. Many factors determine when and how genes are expressed in a given cell. Even the type of chromosome a gene is located on, like whether it is a sex chromosome or not, can determine its expression pattern, as can mutations or changes in DNA sequence and other external factors. In prokaryotes, gene expression is regulated primarily at the level of transcription, when DNA is copied into RNA. However, eukaryotes have evolved regulatory mechanisms in gene expression at multiple levels. In all cases, regulation of gene expression determines the type and amount of protein produced in the cell. Errors in regulatory processes can result in many human diseases and conditions, including cancer.
Gene expression regulation occurs at different points in prokaryotes and eukaryotes. Prokaryotic organisms express their entire genome in every cell, but not necessarily all at the same time. In general, a gene is expressed only when its specific protein product is needed. Remember that each cell in an organism carries the same DNA as every other cell. Yet cells of eukaryotic organisms each express a unique subset of DNA depending on cell type. To express a protein, DNA is first transcribed into RNA, which is then translated into proteins. In prokaryotic cells, transcription and translation occur almost simultaneously. In eukaryotic cells, transcription occurs in the nucleus, separate from the translation that occurs in the cytoplasm along ribosomes attached to endoplasmic reticulum. As stated above, gene expression in prokaryotes is regulated at the level of transcription, whereas in eukaryotes, gene expression is regulated at multiple levels, including the epigenetic (DNA), transcriptional, pre- and post-transcriptional, and translational levels.
The science of epigenetics studies heritable changes in the genome that do not affect the underlying DNA gene sequences.
The content presented in this section supports the learning objectives outlined in Big Idea 3 of the AP® Biology Curriculum Framework. The AP® learning objectives merge essential knowledge content with one or more of the seven science practices. These objectives provide a transparent foundation for the AP® Biology course, along with inquiry-based laboratory experiences, instructional activities, and AP® exam questions.
|Big Idea 3||Living systems store, retrieve, transmit and respond to information essential to life processes.|
|Enduring Understanding 3.B||Expression of genetic information involves cellular and molecular mechanisms.|
|Essential Knowledge||3.B.1 Gene regulation results in differential gene expression, leading to cell specialization|
|Science Practice||7.1 The student can connect phenomena and models across spatial and temporal scales.|
|Learning Objective||3.18 The student is able to describe the connection between the regulation of gene expression and observed differences between different kinds of organisms.|
For a cell to function properly, necessary proteins must be synthesized at the proper time. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.
Although genetic differences between species and between individuals within a species are often responsible for phenotypic differences, another mechanism that can create phenotypic differences is differences in gene expression. For example, although every cell in an organism contains the same genes, the bone cells in the organism appears different from the fat cells due to differences in which genes are expressed by which cell. Similarly, although mice and humans share approximately 97.5% of their genes, they are very different organisms because different genes are turned on at different times during development and in different cells. Even organisms that share 100% identity in their genomes (a.k.a clones) can eventually appear different if they express their genes differently in response to different environmental conditions, for example. Even among humans, identical twins can possess different birthmarks, wrinkles, or other features that arise during development sometimes due to differential gene expression.
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases.
Ask students what genes are present in the DNA in a muscle cell and skin cell. Ask them if the same genome is present in every cell in the body, how do the cells have different properties. For example, discuss red blood cells, which lose their nucleus during development. This video gives an overview of gene regulation in prokaryotes and eukaryotes.
Prokaryotic versus Eukaryotic Gene Expression
To understand how gene expression is regulated, we must first understand how a gene codes for a functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in slightly different manners.
Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesize a protein, the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription. All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level.
Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA. The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. The processes of transcription and translation are physically separated by the nuclear membrane; transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. The regulation of gene expression can occur at all stages of the process (Figure 16.2). Regulation may occur when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors (epigenetic level), when the RNA is transcribed (transcriptional level), when the RNA is processed and exported to the cytoplasm after it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational level), or after the protein has been made (post-translational level).
Figure 16.2Prokaryotic transcription and translation occur simultaneously in the cytoplasm, and regulation occurs at the transcriptional level. Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, and during protein translation, which takes place in the cytoplasm. Further regulation may occur through post-translational modifications of proteins.
The differences in the regulation of gene expression between prokaryotes and eukaryotes are summarized in Table 16.1. The regulation of gene expression is discussed in detail in subsequent modules.
Differences in the Regulation of Gene Expression of Prokaryotic and Eukaryotic Organisms
|Prokaryotic organisms||Eukaryotic organisms|
|Lack nucleus||Contain nucleus|
|DNA is found in the cytoplasm||DNA is confined to the nuclear compartment|
|RNA transcription and protein formation occur almost simultaneously||RNA transcription occurs prior to protein formation, and it takes place in the nucleus. Translation of RNA to protein occurs in the cytoplasm.|
|Gene expression is regulated primarily at the transcriptional level||Gene expression is regulated at many levels (epigenetic, transcriptional, nuclear shuttling, post-transcriptional, translational, and post-translational)|
DNA, Genetics, and Evolution
By gene expression we mean the transcription of a gene into mRNA and its subsequent translation into protein. Gene expression is primarily controlled at the level of transcription, largely as a result of binding of proteins to specific sites on DNA. In 1965 Francois Jacob, Jacques Monod, and Andre Lwoff shared the Nobel prize in medicine for their work supporting the idea that control of enzyme levels in cells is regulated by transcription of DNA. occurs through regulation of transcription, which can be either induced or repressed. These researchers proposed that production of the enzyme is controlled by an "operon," which consists a series of related genes on the chromosome consisting of an operator, a promoter, a regulator gene, and structural genes.
- The structural genes contain the code for the proteins products that are to be produced. Regulation of protein production is largely achieved by modulating access of RNA polymerase to the structural gene being transcribed.
- The promoter gene doesn't encode anything; it is simply a DNA sequence that is initial binding site for RNA polymerase.
- The operator gene is also non-coding; it is just a DNA sequence that is the binding site for the repressor.
- The regulator gene codes for synthesis of a repressor molecule that binds to the operator and blocks RNA polymerase from transcribing the structural genes.
The operator gene is the sequence of non-transcribable DNA that is the repressor binding site. There is also a regulator gene, which codes for the synthesis of a repressor molecule hat binds to the operator
- Example of Inducible Transcription: The bacterium E. coli has three genes that encode for enzymes that enable it to split and metabolize lactose (a sugar in milk). The promoter is the site on DNA where RNA polymerase binds in order to initiate transcription. However, the enzymes are usually present in very low concentrations, because their transcription is inhibited by a repressor protein produced by a regulator gene (see the top portion of the figure below). The repressor protein binds to the operator site and inhibits transcription. However, if lactose is present in the environment, it can bind to the repressor protein and inactivate it, effectively removing the blockade and enabling transcription of the messenger RNA needed for synthesis of these genes (lower portion of the figure below).
- Example of Repressible Transcription: E. coli need the amino acid tryptophan, and the DNA in E. coli also has genes for synthesizing it. These genes generally transcribe continuously since the bacterium needs tryptophan. However, if tryptophan concentrations are high, transcription is repressed (turned off) by binding to a repressor protein and activating it as illustrated below.
Control of Gene Expression in Eukaryotes
Eukaryotic cells have similar mechanisms for control of gene expression, but they are more complex. Consider, for example, that prokaryotic cells of a given species are all the same, but most eukaryotes are multicellular organisms with many cell types, so control of gene expression is much more complicated. Not surprisingly, gene expression in eukaryotic cells is controlled by a number of complex processes which are summarized by the following list.
- After fertilization, the cells in the developing embryo become increasingly specialized, largely by turning on some genes and turning off many others. Some cells in the pancreas, for example, are specialized to synthesize and secrete digestive enzymes, while other pancreatic cells (β-cells in the islets of Langerhans) are specialized to synthesis and secrete insulin. Each type of cell has a particular pattern of expressed genes. This differentiation into specialized cells occurs largely as a result of turning off the expression of most genes in the cell; mature cells may only use 3-5% of the genes present in the cell's nucleus.
- Gene expression in eukaryotes may also be regulated through by alterations in the packing of DNA, which modulates the access of the cell's transcription enzymes (e.g., RNA polymerase) to DNA. The illustration below shows that chromosomes have a complex structure. The DNA helix is wrapped around special proteins called histones, and this are wrapped into tight helical fibers. These fibers are then looped and folded into increasingly compact structures, which, when fully coiled and condensed, give the chromosomes their characteristic appearance in metaphase.
- Similar to the operons described above for prokaryotes, eukaryotes also use regulatory proteins to control transcription, but each eukaryotic gene has its own set of controls. In addition, there are many more regulatory proteins in eukaryotes and the interactions are much more complex.
- In eukaryotes transcription takes place within the membrane-bound nucleus, and the initial transcript is modified before it is transported from the nucleus to the cytoplasm for translation at the ribosome s. The initial transcript in eukaryotes has coding segments (exons) alternating with non-coding segments (introns). Before the mRNA leaves the nucleus, the introns are removed from the transcript by a process called RNA splicing (see graphic & video below), and extra nucleotides are added to the ends of the transcript; these non-coding "caps" and "tails" protect the mRNA from attack by cellular enzymes and aid in recognition by the ribosomes.
- Variation in the longevity of mRNA provides yet another opportunity for control of gene expression. Prokaryotic mRNA is very short-lived, but eukaryotic transcripts can last hours, or sometimes even weeks (e.g., mRNA for hemoglobin in the red blood cells of birds).
- The process of translation offers additional opportunities for regulation by many proteins. For example, the translation of hemoglobin mRNA is inhibited unless iron-containing heme is present in the cell.
- There are also opportunities for "post-translational" controls of gene expression in eukaryotes. Some translated polypeptides (proteins) are cut by enzymes into smaller, active final products. as illustrated in the figure below which depicts post-translational processing of the hormone insulin. Insulin is initially translated as a large, inactive precursor; a signal sequence is removed from the head of the precursor, and a large central portion (the C-chain) is cut away, leaving two smaller peptide chains which are then linked to each other by disulfide bridges.The smaller final form is the active form of insulin.
- Gene expression can also be modified by the breakdown of the proteins that are produced. For example, some of the enzymes involved in cell metabolism are broken down shortly after they are produced; this provides a mechanism for rapidly responding to changing metabolic demands.
- Gene expression can also be influenced by signals from other cells. There are many examples in which a signal molecule (e.g., a hormone) from one cell binds to a receptor protein on a target cell and initiates a sequence of biochemical changes (a signal transduction pathway) that result in changes within the target cell. These changes can include increased or decreased transcription as illustrated in the figure below.
- The RNA Interference system (RNAi) is yet another mechanism by which cells control gene expression by shutting off translation of mRNA. RNAi can also be used to shut down translation of viral proteins when a cell is infected by a virus. The RNAi system also has the potential to be exploited therapeutically.
Some RNA virus will invade cells and introduce double-stranded RNA which will use the cells machinery to make new copies of viral RNA and viral proteins. The cell's RNA interference system (RNAi) can prevent the viral RNA from replicating. First, an enzyme nicknamed "Dicer" chops any double-stranded RNA it finds into pieces that are about 22 nucleotides long. Next, protein complexes called RISC (RNA-induced Silencing Complex) bind to the fragments of double-stranded RNA, winds it, and then releases one of the strands, while retaining the other. The RISC-RNA complex will then bind to any other viral RNA with nucleotide sequences matching those on the RNA attached to the complex. This binding blocks translation of viral proteins at least partially, if not completely. The RNAi system could potentially be used to develop treatments for defective genes that cause disease. The treatment would involve making a double-stranded RNA from the diseased gene and introducing it into cells to silence the expression of that gene. For an illustrated explanation of RNAi, see the short, interactive Flash module at http://www.pbs.org/wgbh/nova/body/rnai-explained.html
The RNA interference system is also explained more completely in the video below from Nature Video.
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Pogil control expression of gene
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