27.1: Regulation of Gene Expression in Bacteria

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Learning Goals (ChatGPT o3-mini)

• Explain the Importance of Gene Regulation:

  • Describe how identical genomes can produce diverse phenotypes in multicellular organisms and enable adaptive responses in single-celled organisms.
  • Discuss why precise control of gene expression is essential for cellular differentiation, metabolism, and overall homeostasis.

• Differentiate Between Structural and Regulatory Genes:

  • Identify the roles of structural genes (encoding proteins or enzymes) and regulatory genes (encoding transcription factors and other regulators) in controlling gene expression.

• Understand Operon Organization in Prokaryotes:

  • Explain the concept of the operon, including the organization of structural genes under a single promoter and regulatory region.
  • Compare constitutively expressed operons with those that are inducible or repressible.

• Analyze Specific Examples of Operon Regulation:

  • Describe the mechanism of the trp operon, focusing on how the trp repressor binds to the operator in the presence of tryptophan to block transcription.
  • Explain the regulation of the lac operon, including the roles of the lac repressor, inducer molecules (e.g., allolactose, IPTG), and the CAP-cAMP complex in activating transcription when glucose is low.

• Examine Global Regulatory Mechanisms in Bacteria:

  • Define regulons and discuss how they allow the simultaneous control of multiple operons in response to environmental changes.
  • Explain the role of alarmones such as pppGpp in the stringent response, and how they adjust gene expression during nutrient stress.

• Describe Quorum Sensing and Population-Level Regulation:

  • Explain the basic components of quorum sensing systems (signal synthase, signal receptor, and signal molecules) and their role in coordinating bacterial behavior, including virulence, biofilm formation, and secondary metabolism.
  • Analyze how quorum sensing enables bacteria to alter gene expression based on population density.

• Understand the Role of Alternate σ Factors:

  • Describe how the use of different σ subunits by bacterial RNA polymerase allows for rapid and global shifts in gene expression in response to environmental signals.
  • Discuss examples of σ factor switching in processes such as sporulation.

• Explore Post-Initiation Regulatory Mechanisms:

  • Define attenuation and explain how leader sequences and the formation of alternative RNA secondary structures can control transcription termination.
  • Describe riboswitches, including how they bind small intracellular molecules to influence mRNA structure and regulate transcription or translation.

These learning goals are intended to provide you with a comprehensive framework for understanding how gene expression is dynamically regulated in response to both internal signals and external environmental cues.

Introduction

Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two pure bacterial cultures inoculated from the same starting colony contain the same DNA, except for changes that arise from spontaneous mutations. If each cell in a multicellular organism has the same DNA, then how is it that cells in different parts of the organism’s body exhibit different characteristics? Similarly, how is it that the same bacterial cells within two pure cultures exposed to different environmental conditions can exhibit different phenotypes? In both cases, each genetically identical cell does not "turn on" or express the same set of genes. Only a subset of proteins in a cell at a given time is expressed.

Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms enables cellular differentiation, in single-celled organisms like prokaryotes, it primarily ensures that a cell’s resources are not wasted on making proteins that the cell does not need at that time.

Elucidating the mechanisms that control gene expression is crucial to understanding human health. Malfunctions in this process in humans lead to the development of cancer and other diseases. Understanding the interaction between the gene expression of a pathogen and that of its human host is crucial for understanding a particular infectious disease. Gene regulation involves a complex web of interactions within a given cell among signals from the cell’s environment, signaling molecules within the cell, and the cell’s DNA. These interactions result in the expression of specific genes and the suppression of others, depending on the circumstances.

Prokaryotes and eukaryotes share some similarities in their mechanisms for regulating gene expression; however, gene expression in eukaryotes is more complex due to the temporal and spatial separation between the processes of transcription and translation. Thus, although most regulation of gene expression occurs through transcriptional control in prokaryotes, regulation of gene expression in eukaryotes occurs at the transcriptional level and post-transcriptionally (after the primary transcript has been made).

In bacteria and archaea, structural proteins with related functions are typically encoded together within the genome in a block known as an operon. They are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript, as shown in Figure \(\PageIndex{1}\). In this way, the regulation of transcription for all the structural genes encoding the enzymes that catalyze the many steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time or none will be required. For example, in E. coli, all the structural genes that encode enzymes needed to utilize lactose as an energy source are clustered together in the lactose (or lac) operon, under the control of a single promoter, the lac promoter. French scientists François Jacob (1920–2013) and Jacques Monod, at the Pasteur Institute, were the first to demonstrate the organization of bacterial genes into operons through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine in 1965.

Figure \(\PageIndex{1}\): Schematic Representation of an Operon. In prokaryotes, structural genes of related functions 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. Parker, N., et. al. (2019) Microbiology. Openstax

Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region. The regulatory region encompasses the promoter and the surrounding region to which transcription factors and proteins encoded by regulatory genes can bind. Transcription factors influence the binding of RNA polymerase to the promoter, allowing it to progress and transcribe structural genes. A repressor is a transcription factor that suppresses the 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 the binding of RNA polymerase 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.

In prokaryotes, there are examples of operons whose gene products are required consistently and whose expression, therefore, is not regulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, other prokaryotic operons are expressed only when needed and are regulated by repressors, activators, and inducers.

Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby preventing the transcription of the structural genes. Such operons are classified as either repressible operons or inducible operons. Repressible operons, like the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However, when the product of the biosynthetic pathway begins to accumulate in the cell, eliminating the need for the cell to continue producing more, the expression of the operon is repressed. Conversely, inducible operons, such as the lac operon of E. coli, often contain genes encoding enzymes involved in a pathway that metabolizes a specific substrate, like lactose. These enzymes are only required when that substrate is available; thus, expression of the operons is typically induced only in the presence of the substrate.

The trp Operon - A Repressible Operon

E. coli can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each other in the trp operon, as shown in Figure \(\PageIndex{2}\). When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur, and tryptophan is not synthesized.

When tryptophan is not present in the cell, the repressor binds to the operator by itself; therefore, the operon is active, and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping the expression of the operon. Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.

Figure \(\PageIndex{2}\): The Trp Operon. Parker, N., et. al. (2019) Microbiology. Openstax

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

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of the E. Coli Trp repressor-operator complex (1TRO).

Figure \(\PageIndex{3}\): E. Coli Trp repressor-operator complex (1TRO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...tjbYwr47gVZjn8

The Trp repressor is depicted as a dimer, with one subunit shown in gray and the other in gold. The backbone of the two DNA strands is shown in spacefill magenta and cyan, except where the bases on the major groove interact with the Trp repressor. The tryptophan in each of the proton monomers is shown in spacefill with CPK colors and labeled. Noncovalent interactions (hydrogen bonds and salt bridges) between the protein and DNA are shown with dotted lines. 6 water-mediated hydrogen bonds to phosphate are not shown. Note that there are a few H bonds between the protein and base hydrogen bond donors and acceptors, suggesting that the repressor might bind specifically through geometric interactions with the backbone, along with the water-mediated hydrogen bonds.

The Lac Operon: An Inducible Operon

The lac operon is an example of an inducible operon that is also subject to activation in the absence of glucose. The lac operon encodes three structural genes, lacZ, lacY, and lacA, necessary to acquire and process the disaccharide lactose from the environment, as shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Biological Activity of the lac Operon.Figure modified from Esmaeili, A., et. al. (2015) BMC Bioinformatics 16:311

Panel (A) shows a schematic representation of the lac operon in E. coli. The lac operon has three structural genes, lacZ, lacY, and lacA, that encode for β-galactosidase, permease, and galactoside acetyltransferase, respectively. The promoter (p) and operator (o) sequences that control the expression of the operon are shown. Upstream of the lac operon is the lac repressor gene, lacI, controlled by the lacI promoter (p).

Panel (B) shows the lac repressor inhibition of the lac operon gene expression in the absence of lactose. The lac repressor binds to the operator sequence of the operon and prevents the RNA polymerase enzyme, bound to the promoter (p), from initiating transcription.

Panel (C) shows that in the presence of lactose, some of the lactose is converted into allolactose, which binds and inhibits the activity of the lac repressor. The lac repressor-allolactose complex cannot bind to the operator region of the operon, thereby freeing RNA polymerase and allowing for the initiation of transcription. Expression of the lac operon genes enables the breakdown and utilization of lactose as a food source within the organism.

The lacZ gene encodes the β-galactosidase (β-gal) enzyme responsible for the hydrolysis of lactose into simple sugars glucose and galactose, as shown in Figure \(\PageIndex{5}\). The β-gal enzyme can also mediate the breakdown of the alternate substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) (panel B). The breakdown product, 5-bromo-4-chloro-3-hydroxyindole – 1, spontaneously dimerizes to form the intensely blue product, 5,5′-dibromo-4,4′-dichloro-indigo – 2. Thus, Xgal has been a valuable research tool, not only in the study of β-gal enzymatic activity but also in the development of the commonly used blue-white DNA cloning system, which utilizes the β-gal enzyme as a marker in molecular cloning experiments.

Figure \(\PageIndex{5}\): Reactions Controlled by the Expression of the Lac Operon. Andreas Piehler, Yikrazuul, and NUROtikerr Panel (A) shows the expression of the β-galactosidase enzyme enables the breakdown of lactose into simple sugars, glucose, and galactose for E. coli to use as a food resource. Panel (B) shows that the β-galactosidase enzyme also mediates the breakdown of the non-native substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal). Breakdown product (1) 5-bromo-4-chloro-3-hydroxyindole quickly dimerizes into the intensely blue product (2) 5,5′-dibromo-4,4′-dichloro-indigo, making it a useful tool for molecular biology. Panel (C) shows that β-D-1-thiogalactopyranoside (IPTG) can serve as a non-native inducer of the lac operon. It mimics the structure of lactose and binds with the Lac Repressor.

The lac operon contains two more genes, in addition to lacZ (Fig. 4). The lacY gene encodes a permease that increases the uptake of lactose into the cell, and lacA encodes a galactoside acetyltransferase (GAT) enzyme. The exact function of GAT during lactose metabolism has not been conclusively elucidated, but acetylation is thought to play a role in the transport of the modified sugars.

For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be energetically wasteful to produce the enzymes necessary for lactose processing if lactose were not available.

In the absence of lactose, the lacI gene is constitutively expressed, resulting in the production of the lac repressor protein (Fig. 28.2.3B). The lac repressor binds to the operator region of the lac operon and physically prevents RNA polymerase from transcribing the structural genes (Fig. 28.2.3B). However, when lactose is present, the lactose inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and altering its shape so that it can no longer bind to the operator DNA (Fig. 28.2.3 C). Removal of the repressor in the presence of lactose enables RNA polymerase to proceed through the operator region and initiate transcription of the lac structural genes. In addition to lactose, laboratory experiments have revealed that the non-natural compound Isopropyl β-D-1-thiogalactopyranoside (IPTG) can also bind to the lac repressor and induce the expression of the lac operon (panel C). Similar to Xgal, this compound has also been used as a research tool for molecular cloning.

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the lactose operon repressor and its complexes with DNA (1LBG).

Figure \(\PageIndex{6}\): Lactose operon repressor and its complexes with DNA (1LBG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...a79MvrAHGpJk99

The resolution of the structure above was insufficient to show the amino acid side chains. Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the NMR solution structure of the dimer of LAC repressor DNA-binding domain complexed to its natural operator O1 (2KEI).

Figure \(\PageIndex{7}\): NMR solution structure of Dimer of LAC repressor DNA-binding domain complexed to its natural operator O1 (2KEI). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...p48VrPFXg6WgD9

Note the presence of white spheres representing hydrogen atoms (these do not appear in crystal structures but do in NMR structures). Color coding is the same as above. Zoom in to see specific interactions between the protein and the exposed DNA base hydrogen bond donors and acceptors. The complex of O1 and O2 exhibits similar specific and nonspecific contacts, which is expected given that the lambda repressor has a similar affinity for these two operator sites. In contrast, one side of the O3 complex exhibits a loss of protein-DNA interactions, consistent with its lower affinity for the operator O3.

The Lac Operon: Activation by Catabolite Activator Protein

Bacteria typically can use a variety of substrates as carbon sources. However, because glucose is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted. Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed only when the alternative substrate is available. In the 1940s, Jacques Monod was the first to demonstrate a preference for certain substrates over others through his studies of E. coli’s growth when cultured in the presence of two different substrates simultaneously. Such studies generated diauxic growth curves, as shown in Figure \(\PageIndex{8}\). Although the preferred substrate glucose is used first, E. coli grows quickly, and the enzymes for lactose metabolism are absent. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose. Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.

Figure \(\PageIndex{8}\): Utilization of Glucose in E. Coli. When grown in the presence of two substrates, E. coli uses the preferred substrate (in this case, glucose) until it is depleted. Then, enzymes needed for the metabolism of the second substrate are expressed and growth resumes, although at a slower rate. Parker, N., et. al. (2019) Microbiology. Openstax

The ability to switch from glucose use to another substrate, like lactose, is a consequence of the activity of an enzyme called Enzyme IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism, and EIIA becomes phosphorylated. Phosphorylated EIIA activates adenylyl cyclase, an enzyme that converts some of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of AMP and an important signaling molecule involved in glucose and energy metabolism in E. coli, as shown in Figure \(\PageIndex{9}\). As a result, cAMP levels begin to rise in the cell. This is an indicator to the cell that overall energy levels are low and that ATP is being depleted.

Figure \(\PageIndex{9}\): Conversion of ATP to cAMP. When ATP levels decrease due to the depletion of glucose, some remaining ATP is converted to cAMP by adenylyl cyclase. Thus, increased cAMP levels signal glucose depletion. Figure from: Parker, N., et. al. (2019) Microbiology. Openstax

The lac operon also plays a crucial role in this switch from glucose to lactose utilization. When glucose is scarce, the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon, as shown in Figure \(\PageIndex{10}\). In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. The binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present (removing the lac repressor protein), and glucose levels must be depleted (allowing the binding of an activating protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. Because of low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate the transcription of these operons.

Figure \(\PageIndex{10}\): Effect of CAP on the Lac Operon. (a) In the presence of cAMP, CAP binds to the promoters of operons, like the lac operon, that encode genes for enzymes for the use of alternative substrates. (b) For the lac operon to be expressed, there must be activation by cAMP-CAP as well as the removal of the lac repressor from the operator. Parker, N., et. al. (2019) Microbiology. Openstax

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the Catabolite activator protein CAP-DNA complex with bound cAMP (2CGP).

Figure \(\PageIndex{11}\): Catabolite activator protein CAP-DNA complex with bound cAMP (2CGP). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...z6mLhBpPmPMsC6

Global Responses of Prokaryotes

In prokaryotes, several higher levels of gene regulation can control the transcription of many related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a regulon.

Alarmones

When sensing impending stress, prokaryotes alter the expression of a wide range of operons to respond in a coordinated manner. They do this through the production of alarmones, which are small intracellular nucleotide derivatives, such as guanosine pentaphosphate (pppGpp), as shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Structure of Guanosine Pentaphosphate (pppGpp). Yikrazuul

Alarmones change which genes are expressed and stimulate the expression of specific stress-response genes. For example, pppGpp signaling is involved in the stringent response in bacteria, causing the inhibition of RNA synthesis when there is a shortage of amino acids present. This causes translation to decrease, and the amino acids present are therefore conserved. Furthermore, pppGpp causes the upregulation of many other genes involved in stress response, such as those for amino acid uptake from the surrounding media and biosynthesis.

The use of alarmones to alter gene expression in response to stress appears to be important in pathogenic bacteria, as well. On encountering host defense mechanisms and other harsh conditions during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling. Knowledge of these responses is crucial for understanding the infection process of many pathogens and for developing therapies to counteract this process.

Quorum Sensing

Quorum sensing (QS) is an intercellular communication mechanism of bacteria used to coordinate the activities of individual cells at the population level in response to surroundings through the production and perception of diffusible signal molecules such as Acyl Homoserine Lactones or small signaling peptides, as shown in Figure \(\PageIndex{13}\). The signal synthase, signal receptor, and signal molecules are three essential elements of the basic QS circuit machinery. Genes encoding signal-generating proteins are also included among the QS target genes. This forms an autoinduction feedback loop to modulate the generation of signal molecules. Several bacterial behaviors, including the expression of virulence factors, the production of secondary metabolites, biofilm formation, motility, and luminescence, are regulated by quorum sensing (QS). Through complex regulatory networks, bacteria are capable of expressing corresponding genes according to their population size and of behaving in a coordinated manner.

Figure \(\PageIndex{13}\): Examples of Quorum Sensing Pathways. Verbeke, F., et.al. (2017) Frontiers in Neuroscience 11:183.

The left panel shows the typical Gram-negative quorum-sensing mechanism. Acyl homoserine lactone molecules, synthesized by LuxI, passively pass the bacterial cell membrane and when a sufficient concentration is reached (threshold level) activate the intracellular LuxR which subsequently activates target gene expression in a coordinated way. Note that a single cell is shown for simplicity. However, acyl homoserine lactones will commonly diffuse and target neighboring cells within the colony to mediate a communal or population response within the bacterial colony.

The right panel illustrates that quorum-sensing peptides are synthesized by bacterial ribosomes as propeptide proteins and undergo post-translational modifications during excretion via active transport. The quorum-sensing peptides bind to membrane-associated receptors, which become autophosphorylated and activate intracellular response regulators through phosphotransfer. These phosphorylated response regulators induce increased expression of their target genes.

For example, some microbial species, such as Staphylococcus aureus, can encase their community within a self-produced matrix of hydrated extracellular polymeric substances that include polysaccharides, proteins, nucleic acids, and lipid molecules. These encasements are known as biofilms. The formation of the biofilm on solid surfaces is a step-wise process comprising several stages, as shown in Figure \(\PageIndex{14}\). It begins with the conditioning of the surface through coating with macromolecules from the surrounding aqueous environment, which enables the initial reversible adhesion of microorganisms. The next step involves the formation of stronger, irreversible attachments to the surface, followed by the proliferation and aggregation of microorganisms into multicellular and multilayered clusters that actively produce an extracellular matrix. Some cells in the mature biofilms continuously detach and separate from the aggregates, representing a continuous source of planktonic bacteria that can subsequently spread and form new microcolonies.

Figure \(\PageIndex{14}\) Schematic drawing of biofilm formation. Rukavina, Z., and Vanic, Z. (2016) Pharmaceutics 8(2):18.

Biofilms are a common cause of chronic, nosocomial (originating in a hospital), and medical device-related infections because they can develop on both vital and necrotic tissue, as well as on the inert surfaces of various implanted materials. Moreover, biofilms are linked with high-level resistance to antimicrobials, frequent treatment failures, and increased morbidity and mortality. As a consequence, biofilm infections and accompanying diseases have become a major health concern and a serious challenge for both modern medicine and pharmacy. A rough estimate suggests that more than 60% of hospital-associated infections are attributable to biofilms formed on indwelling medical devices, resulting in over one million cases of infected patients annually and exceeding $1 billion in hospitalization costs per year in the USA.

Biofilm infections share several common characteristics: slow development in one or more focal points, delayed clinical manifestation, and persistence for months or years, typically with alternating periods of acute exacerbations and periods of clinical symptom absence. Although they are less aggressive than acute infections, their treatment is more challenging. There is up to a 1000-fold decrease in the susceptibility of biofilms to antimicrobial agents and disinfectants, as well as resistance to the host immune response. Thus, ways to reduce or inhibit biofilm formation are highly sought. The majority of the proposed biofilm-control methods focus on: (i) prevention and minimization of biofilm formation by selection and surface modifications of anti-adhesive materials; (ii) debridement techniques including ultrasound and surgical procedures; (iii) disruption of biofilm QS-signaling system; or (iv) achieving proper drug penetration and delivery to formed biofilms by the use of an electromagnetic field, ultrasound waves, photodynamic activation or specific drug delivery systems.

Alternate σ Factors

Since the σ subunit of bacterial RNA polymerase confers specificity on which promoters should be transcribed, altering the σ factor used is another way for bacteria to quickly and globally change which regulons are transcribed at a given time. The σ factor recognizes sequences within a bacterial promoter, so different σ factors will each recognize slightly different promoter sequences. In this way, when the cell senses specific environmental conditions, it may respond by changing which σ factor it expresses, degrading the old one, and producing a new one to transcribe the operons encoding genes whose products will be useful under the new environmental condition. For example, in sporulating bacteria of the genera Bacillus and Clostridium (which include many pathogens), a group of σ factors regulates the expression of the numerous genes required for sporulation in response to sporulation-stimulating signals.

Prokaryotic Attenuation and Riboswitches

Although most gene expression in prokaryotes is regulated at the level of transcription initiation, some mechanisms control both the completion of transcription and translation concurrently. Since their discovery, these mechanisms have been shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes.

One such regulatory system is attenuation, whereby secondary stem-loop structures formed within the 5’ end of an mRNA, as it is being transcribed, determine whether transcription to complete the synthesis of this mRNA will occur and whether this mRNA will be used for translation. Beyond the transcriptional repression mechanism already discussed, attenuation also controls the expression of the trp operon in E. coli as shown in Figure \(\PageIndex{15}\). The trp operon regulatory region contains a leader sequence called trpL between the operator and the first structural gene, which has four stretches of RNA that can base pair with each other in different combinations. When a terminator stem-loop forms, transcription terminates, releasing RNA polymerase from the mRNA. However, when an antiterminator stem-loop forms, it prevents the formation of the terminator stem-loop, allowing RNA polymerase to transcribe the structural genes.

Figure \(\PageIndex{15}\): Attenuation of Transcription and Translation. Parker, N., et. al. (2019) Microbiology. Openstax

When tryptophan is plentiful, translation of the short leader peptide encoded by trpL proceeds, the terminator loop between regions 3 and 4 forms, and transcription terminates. When tryptophan levels are depleted, translation of the short leader peptide stalls at region 1, allowing regions 2 and 3 to form an antiterminator loop, which enables RNA polymerase to transcribe the structural genes of the trp operon.

A related mechanism of concurrent regulation of transcription and translation in prokaryotes is the use of a riboswitch, a small region of noncoding RNA found within the 5’ end of some prokaryotic mRNA molecules, as shown in Figure \(\PageIndex{16}\). A riboswitch may bind to a small intracellular molecule to stabilize certain secondary structures of the mRNA molecule. The binding of the small molecule determines which stem-loop structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.

Figure \(\PageIndex{16}\): Riboswitch Form and function. Riboswitches found within prokaryotic mRNA molecules can bind to small intracellular molecules, stabilizing certain RNA structures, and influencing either the completion of the synthesis of the mRNA molecule itself (left) or the protein made using that mRNA (right). Parker, N., et. al. (2019) Microbiology. Openstax

Riboswitches found within prokaryotic mRNA molecules can bind to small intracellular molecules, stabilizing specific RNA structures and influencing either the completion of the mRNA synthesis process itself (left) or the protein produced using that mRNA (right).

Figure \(\PageIndex{17}\) shows interactive iCn3D models of a series of bacterial riboswitches. They are described in the legend below.

Guanine-responsive riboswitch bound to metabolite hypoxanthine (4FE5) A. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Nh1Sv7oSSyC6Z6	Divalent cation-sensing regulatory RNA (2QBZ) B. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VbU9Vf6DUA4VE8
Cyclid-di-GMP RNA riboswitch (3IRW) C. (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...wVntbdScP3VCF8	GlmS ribozyme bound to glucosamine-6-phosphate (2Z75) D. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...w9T4K9ffK1NU27

A: Guanine-responsive riboswitch bound to metabolite hypoxanthine (4FE5) - Hypoxanthine, involved in purine metabolism, is shown bound to RNA representing the 5' untranslated region of the xanthine phosphoribosyltransferase (xbt)/ xanthine-specific purine permease (pbux) genes that lead to transcription termination.

B: The M-box in mycobacterial genes regulating Mg²⁺ transport binds divalent cations. They are transcribed under low Mg²⁺ concentrations. Salt bridges (ion-ion interactions) are shown in cyan, and pi-cation interactions in red dotted lines

C: Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger in bacteria and regulates many cellular processes, including the formation of biofilms. The riboswitch shown here is from Vibrio cholerae. The U1 small nuclear ribonucleoprotein A is shown in cyan. Figure \(\PageIndex{18}\) shows a cartoon of the actual riboswitch in the 5' untranslated region of target genes

Figure \(\PageIndex{18}\)" Consensus secondary structure and sequence conservation of Cyclic di-GMP-I riboswitch

D. This ribozyme is in the 5′ untranslated region of glucosamine-6-phosphate synthase mRNA. The protein enzyme, 2. This protein enzyme catalyzes the conversion of fructose 6-phosphate and glutamine to glucosamine 6-phosphate (GlcN6P) and glutamate. The glmS ribozyme in the 5'-untranslated region cleaves itself on binding GlcN6P. This self-cleavage is inhibited by glucose 6-phosphate (Glc6P). Hence, high levels of the gene product for the synthase lead to cleavage of its own mRNA. The glmS ribozyme RNA is shown in gray, and its substrate RNA is shown in cyan.

Summary

This chapter provides an in‐depth exploration of the mechanisms by which cells regulate gene expression, addressing how genetically identical cells can exhibit distinct phenotypes. The material highlights the importance of both local (operon-level) and global regulatory strategies, integrating insights from prokaryotic and eukaryotic systems.

Key Concepts and Themes

• Foundations of Gene Regulation:
  Although every cell in an organism contains the same DNA, the selective expression of only a subset of genes at any given time enables cells to differentiate and respond to environmental cues. This regulation is crucial for both multicellular development and the efficient use of resources in single-celled organisms.

• Operon Organization in Prokaryotes:
  In bacteria, genes encoding proteins with related functions are often organized into operons—clusters of structural genes controlled by a single promoter and regulatory region. This organization allows for the coordinated expression of entire biochemical pathways.

  • Repressible Operons: The trp operon serves as a model where gene expression is turned off when tryptophan levels are high, as tryptophan binds to the trp repressor, enabling it to block transcription.
  • Inducible Operons: Conversely, the lac operon is activated only in the presence of lactose (and in the absence of glucose), with the lac repressor preventing transcription in the absence of the inducer. The CAP-cAMP complex further enhances transcription when energy levels are low.

• Regulatory Elements and Transcription Factors:
  Regulatory regions, including promoters and operators, serve as binding sites for transcription factors such as repressors and activators. These factors modulate the ability of RNA polymerase to initiate transcription, either by hindering its binding or by facilitating its recruitment.

• Global Regulatory Mechanisms:
  Beyond the control of individual operons, bacteria coordinate the expression of multiple genes and operons through regulons. Key regulatory molecules, such as alarmones (e.g., pppGpp), enable cells to adjust gene expression in response to stress (the stringent response). Additionally, quorum sensing allows bacteria to communicate via diffusible signals, coordinating collective behaviors like biofilm formation and virulence.

• Alternative σ Factors:
  Bacterial RNA polymerase employs different σ subunits to recognize distinct promoter sequences. By switching σ factors, cells can rapidly reprogram gene expression in response to environmental changes, such as during sporulation or stress responses.

• Post-Initiation Regulation – Attenuation and Riboswitches:
  Some operons are further regulated by mechanisms that modulate transcription elongation. Attenuation, exemplified by the trp operon, uses alternative RNA secondary structures in a leader sequence to either terminate or allow transcription. Riboswitches, on the other hand, are small regulatory RNA elements that bind specific metabolites; their conformational changes can affect either transcription termination or translation initiation.

Conclusion

Overall, this chapter underscores that gene regulation is a multifaceted process critical for cellular function and adaptation. By coordinating the expression of structural and regulatory genes through operon organization, transcription factor binding, global signaling molecules, and RNA-based regulatory mechanisms, cells can finely tune their proteomic output in response to internal and external signals. Understanding these processes is fundamental not only to basic biology but also to addressing health-related issues such as cancer, infectious diseases, and antibiotic resistance.

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