Chapter 15 Regulation of Gene Expression in Bacteria.

Figure 15.3

All three structural genes are transcribed as a single unit, resulting in a so-called polycistronic mRNA (cistron refers to the part of a nucleotide sequence coding for a single gene). This results in the coordinate regulation of all three genes, since a single-message RNA is simultaneously translated into all three gene products.

In addition to regulating gene expression in response to environmental conditions, bacteria also must respond to attacks from bacteriophages-the viruses that infect them. Later in this chapter we will explore the regulation of a genetic system called _______________ that serves as an immune system to fight invading bacteriophage DNA sequences.

CRISPR-Cas

While it was once thought that bacteria were not advanced enough to possess such immunity, we now know that bacteria possess an immune system called ____________ that can be adapted to fight specific types of phage by preventing phage gene expression and subsequent phage reproduction.

CRISPR-Cas

______________ is an adaptive immune system in bacteria

CRISPR-Cas

The well studied CRISPR-Cas system of Streptococcus pyogenes uses a single nuclease called _____ for target interference while many other species require a large complex of Cas proteins

Cas9

What happens if all the lactose present is metabolized?

If all lactose is metabolized, none is available to bind to the repressor, which is again free to bind to operator DNA and to repress transcription.

T/F Bacteria have an adaptive immune system that uses an RNA-guided nuclease to cleave invading viral DNA in a sequence-specific manner

T

The regulation of gene expression has been extensively studied in bacteria, particularly in E.coli. T/F Geneticists have learned that highly efficient genetic mechanisms have evolved in these organisms to turn transcription of specific genes on and off, depending on the cell's metabolic need for the respective gene products. T/F Not only do bacteria respond to changes in their environment, but they also regulate gene activity associated with a variety of cellular activities, including the replication, recombination, and repair of their DNA, and with cell division.

T T

T/F many other bacterial operons use attenuation to control gene expression

T. These include operons that encode enzymes involved in the biosynthesis of amino acids such as threonine, histidine, leucine, and phenylalanine.

Figure 15.1

The discovery of a regulatory gene and a regulatory site that are part of the gene cluster was paramount to the understanding of how gene expression is controlled in the system. Neither of these regulatory elements encodes enzymes necessary for lactose metabolism-the function of the three genes in the cluster. The three structural genes and the adjacent regulatory site constitute the lactore (lac) operon. Together, the entire gene cluster functions in an integrated fashion to provide a rapid response to the presence or absence of lactose. Mapping studies by Lederberg established that all three genes are closely linked or contiguous to one another on the bacterial chromosome, in the order Z-Y-A.

Bertrand and his colleagues observed that, when tryptophan is present and the trp operon is repressed, initiation of transcription still occurs at a low level but is subsequently terminated at a point about 140 nucleotides along the transcript. They call this process _____________, as it "weakens or impairs" expression of the operon.

attenuation

RNAs are sometimes involved in the regulation of gene expression in bacteria, including the process of ___________, and the involvement of _____________

attuenuation riboswitches

Further investigation showed that a series of enzymes encoded by ______ contiguous genes on the E.coli chromosome are involved in tryptophan synthesis. These genes are part of an _________, and in the presence of tryptophan, all are coordinately ________ and none of the enzymes are produced.

five operon repressed

Lactose metabolism in E.coli is regulated by an ____________ system

inducible

Research provided insights into the way in which gene activity is repressed when lactose is absent but induced when it is available. In the presence of lactose, the concentration of the enzymes responsible for its metabolism increases rapidly from a few molecules to thousands per cell. The enzymes responsible for lactose metabolism are thus ______________, and lactose serves as the ____________

inducible inducer

While bacteria have evolved mechanisms to ward off phage infection, most of these mechanisms provide _______ immunity because they are not tailored to a specific phage

innate immunity For example, some bacteria possess mechanisms to prevent phage binding to the cell surface, block phage DNA from entering the cell, or induce suicide in infected cells to prevent the spread of infection.

In contrast, when tryptophan is absent or present in very low concentrations, transcription is initiated but is not subsequently terminated, instead...

instead continuing beyond the leader sequence into the structural genes.

The Catabolite-Activating protein (CAP) exerts _______ control over the lac operon

positive

Regulation of the lac operon by catabolite repression results in efficient energy use, because the presence of glucose will override the need for the metabolism of lactose, if lactose is available to the cell. In contrast to the negative regulation conferred by the lac repressor, the action of cAMP-CAP constitutes positive regulation. Thus, a combination of ___________ and _____________ regulatory mechanisms determines transcription levels of the lac operon.

positive and negative

Events at the _______________ determine whether the genes are transcribed into mRNA and thus whether the corresponding enzymes or other protein products may be synthesized from the genetic information in the mRNA.

regulatory region

The Tryptophan (trp) operon in E.coli is a __________ gene system.

repressible

Unlike the lac operon, the trp operon is ____________. In the presence of _________, the repressor binds to the regulatory region of the trp operon (trpO) and _________ transcription initiation.

repressible tryptophan represses

To study the genes coding for these three enzymes, researchers isolated numerous mutations that lacked the function of one or the other enzyme. Such lac- mutants were first isolated and studied by Jousha Lederberg. Mutant cells that fail to produce active β-galactosidase (lacZ-) or permease (lacY-) are..

unable to use lactose as an energy source

The operon model is a good one because it leads to three major predictions that can be tested to determine its validity. The major predictions to be tested are that (1) the I gene produces a diffusible product (that is, a trans-acting product), (2) the O region is involved in regulation but does not produce a product (it is cis-acting), and (3) the O region must be adjacent to the structural genes to regulate transcription. The creation of a partially diploid bacteria allows us to assess these assumptions, particularly those that predict the presence of trans-acting regulatory elements. How?

For example, the F plasmid may contain chromosomal genes, in which case it is designated F'. When an F- cell acquires such a plasmid, it contains its own chromosome plus one or more additional genes present in the plasmid. This host cell is thus a merozygote, a cell that is diploid for certain added genes (but not for the rest of the chromosome). The use of such a plasmid makes it possible, for example, to introduce an I^+ gene into a host cell whose genotype is I-, or to introduce an O^+ region into a host cell of genotype O^C. Adding an I^+ gene to an I^- cell should restore inducibility, because the normal wild-type repressor, which is a trans-acting factor, would be produced by the inserted I^+ gene. In contrast, adding an O^+ region to an O^C cell should have no effect on constitutive enzyme production, since regulation depends on an O^+ region being located immediately adjacent to the structural genes-that is, O^+ is a cis-acting element

How does lactose stimulate transcription of the lac operon and induce the synthesis of the enzymes for which it codes? A partial answer came from the studies using gratuitous inducers, chemical analogs of lactose such as the sulfur-containing analog isopropylthiogalactoside (IPTG). How were gratuitous inducers used and what did they show?

Gratuitous inducers behave like natural inducers, but they do not serve as substrates for the enzymes that are subsequently synthesized. Their discovery provides strong evidence that the primary induction event does not depend on the interaction between the inducer and the enzyme.

Another prediction of the operon model is that certain mutations in the I gene should have the opposite effect of I-. That is, instead of being constitutive because the repressor cannot bind the operator, mutant repressor molecules should be produced that cannot interact with the inducer, lactose. Thus, these repressors would always bind to the operator sequence, and the structural genes would be permanently repressed. In cases like this, the presence of an additional I^+ gene would have little or no effect on repression. In fact, such a mutation, ___, was discovered wherein the operon,as predicted, is "superrepressed," as shown in part D of table 15.1 and in the figure on the backside of the card Figure 15.7.

I^S An additional I^+ gene does not effectively relieve repression of gene activity. These observations are consistent with the idea that the repressor contains separate DNA-binding domains and inducer-binding domains.

When grown in minimal medium, wild-type E.coli produce the enzymes necessary for the biosynthesis of amino acids as well as many other essential macromolecules. Focusing his studies on the amino acid tryptophan and the enzyme tryptophan synthetase, Monod discovered that if tryptophan is present in sufficient quality in the growth medium...

If tryptophan is present in sufficient quality in the growth medium, the enzymes necessary for its synthesis are not produced. It is energetically advantageous for bacteria to repress expression of genes involved in tryptophan synthesis when ample tryptophan is present in the growth medium.

Figure 15.14 The mechanism of CRISPR-Cas adaptive immunity in bacteria

In addition to the CRISPR loci, adaptive immunity is dependent on a set of adjacent CRISPR-associated (cas) genes. The cas genes encode a wide variety of proteins such as DNases, RNases, and proteins of unknown function . The CRISPR-Cas mechanism includes three steps outline in the figure. 1. The first step is known as spacer acquisition. Invading phage DNA is cleaved into small fragments, which are directly inserted into the CRISPR locus to become new spacers. The Cas1 nuclease and an associated Cas2 protein are required for spacer acquisition. New spacers are inserted proximal to the leader sequence of the CRISPR locus, with older spacers being located progressively more distal. When new spacers are added, repeat sequences are duplicated such that each spacer is flanked by repeats. 2. In the second step, CRISPR loci are transcribed, starting at the promoter within the leader. These long transcripts are then processed into short CRISPR-derived RNAs (crRNAs)(or the "guide" RNA), each containing a single spacer and repeat sequences (on either or both sides). This step is called crRNA biogenesis. 3. The third step is referred to as target interference. Mature crRNAs associate with Cas nucleases and recruit them to complementary sequences in invading phage DNA. The Cas nucleases then cleave the viral DNA, thus neutralizing infection. In short, CRISPR-Cas is a simple system whereby bacteria incorporate small segments of viral DNA into their own genome and then express them as short crRNAs that guide a nuclease to cleave complementary viral DNA. Thus, CRISPR-Cas uses viral DNA sequences to specifically fight that same virus.

What, then, is the role of lactose in induction? The answer to this question requires the study of another class of mutations described as constitutive mutations. Explain these.

In cells bearing these types of mutations, enzymes are produced regardless of the presence or absence of lactose.

Figure 15.8

In the absence of glucose and under inducible conditions, CAP exerts positive control by binding to the CAP site, facilitating RNA-polymerase binding at the promoter, and thus transcription. Therefore, for maximal transcription of the structural genes to occur, the repressor must be bound by lactose (so as not to repress operon expression) and CAP must be bound to the CAP-binding site. This leads to the central question about CAP: How does the presence of glucose inhibit CAP binding? The answer involves still another molecule, cyclic adenosine monophosphate (cAMP), which is a nucleotide with an adenine base, a ribose sugar, and a single phosphate bound to the sugar at both the 5' and 3' positions. In order to bind to the promoter, CAP must first be bound to cAMP. The level of cAMP is itself dependent on an enzyme, adenyl cyclase, which catalyzes the conversion of ATP to cAMP. The role of glucose in catabolite repression is now clear. It inhibits the activity of adenyl cyclase, causing a decline in the level of cAMP in the cell. Under this condition, CAP cannot form the cAMP-CAP complex essential to the positive stimulation of transcription of the lac operon.

Figure 15.6 The response of the lac operon in the absence of lactose when a cell bears either the I- or the O^C mutation.

In the case of the I- mutant, seen in (a), the repressor protein is altered or absent and cannot bind to the operator region, so the structural genes are always turned on . In the case of the O^C mutant (b), the nucleotide sequence of the operator DNA is altered and will not bind with a normal repressor molecule. The result is the same: The structural genes are always transcribed.

The idea that microorganisms regulate the synthesis of their gene products is not a new one. As early as 1900, it was shown that when lactose (a galactose and glucose-containing disaccharide) is present in the growth medium of yeast, the organisms synthesize enzymes required for lactose metabolism. When lactose is absent, synthesis diminishes to a basal level. Soon thereafter investigators generalized that bacteria adapt to their environment, producing certain enzymes only when specific chemical substrates are present. These enzymes are referred to as ____________________. In contrast, enzymes that are produced continuously, are called _____________________

Inducible enzymes (are these still produced at the basal level?) constitutive enzymes

Figure 15.10 How attenuation works

It was proposed that the initial DNA sequence that is transcribed gives rise to an mRNA sequence that has the potential to fold into mutually exclusive stem-loop structures referred to as hairpins. If tryptophan is scarce, an mRNA secondary structure referred to as the antiterminator hairpin is formed. Transcription proceeds past the antiterminator hairpin region, and the entire mRNA is subsequently produced. Alternatively, in the process of excess tryptophan, the mRNA structure that is formed is referred to as a terminator hairpin, and transcription is almost always terminated prematurely, just beyond a region called the attenuator. A key point in this model is that the transcript of the leader sequence (see figure 15.9) must be translated for the antiterminator hairpin to form. This leader transcript contains two triplets (UGG) that encode tryptophan, and these are present just downstream of the AUG sequence that signals the initiation of translation by ribosomes. When adequate tryptophan is present, charged tRNA^trp is present in the cell, whereby ribosomes translate these UGG triplets, proceed through the attenuator, and allow the terminator hairpin to form, and the operon is not transcribed. If cells are starved of tryptophan, charged tRNA^trp will be unavailable and ribosomes "stall" during translation of the UGG triplets. Thus, the antiterminator hairpin forms within the leader transcript, and as a result, transcription proceeds, leading to expression of the entire set of structural genes.

Figure 15.5 (read all)

Jacob and Monod proposed a hypothetical mechanism involving negative control that they called the operon model, in which a group of genes is regulated and expressed together as a unit. The lac operon they proposed consists of the Z, Y, and A structural genes, as well as the adjacent sequences of DNA referred to as the operator region. They argued that the lacI gene regulates transcription of the structural genes by producing a repressor molecule and that the repressor is allosteric, meaning that the molecule reversibly interacts with another molecule, undergoing both a conformational change in three-dimensional shape and a change in chemical activity. They suggested that the repressor normally binds to the DNA sequence of the operator region. When it does so, it inhibits the action of RNA polymerase, effectively repressing the transcription of the structural genes (b). However, when lactose is present, this sugar binds to the repressor and causes an allosteric (conformational) change. The change alters the binding site of the repressor, rendering it incapable of interacting with operator DNA (c) In the absence of the repressor-operator interaction, RNA polymerase transcribes the structural genes, and the enzymes necessary for lactose metabolism are produced. Because transcription occurs only when the repressor fails to bind to the operator region, regulation is said to be under negative control. To summarize, the operon model invokes a series of molecular interactions between proteins, inducers, and DNA to explain the efficient regulation of structural gene expression. In the absence of lactose, the enzymes encoded by the genes are not needed and the expression of genes encoding these enzymes is repressed. When lactose is present, it indirectly induces the activation of the genes by binding with the repressor.

Figure 15.9

Support for the concept of a repressible operon was soon forthcoming, based primarily on the isolation of two distinct categories of constitutive mutations. The first class, trpR-, maps at a considerable distance from the repressor. Presumably, the mutation inhibits either the repressor's interaction with tryptophan or repressor formation entirely. Whichever the case, repression never occurs in cells with the trpR- mutation. -As expected, if the trpR+ gene encodes a functional repressor molecule, the presence of a copy of this gene will restore repressibility. The second constitutive mutation is analogous to that of the operator of the lactose operon, because it maps immediately adjacent to the structural genes. Furthermore, the insertion of a plasmid bearing a wild-type operator into mutant cells does not restore repression. This is what would be predicted if the mutant operator no longer interacts with the repressor-tryptophan complex. The five contiguous structural genes (trpE, D, C, B, and A) are transcribed as a polycistronic message irecting translation of the enzymes that catalyze the biosynthesis of tryptophan. As in the lac operon, a promoter region (trpP) represents the binding site for RNA polymerase, and an operator region (trpO) is bound by the repressor. In the absence of binding, transcription is initiated within the trpP-trpO region and proceeds along a leader sequence 162 nucleotides prior to the first structural gene (trpE). Within that leader sequence, still another regulatory site has been demonstrated, called an attenuator-the subject of next section.

T/F In both types of constitutive mutants, the enzymes are produced continually, inducibility is eliminated, and gene regulation has been lost.

T

T/F In theory, either type of control or a combination of the two can govern inducible or repressible systems.

T

T/F The location of the regulatory region is almost always upstream (5') of the gene cluster it controls.

T

T/F Although most bacterial gene products are present continuously at a basal level (a few copies), the concentration of these products can increase dramatically when required.

T Clearly, fundamental regulatory mechanisms must exist to control the expression of the genetic information.

Figure 15.11

The mechanism of riboswitch regulation involves short ribonucleotide sequences (or elements) present in the 5'-untranslated regions of mRNAs (UTRs). These RNA elements are capable of binding with small molecule ligands, such as metabolites, whose synthesis or activity is controlled by the genes encoded by the mRNA. Such binding causes a conformational change in one domain of the riboswitch element, which induces another change at a second RNA domain, most often creating a transcription terminator structure. This terminator structure interfaces directly with the transcriptional machinery and shuts it down. Riboswitches can recognize a broad range of ligands, including amino acids, purines, vitamin cofactors, amino sugars, and metal ions. The two important domains within a riboswitch are the ligand-binding site, called the aptamer, and the expression platform, which is capable of forming the terminator structure. The 5'-UTR of an mRNA is shown of the left side of the figure in the absence of the ligand (metabolite). RNA polymerase has transcribed the unbound ligand-binding site, and in the default conformation, the expression domain adopts an antiterminator conformation. Thus transcription continues through the expression platform and into the coding region. On the right side of the figure, the presence of the ligand on the ligand-binding site induces an alternative conformation in the expression platform, creating the terminator conformation. RNA polymerase is effectively blocked, and transcription ceases.

Under negative control, what happens?

Under negative control, genetic expression occurs unless it is shut off by some form of a regulator molecule.

Under positive control, what happens?

Under positive control, transcription occurs only if a regulator molecule directly stimulates RNA production.

Results of such experiments described above are shown in table 15.1

Z represents the structural genes (and the inserted genes are listed after the designation F'). In both cases described above, the model is upheld (part b). Part c of the table shows the reverse experiment, where either an I- gene or an O^C region is added to cells of normal inducible genotypes. As the model predicts, inducibility is maintained in these partial diploids.

In contrast, _________ immunity refers to a defense mechanism whereby past exposure to a pathogen stimulates a robust defense against future expose to the same pathogen.

adaptive immunity For example, adaptive immunity in humans enables vaccines to provide protection against specific disease-causing viruses.

To account for repression, Jacob and Monod suggested the presence of a normally inactive repressor that alone cannot interact with the operator of the operon. However, the repressor is an ________ molecule that can bind to tryptophan.

allosteric

Both I- and O^C constitutive mutations interfere with these molecular interactions, allowing...

allowing continuous transcription of the structural genes.

As described earlier, the role of β-galactosidase is to cleave lactose into its components, glucose and galactose. Then, to be used by the cell, the galactose, too, must be converted to glucose. What if the cell found itself in an environment that contained ample amounts of both lactose and glucose? Given that glucose is the preferred carbon source for E.coli it would not be energetically efficient for a cell to induce transcription of the lac operon, since what it really needs-glucose-is already present. As we will see next, still another molecular component, called the ___________________, is involved in diminishing the expression of the lac operon when glucose is present. This inhibition is called _________________

catabolite-activating protein (CAP) catabolite repression

Because the regulatory region is on the same strand as those genes, we refer to it as a ______________

cis-acting site

A CRISPR is a genomic locus in bacteria that contains Figure 15.13

clustered regularly interspaced short palindromic repeats. CRISPR loci were first identified in 1987 in the escherichia coli genome based on a simple description of repeated DNA sequences with nonrepetitive spacer sequences between them. The spacers remained a mystery until 2005 where three independent studies demonstrated that CRISPR spacer sequences were identical to fragments of phage genomes. This insight led to speculation that viral sequences within CRISPR loci serve as a "molecular memory" of previous viral attacks.

As tryptophan participates in repression, it is referred to as a ____________ in this regulatory scheme

corepressor

Bacteria regulate gene expression in response to ___________________

environmental conditions

Previous chapters have discussed how DNA is organized into genes, how genes store genetic information, and how this information is expressed through the process of transcription and translation. We now consider one of the most fundamental questions in molecular genetics: How is gene expression regulated? It is now clear the gene expression varies widely in bacteria under different _______________ conditions

environmental. For example, detailed analysis of proteins in Escherichia coli shows that concentrations of the 4000 or so polypeptide chains encoded by the genome vary widely. Some proteins may be present in as few as 5 to 10 molecules per cell, whereas others, such as ribosomal proteins and the many proteins involved in the glycolytic pathway, are present in as many as 100,000 copies per cell.

The lacZ gene encodes β-galactosidase, an enzyme whose primary role is to..

is to convert the disaccharide lactose to the monosaccharides glucose and galactose. This conversion is essential if lactose is to serve as the primary energy source in glycolysis.

There are three structural genes in the lac operon. What are they

lacZ gene lacY gene lacA gene

attenuation occurs in a ________ sequence that contains an __________ region

leader attenuator

Cis-acting regulatory regions are bound by

molecules that control transcription of the gene cluster. Such molecules are called trans-acting factors.

When tryptophan is present, the resultant complex of repressor and tryptophan attains a new conformation that binds to the operator, repressing transcription. Thus, when tryptophan, the end product of this anabolic pathway, is present, the system is repressed and enzymes are not made. Since the regulatory complex inhibits transcription of the operon, this repressible system is under _________ control.

negative

Genes involved in the metabolism of lactose are coordinately regulated by a _______ control system that responds to:

negative control system that responds to the presence or absence of lactose.

Regulation, whether of the inducible or repressible type, may be under either _____________ or _____________ control

negative or positive control

Gene regulation in bacteria can also occur through the interactions of regulatory molecules with specific regions of a nascent mRNA, after transcription has been initiated. The binding of these regulatory molecules alters the secondary structure of the mRNA, leading to premature transcription termination or repression of translation. We will discuss two types of regulation involving RNA-attenuation and riboswitches.

okay

A second set of constitutive mutations producing effects identical to those of lacI- is present in a region immediately adjacent to the structural genes. This class of mutations, designated by lacO^C, is located in the _____________ of the operon.

operator region

In bacteria, genes that code for enzymes with related functions (for example, the set of genes involved with lactose metabolism) tend to be organized in clusters on the bacterial chromosome, and transcription of these genes is often under the coordinated control of a single regulatory region. Such clusters, including their adjacent regulatory sequences, are called _______________.

operons

The second gene, lacY, specifies the primary structure of ____________, an enzyme that facilitates the entry of lactose into the bacterial cell.

permease

More recent investigation has revealed a contrasting system, whereby the presence of a specific molecule inhibits gene expression. Such molecules are usually end products of biosynthetic pathways. For example, utilizing a multistep metabolic pathway, the amino acid tryptophan can be synthesized by bacterial cells. If a sufficient supply of tryptophan is present in the environment or culture medium, then it is inefficient for the organism to expend energy to synthesize the enzymes necessary for tryptophan production. A mechanism has therefore evolved whereby tryptophan plays a role in repressing the transcription of mRNA needed for producing tryptophan-synthesizing enzymes. In contrast to the inducible system controlling lactose metabolism, the system governing tryptophan expression is said to be _______________________

repressible

Studies of the constitutive mutation lacI- mapped the mutation to a site on the bacterial chromosome close to, but not part of the structural genes lacZ, lacY, and lacA. This mutation led researchers to discover the lacI gene, which is appropriately called a _________

repressor gene.

Since the elucidation of attenuation in the trp operon, numerous cases of gene regulation that also depend on alternative forms of mRNA secondary structure have been documented. These are examples of what are more generally referred to as __________________

riboswitches

Genes coding for the primary structure of an enzyme are called ____________________

structural genes

Binding of a __________________ at a ___________________ can regulate the gene cluster either negatively (by turning off transcription) or positively (by turning on transcription of genes in the cluster).

trans-acting element cis-acting site

The third gene, lacA, codes for the enzyme ______________

transacetylase. While its physiological role is still not completely clear, it may be involved in the removal of toxic by-products of lactose digestion from the cell.