contents Principles of Biology 51 Prokaryotic Gene Regulation Prokaryotic gene regulation mechanisms allow bacteria to quickly adapt to their environments.
Bacterial strains. Scanning electron micrograph of a variety of species of bacteria from the human intestine. Magnification is 8,000x. David M. Phillips/Science Source.
Topics Covered in this Module
Gene Regulation in Bacteria
Major Objectives of this Module
Describe the organization of bacterial DNA into operons. Explain the function of RNA polymerase. Describe the role of riboswitches in regulating gene expression.
page 260 of 989 3 pages left in this module
contents Principles of Biology
51 Prokaryotic Gene Regulation Gene Regulation in Bacteria Prokaryotes, such as bacteria, lack a nuclear membrane and are generally unicellular organisms. The lack of membrane-bound organelles means that processes involved in genetic expression or regulation occur without physical separation (Figure 1). In order for genes to be expressed at the right time and location, gene expression must be regulated carefully.
Figure 1: Coupled transcription and translation in E. coli. The long fiber running from left to right is a segment of the E. coli chromosome. Transcription is occurring at multiple points along the DNA where RNA polymerase attaches. mRNA is transcribed as the polymerase moves along the DNA from left to right. Translation begins even while transcription is still progressing; the ribosomes attach to the nascent mRNA strands and assemble amino acids into polypeptide chains as they move toward the DNA strand. Professor Oscar Miller/Science Source.
RNA polymerases in prokaryotes and eukaryotes differ. Prokaryotes use a single type of RNA polymerase, but eukaryotes have at least three different types of RNA polymerase. All of the genetic information contained within prokaryotes and eukaryotes is considered their genome. In some cases, the cells interact with each other or their environment to regulate gene expression.
Let's focus on a classical example of prokaryotic gene expression. Escherichia coli is a bacterial species that is common in the human large intestine, consuming nutrients provided by the host. Individual E. coli require a continuous supply of certain amino acids, such as tryptophan, to survive. However, tryptophan is not always available in the intestinal environment. As a result, E. coli is capable of synthesizing tryptophan itself by activating a metabolic pathway when it is unavailable in the environment. When tryptophan again becomes available in the environment, the cell can conserve energy by halting synthesis and using environmental tryptophan instead (Figure 2). This is a way that bacteria adjust their metabolism to environmental changes.
Figure 2: Feedback inhibition in the E. coli tryptophan biosynthesis pathway. In E. coli, when tryptophan levels are low, tryptophan is synthesized from a precursor by three enzyme complexes. When tryptophan is abundant in the bacterium, the biosynthetic pathway undergoes feedback inhibition. Tryptophan binds to the first enzyme complex, inhibiting the pathway from producing additional tryptophan. © 2014 Nature Education All rights reserved. Figure Detail
Bacterial genes are organized into operons. An operon is a set of genes that are transcribed together as a unit and under the control of a single set of regulators. A basic regulatory region consists of an operator and a promoter. The promoter is the region of the DNA that has a specific sequence that the RNA polymerase binds to for initiation of transcription. The ability of RNA polymerase to access the promoter is regulated by the operator and transcription factors. An operator is a specific sequence within the DNA that binds transcription factors to turn transcription on or off. The operator is found within the promoter or between the promoter and the coding region of the gene. The operator, promoter and coding regions of the genes make up the operon. The operon can be turned off by a repressor, a transcription factor that binds to the operator and inhibits RNA polymerase from binding to the promoter. Blocking RNA polymerase from accessing the promoter prevents transcription from taking place. Repressor proteins have a specific binding site on an operator, and therefore, have no effect on any other operator sites within the given genome. The regulatory genes that encode repressor proteins are continuously expressed at a low rate. The operons regulated by the repressors are not permanently shut down because the binding of a repressor to its operator is reversible. A majority of regulatory proteins are allosteric proteins. Allosteric proteins are proteins that change their shape when bound to a specific molecule called an allosteric effector. This change in shape may activate the allosteric protein to bind the operator sequence and shut down expression of the operon or in other cases to let go of the operator and turn on expression of the operon.
An example of this gene regulation can be seen with the lac operon, which codes for three proteins (Figure 3). Import and metabolism of lactose is controlled by these lac operon-encoded proteins. The presence and absence of lactose regulates the expression of the lac operon. When lactose is absent from the environment, the lactose repressor protein, LacI, will bind the operator and repress the lac operon by preventing RNA polymerase from accessing the promoter. The allosteric effector of LacI is allolactose, an isomer of lactose. When lactose is present, the cell modifies some of it into allolactose. In turn, allolactose binds to LacI and changes its conformation so that it can no longer bind to the operator. Without the LacI repressor in the way, the transcription of the three lac genes can proceed. This is an example of gene expression regulation that allows the organism to adapt to environmental changes quickly and efficiently.
Figure 3: The lac operon of E. coli. In this operon, expression is controlled by a repressor, an operator and RNA polymerase. The lac operon of E. coli is a segment of DNA that includes a promoter (P), an operator (O), and the three structural genes (lacZ, lacY, and lacA) that code for proteins involved in lactose import and lactose metabolism. In the absence of lactose (a), the lactose repressor protein, LacI (I), which is encoded by the lacI gene, binds to the operator and inhibits transcription of the lac operon. The repressor protein prevents RNA polymerase from transcribing lacZ, lacY, and lacA. In the presence of lactose (b), some lactose is modified to allolactose, which binds to the allosteric site of the LacI repressor protein. This binding changes the conformation of LacI, preventing it from binding to the operator. The release of LacI from the operator allows RNA polymerase to transcribe lacZ, lacY, and lacA. © 2013 Nature Education All rights reserved.
Gene regulation can be positive or negative. In negative gene regulation, the primary function of a regulatory protein is to inhibit the expression of a gene. Operons controlled by negative gene regulation can be categorized into repressible operons or inducible operons depending on the effect of the allosteric effector on repressor-operator binding. In repressible operons, the allosteric effector causes the repressor to inhibit the expression of the operon. An example of this is tryptophan and the trp operon (Figure 4). Without tryptophan as an allosteric effector, the repressor cannot bind to the operator, and transcription of the trp operon is allowed to proceed. When tryptophan is present, however, it binds to the repressor, causing a conformational change that allows it to bind to the operator and block transcription of the trp operon. In effect, the operon is repressible by addition of tryptophan to the environment. In contrast, inducible operons inhibit the expression of the operon when the allosteric effector is absent. However, the expression of the operon is turned on, or induced, when the allosteric effector is present and binds to the regulatory protein. An example of this is the LacI repressor and allolactose of the lac operon. Without the allosteric effector allolactose, LacI remains bound to the operator, preventing lac operon transcription. However, the presence of lactose (and therefore allolactose) changes the conformation of LacI so that it releases from the operator and permits lac operon transcription. A third type of gene regulation is positive gene regulation, in which the interaction between a protein and its allosteric effector results in the activation of gene expression. The lac operon also includes an example of positive gene regulation. In E. coli, the metabolite cyclic AMP (cAMP) accumulates to high levels when glucose levels in the cell are low. Conversely, cAMP levels fall when cellular glucose levels increase. cAMP is the allosteric effector for an allosteric regulatory protein called catabolite activator protein (CAP). In the absence of cAMP, CAP is inactive, but when CAP is bound to cAMP, it changes into an active conformation, which is able to bind a specific DNA sequence called the activator sequence. This activator sequence is located upstream of the lac promoter. When the activator sequence is not bound by CAP-cAMP, the affinity of RNA polymerase for the adjacent promoter is low, but when CAP-cAMP is bound to the activator sequence, the affinity of RNA polymerase for the promoter increases, increasing the expression of the lac operon genes. Therefore, in the absence of glucose, cAMP levels are high, and lac operon expression is also high. In the presence of glucose, however, cAMP levels are low, and therefore lac operon expression is low as well.
The overall benefit of negative and positive gene regulation of the lac operon is that the expression levels of the lactose metabolism genes are directly influenced by the nutritional environment of the cell. That is, when lactose is absent, nucleotides, amino acids and energy are not wasted in expressing the lactose metabolism genes. The inducible nature of the lac operon helps ensure that the operon is not expressed unless lactose is present to induce its expression. Furthermore, even if lactose is present, it would be wasteful to express the lactose metabolism genes at maximal levels if other energy sources — such as glucose — had not yet been exhausted. This wasteful potential outcome is avoided thanks to positive gene regulation by the CAP-cAMP system, which helps ensure that lac operon expression is maximized only when glucose is absent and lactose is present.
Figure 4: Regulation of the trp operon. The trp operon regulates the expression of five genes in response to tryptophan levels inside the cell. The trpR gene, regulated separately from the trp operon, encodes the Trp repressor protein. In the absence of tryptophan (a), the Trp repressor is unable to bind to the operator of the trp operon. As a result, RNA polymerase binds to the promoter and transcribes five genes — trpE, trpD, trpC, trpB, and trpA — which encode enzymes required for tryptophan synthesis. Tryptophan levels in the cell increase as a result. Once tryptophan levels exceed a critical concentration (b), tryptophan molecules bind to the allosteric sites of the Trp repressor, changing its conformation. In its new conformation, the repressor binds to the operator, blocking RNA polymerase from binding to the promoter and preventing the expression of the five trp genes. © 2014 Nature Education All rights reserved.
Bacterial transcription is regulated by a single type of RNA polymerase. All transcription in bacteria is carried out by a single RNA polymerase, which has five core subunits and a sigma subunit. RNA polymerase binds to specific sequences on the DNA called promoters. What does the sigma subunit do? In 1969, Andrew Travers and Richard Burgess discovered the role of RNA polymerase sigma. They found that in in vitro assays of bacterial transcription with template DNA and purified RNA polymerase, transcription did not start at specific sites. In vivo, the RNA polymerase did start at specific sites, even with a minimal amount of polymerase. This was because sigma factor was not purified with the core RNA polymerase that was used in the in vitro assays. Without sigma factor, the core RNA polymerase made errors in start point choice, which led to RNA synthesis at inappropriate sites. Travers and Burgess concluded that sigma factors were necessary for specificity in the initiation of transcription (Figure 5). Figure 5: The sigma cycle. Sigma factors are necessary for specificity in the initiation of transcription. The core RNA polymerase joins with the sigma factor and attaches to the DNA. After RNA synthesis is initiated, RNA polymerase continues transcription, but the sigma factor is released and can bind to other RNA polymerases. © 2011 Nature Education All rights reserved. Figure Detail
Bacterial sporulation of Bacillus subtilis provides a good example of sigma factors that specifically bind specific promoters. These bacteria exist in both the vegetative state and endospore state. During endospore formation, specific genes are expressed, but they are not expressed during the vegetative state. The first genes for sporulation are turned on after the expression of a gene that encodes an alternative sigma factor. Different sigma factors are subsequently expressed to activate new genes that are needed later in the sporulation. Each of these sigma factors recognizes promoters of the genes in its group. By using the same core RNA polymerase with different sigma factor subunits, the organism is able to regulate expression of different sets of genes.
Riboswitches frequently regulate gene expression in bacteria. Some mRNA transcripts contain riboswitches, or regions of RNA that can switch conformations depending on the presence or absence of ligands such as metabolites or metal ions. In bacterial mRNA, riboswitches are commonly found in the 5′ untranslated region (5′-UTR) preceding the start codon (Figure 6). Similar to the interaction between an allosteric effector and its allosteric protein, the binding of a riboswitch to its appropriate ligand changes the conformation of the mRNA molecule in a variety of ways that can influence the expression levels of the genes contained in the mRNA. Figure 6: Bacterial mRNA with a riboswitch. Riboswitches are typically found in the 5′ untranslated region of an mRNA transcript. In this type of riboswitch, the conformation of the mRNA determines whether the remainder of the mRNA is transcribed past the riboswitch region. © 2013 Nature Education All rights reserved. Figure Detail Because RNA is primarily single stranded, it can fold into secondary and tertiary structures, much like proteins can. This ability of RNA to adopt three- dimensional structures allows mRNA to possess binding sites for other molecules and switch between conformations depending on whether these binding sites are occupied. The aptamer of a riboswitch is the region that binds to a specific ligand, such as enzyme cofactors, nucleotide precursors, amino acids or metal ions. When the aptamer binds the appropriate ligand, it induces a conformational change in a nearby region of mRNA known as the expression platform. Conformational changes in the expression platform can have a variety of effects on gene expression. One example involves a mechanism of transcription termination that depends on the formation of a stem-loop structure. In some genes, the DNA template contains a special sequence such that the mRNA transcribed from it folds back on itself and forms a hairpin loop. The hairpin is created by base pairing of nearby complementary G and C bases within the mRNA. The region immediately following the hairpin is rich in U bases in the mRNA — and therefore A bases in the corresponding DNA template. The formation of the stronger GC base pairs in the hairpin, combined with the weaker UA base pairs between the mRNA and the DNA template, separates the mRNA from the DNA and terminates transcription. The stem-loop structure can form the basis of an expression platform that determines whether an mRNA is transcribed to completion (Figure 7). If the ligand for the riboswitch is absent, the stem-loop structure does not form, and transcription proceeds normally. However, if the ligand is present and binds to the aptamer, the conformation of the expression platform changes such that the stem-loop structure forms and prematurely terminates transcription. In effect, the presence of the ligand prevents mRNA past the riboswitch from being transcribed. In other riboswitches, the converse may be true — the absence of the ligand results in transcription termination, and the presence of the ligand is required for normal levels of transcription. Figure 7: The aptamer and expression platform of a riboswitch. The riboswitch changes between different conformations depending on the presence or absence of a metabolite ligand (M). In this example, if the ligand is not bound (left), a hairpin loop forms, but it is too far from the run of U bases to behave as a transcription termination signal. The remainder of the mRNA is transcribed in this case. If a ligand binds to the aptamer (right), the entire riboswitch changes conformation such that the expression platform is folded into a hairpin loop adjacent to the run of U bases. This is the stem-loop structure that prematurely aborts transcription and prevents the expression of the genes in the remainder of the mRNA. © 2013 Nature Education All rights reserved. Because bacteria lack a nuclear membrane to separate transcription and translation into different areas, translation of polypeptides can occur as soon as mRNA is transcribed. Some types of riboswitches take advantage of the fact that transcription and translation can occur in the same space in bacteria to regulate gene expression at the translational level. For example, some riboswitches can either expose or sequester the ribosome-binding site of the mRNA in response to ligand molecules binding to their aptamers. In this way, an mRNA can be transcribed to completion, but its riboswitch's conformation will decide whether it will actually be translated into protein. Other riboswitches contain codons that influence the position of a ribosome along the 5′-UTR as it translates a short "leader" polypeptide. The ability of the ribosome to pass these codons depends on the availability of specific amino acids. In turn, this ribosome's position determines whether a stem-loop structure forms and whether the full mRNA is transcribed and later translated. In all cases, however, riboswitches are an important way to regulate gene expression in response to chemical cues from the environment.
Biomedical research. The ability of riboswitches to regulate gene expression makes them possible drug targets in biomedical research. Studies of aptamers have shown that RNA can distinguish between structures that are closely related and that they can do this as well as an antibody can. As a result, riboswitches in pathogenic bacteria and fungi are becoming a new target for drug design. In 2006, Rebecca Montange and Robert Batey at the University of Colorado at Boulder found the structure of a riboswitch in bacterial mRNA bound to S-adenosylmethionine. Investigating the complex folded structure of the riboswitch, they identified the structural changes that result from the ligand binding and that prevent transcription. New drugs that target the binding sites that are regulated by the riboswitch could repress transcription, which could kill the bacteria. As riboswitch research continues, with scientists elucidating the three-dimensional structures of more and more riboswitches, researchers will have a greater chance of creating new riboswitch-targeting antimicrobials.
IN THIS MODULE
Gene Regulation in Bacteria Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Controlling inflammation to stop sepsis Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. View | Download
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contents Principles of Biology
51 Prokaryotic Gene Regulation Summary OBJECTIVE Describe the organization of bacterial DNA into operons. Some bacterial genes are organized into operons, in which the genes are under the control of a single promoter and a regulatory region called an operator. Bacteria exhibit three major types of gene regulation. In repressible operons, an allosteric effector triggers the repression of an otherwise expressed operon. In inducible operons, the effector induces the expression of an operon that is repressed in the absence of the effector. In positive gene regulation, the effector increases the expression of the operon above some baseline level.
OBJECTIVE Explain the function of RNA polymerase. RNA polymerase initiates transcription at specific sequences called promoters. Promoter choice by RNA polymerase is regulated by sigma factors. In many operons, the binding of a repressor protein to the operator obstructs the RNA polymerase from binding the promoter, which prevents transcription of the operon genes.
OBJECTIVE Describe the role of riboswitches in regulating gene expression. Riboswitches are mRNA molecules that bind ligands and change conformation as a result. Some of these alternative structures serve as signals to continue or terminate transcription or translation. As a result, riboswitches serve as a way for mRNA molecules to sense the environment and regulate gene expression.
Key Terms allosteric effector A small molecule that binds to the allosteric site of a protein, thereby changing the protein's conformation and activity. allosteric protein A protein whose activity is regulated by the binding of a small regulatory molecule (an allosteric effector) to the allosteric site of the protein, which results in a conformational change.
aptamer The region of a riboswitch that serves as a receptor for a ligand; binding of the ligand to the aptamer triggers conformational changes in the expression platform that influence gene expression. expression platform The region of a riboswitch that changes conformation in response to a ligand molecule binding to the aptamer. inducible operon A negatively regulated operon whose activity is stimulated (induced) by the presence of an allosteric effector; expression of the operon is off when the effector is absent and on when the effector is present. operator A specific DNA sequence within an operon that serves as the binding site for a repressor protein.
operon A cluster of genes that are expressed together and regulated from a single promoter; includes the genes themselves, the promoter and the regulatory sequences, such as the operator. promoter A specific DNA sequence where RNA polymerase binds to the DNA to initiate transcription. repressible operon A negatively regulated operon whose activity is inhibited (repressed) by the presence of an allosteric effector; expression of the operon is on when the effector is absent and off when the effector is present. repressor A regulatory protein that binds to an operator and inhibits RNA polymerase from binding to the promoter. riboswitch A type of regulatory region in RNA that contains a region (the aptamer) that can bind a ligand and a region that changes conformation as a result of ligand binding (the expression platform); involved in regulation of gene expression at the transcriptional and/or translational levels. stem-loop structure A sequence of RNA containing internal GC base pairing such that a hairpin loop forms during its transcription, which serves as a mechanism to terminate continued transcription.
IN THIS MODULE
Gene Regulation in Bacteria Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Controlling inflammation to stop sepsis Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. View | Download
page 262 of 989 1 pages left in this module
contents Principles of Biology
51 Prokaryotic Gene Regulation
Test Your Knowledge
1. Complete the following sentence: Prokaryotes differ from eukaryotes by...
lacking a nucleus. lacking proteins. lacking RNA. lacking DNA. lacking ribosomes.
2. How would a mutation in the operator of the lac operon that prevented the LacI repressor from binding affect the expression of the genes in the lac operon?
The lac operon genes would be expressed continuously. The lac operon genes would not be expressed at all. The mutation would repress lactose. The mutation would inhibit lactose. The lac operon genes would be expressed in a regulated manner.
3. Complete the following sentence: The lac repressor is an allosteric molecule because...
it catalyzes the synthesis of lactose. it lowers the transcription rate. it inhibits the synthesis of lactose. it catalyzes mutations on the operon. it interacts with another molecule to change the repressor's active site.
4. What is the function of the sigma factor in transcription?
to prevent the binding of the polymerase to the promoter to enable specific binding of DNA polymerase to RNA to enable specific binding of promoters to operons to enable specific binding of RNA polymerase to promoters to enable specific binding of RNA to DNA
5. Which environmental condition results in maximal expression levels of the lac operon genes?
low glucose, high lactose high glucose, high lactose low glucose, low lactose high glucose, low lactose All of these conditions will result in the same level of lac operon gene expression. 6. Which mutation is expected to have a similar effect to a mutation in which the LacI repressor is no longer able to bind to the operator?
a mutation in the operator that enables it to bind but not release the LacI repressor a mutation in the operator that prevents recognition by the LacI repressor a mutation in the LacI repressor in which it can still bind the operator but lacks an allosteric site a mutation in the promoter that prevents RNA polymerase from binding None of the answers are correct.
Submit
IN THIS MODULE
Gene Regulation in Bacteria Summary Test Your Knowledge
WHY DOES THIS TOPIC MATTER?
Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.
PRIMARY LITERATURE
Controlling inflammation to stop sepsis Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction. View | Download
page 263 of 989