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31. The Control of Gene Expression in Prokaryotes
Bacteria respond to changes in their environments. A micrograph of the light organ of a newly hatched squid (Euprymna scolopes) is shown on the le:. The light spots are due to colonies of the bacteria Vibrio fischerili that live symbioBcally within these organs. These bacteria become luminescent when they reach an appropriately high density. The density is sensed by the circuit shown on the right in which each bacterium releases a small molecule into the environment. The molecule is subsequently taken up by other bacterial cells, which start a signaling cascade that s@mulates the expression of specific genes.
Even simple prokaryoBc cells must respond to changes in their metabolism or in their environments. Much of this response takes place through changes in gene expression. Genomes comprise thousands of genes. Some of these genes are expressed all the Bme. These genes are subject to consBtuBve expression. Many other genes are expressed only under some circumstances—that is, under a par@cular set of physiological condi@ons. These genes are subject to regulated expression. For example, the level of expression of some genes in bacteria may vary more than a 1000-‐fold in response to the supply of nutrients or to environmental challenges. In this chapter, we will examine gene-‐regula@on mechanisms in prokaryotes, par@cularly E. coli, because many of these processes were first discovered in this organism. In Chapter 32, we will turn to gene-‐regula@on mechanisms in eukaryotes. We shall see both substanBal similariBes and fundamental differences in comparing gene-‐regulatory mechanisms of the two types of organisms.
prokaryotes eukaryotes
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How is gene expression controlled? Gene acBvity is controlled first and foremost at the level of transcripBon. Whether a gene is transcribed is determined largely by the interplay between specific DNA sequences and certain proteins that bind to these sequences. Most o:en, these proteins repress the expression of specific genes by blocking the access of RNA polymerase to their promotors.
Some genes are also controlled at stages beyond the level of transcripBon and we shall examine several mechanisms at these stages. Finally, we will examine several important examples of how gene expression is regulated in response to changes in the concentra@ons of specific molecules in the environment of prokaryo@c cells.
In some cases, however, the proteins can acBvate the expression of specific genes. We shall learn about several different strategies that allow the coordinated regulaBon of sets of genes.
31.1 Many DNA-‐Binding Proteins Recognize Specific DNA Sequences
How do regulatory systems disBnguish the genes that need to be acBvated or repressed from genes that are consBtuBve? A:er all, the DNA sequences of genes themselves do not have any dis@nguishing features that would allow regulatory systems to recognize them. Instead, gene regula@on depends on other sequences in the genome. Regulatory sites are usually binding sites for specific DNA-‐binding proteins, which can s@mulate or repress gene expression. In the presence of the sugar lactose, the bacterium starts to express a gene encoding β-‐galactosidase, an enzyme that can process lactose for use as a carbon and energy source.
The nucleo@de sequence of this site shows a nearly perfect inverted repeat, indica@ng that the DNA in this region has an approximate twofold axis of symmetry. Symmetry in such regulatory sites usually corresponds to symmetry in the protein that binds the site. Symmetry matching is a recurring theme in protein–DNA interacBons.
Lac regulatory site sequence
Arginine residue forms a pair of hydrogen bonds with a guanine residue
Helix-‐turn-‐helix moBf is common to many DNA-‐binding proteins
Are similar strategies uBlized by other prokaryoBc DNA-‐binding proteins?
DNA-‐ binding surfaces of many, but not all, of these proteins consist of a pair of a helices separated by a Bght turn (Figure 31.3).
The recogniBon helix (the second helix) lies in the major groove, where amino acid side chains make contact with the edges of base pairs. In contrast, residues of the first helix par@cipate primarily in contacts with the DNA backbone.
Helix-‐turn-‐helix mo@fs are present on many proteins that bind DNA as dimers, and thus two of the units will be present, one on each monomer.
9
Although the helix-‐turn-‐helix mo@f is the most commonly observed DNA-‐binding unit in prokaryotes, not all regulatory proteins bind DNA through such units. A striking example is provided by the E. coli methionine repressor (Figure 31.4). This protein binds DNA through the inser@on of a pair of β strands into the major groove.
31.2 ProkaryoBc DNA-‐Binding Proteins Bind Specifically to Regulatory Sites in Operons E. coli usually rely on glucose as their source of carbon and energy, even when other sugars are available. However, when glucose is scarce, E. coli can use lactose as their carbon source, even though this disaccharide does not lie on any major metabolic pathways. An essen@al enzyme in the metabolism of lactose is β-‐galactosidase, which hydrolyzes lactose into galactose and glucose.
This reacBon can be conveniently followed in the laboratory through the use of alterna@ve galactoside substrates that form colored products such as X-‐Gal (Figure 31.5).
An E. coli cell growing on a carbon source such as glucose or glycerol contains fewer than 10 molecules of β-‐galactosidase. In contrast, the same cell will contain several thousand molecules of the enzyme when grown on lactose (Figure 31.6). The presence of lactose in the culture medium induces a large increase in the amount of β-‐galactosidase by elici@ng the synthesis of new enzyme molecules rather than by ac@va@ng a preexis@ng but inac@ve precursor.
A crucial clue to the mechanism of gene regula@on was the observaBon that two other proteins are synthesized in concert with β-‐galactosidase— namely, galactoside permease and thiogalactoside transacetylase. The permease is required for the transport of lactose across the bacterial cell membrane (Sec@on 13.3). The transacetylase is not essenBal for lactose metabolism but appears to play a role in the detoxificaBon of compounds that also may be transported by the permease. Thus, the expression levels of a set of enzymes that all contribute to the adaptaBon to a given change in the environment change together. Such a coordinated unit of gene expression is called an operon.
An operon consists of regulatory elements and protein-‐encoding genes The parallel regula@on of β-‐galactosidase, the permease, and the transacetylase suggested that the expression of genes encoding these enzymes is controlled by a common mechanism. François Jacob and Jacques Monod (Nobel, 1965) proposed the operon model to account for this parallel regula@on as well as the results of other gene@c experiments. The gene@c elements of the model are a regulator gene that encodes a regulatory protein, a regulatory DNA sequence called an operator site, and a set of structural genes (Figure 31.7).
The regulator gene encodes a repressor protein that binds to the operator site. The binding of the repressor to the operator prevents transcripBon of the structural genes. The operator and its associated structural genes cons@tute the operon. For the lactose (lac) operon, the i gene encodes the repressor, o is the operator site, and the z, y, and a genes are the structural genes for β-‐galactosidase, the permease, and the transacetylase, respec@vely. The operon also contains a promoter site (denoted by p), which directs the RNA polymerase to the correct transcripBon iniBaBon site. The z, y, and a genes are transcribed to give a single mRNA molecule that encodes all three proteins. An mRNA molecule is known as a polygenic or polycistronic transcript.
The lac repressor protein in the absence of lactose binds to the operator and blocks transcripBon
The lac repressor exists as a tetramer of 37-‐kd subunits with two pairs of subunits coming together to form the DNA-‐binding unit previously discussed. In the absence of lactose, the repressor binds very @ghtly and rapidly to the operator. When the lac repressor is bound to DNA, the repressor prevents RNA polymerase from transcribing the protein-‐coding genes inasmuch as the operator site is directly adjacent to and downstream of the promoter site where the repressor would block the progress of RNA polymerase.
In the absence of lactose, the lactose operon is repressed. How does the lac repressor mediate this repression?
Lac repressor dimer 1EFA.pdb
Protein Data Bank Lac Repressor
How does the lac repressor locate the operator site in the E. coli chromosome? The lac repressor binds 4 x 106 Bmes as strongly to operator DNA as it does to random sites in the genome. This high degree of selecBvity allows the repressor to find the operator efficiently even with a large excess (4.6 x 106 bp) of other sites within the E. coli genome. The dissociaBon constant (Kd) is approximately 0.1 pM (10-‐13 M). The rate constant for associa@on (< 1010 M-‐1 s-‐1) is strikingly high, indica@ng that the repressor finds the operator primarily by diffusing along a DNA molecule (1D search) rather than encountering it from the aqueous medium (3D search). This diffusion has been confirmed by studies that monitored the behavior of fluorescently labeled single molecules of lac repressor inside living E. coli cells.
a(1 – be–kt)
How does the presence of lactose trigger the relief of this repression and, hence, the expression of the lac operon? Interes@ngly, lactose itself does not have this effect; rather, allolactose. Allolactose is thus referred to as the inducer of the lac operon. Allolactose is a side product of the β-‐galactosidase reac@on and is produced at low levels by the few molecules of β-‐galactosidase that are present before induc@on. Some other β-‐galactosides such as isopropylthiogalactoside (IPTG) are potent inducers of β-‐galactosidase expression, although they are not substrates of the enzyme. IPTG is useful in the laboratory as a tool for inducing gene expression in engineered bacterial strains.
Ligand binding can induce structural changes in regulatory proteins
The inducer triggers gene expression by preven@ng the lac repressor from binding the operator. The inducer binds to the lac repressor and thereby greatly reduces the repressor’s affinity for operator DNA.
An inducer molecule binds in the center of the large domain within each monomer. This binding leads to conformaBonal changes that modify the rela@on between the two small DNA-‐binding domains (Figure 31.9). These domains can no longer easily contact DNA simultaneously, leading to a dramaBc reducBon in DNA-‐binding affinity.
Let us recapitulate the processes that regulate gene expression in the lactose operon (Figure 31.10). In the absence of inducer, the lac repressor is bound to DNA in a manner that blocks RNA polymerase from transcribing the z, y, and a genes. Thus, very lihle β-‐galactosidase, permease, or transacetylase are produced.
The addiBon of lactose to the environment leads to the formaBon of allolactose. This inducer binds to the lac repressor, leading to conforma@onal changes and the release of DNA by the lac repressor. With the operator site unoccupied, RNA polymerase can then transcribe the other lac genes and the bacterium will produce the proteins necessary for the efficient use of lactose.
The operon is a common regulatory unit in prokaryotes
Many other gene-‐regulatory networks func@on in ways analogous to the lac operon. For example, genes taking part in purine and, to a lesser degree, pyrimidine biosynthesis are repressed by the pur repressor. This dimeric protein is 31% idenBcal in sequence with the lac repressor and has a similar three-‐dimensional structure. However, the behavior of the pur repressor is opposite that of the lac repressor: whereas the lac repressor is released from DNA by binding to a small molecule, the pur repressor binds DNA specifically, blocking transcripCon, only when bound to a small molecule. Such a small molecule is called a corepressor. For the pur repressor, the corepressor can be either guanine or hypoxanthine. The dimeric pur repressor binds to inverted-‐repeat DNA sites of the form 5’-‐ANGCAANCGNTTNCNT-‐3’, in which the bases shown in bold-‐ face type are par@cularly important.
Examina@on of the E. coli genome sequence reveals the presence of more than 20 such sites, regula@ng 19 operons and including more than 25 genes (Figure 31.11).
Because the DNA binding sites for these regulatory proteins are short, it is likely that they evolved independently and are not related by divergence from an ancestral regulatory site. Once a ligand-‐regulated DNA-‐binding protein is present in a cell, binding sites for the protein may arise by mutaBon adjacent to addiBonal genes. Binding sites for the pur repressor have evolved in the regulatory regions of a wide range of genes taking part in nucleoBde biosynthesis. All such genes can then be regulated in a concerted manner. The organiza@on of prokaryo@c genes into operons is useful for the analysis of completed genome sequences. Some@mes a gene of unknown funcBon is discovered to be part of an operon containing well-‐characterized genes. Such associa@ons can provide powerful clues to the biochemical and physiological func@ons of the uncharacterized gene.
TranscripBon sBmulated by proteins that contact RNA polymerase Glucose: a preferred energy source, When glucose is abundant, the synthesis of these enzymes would be wasteful. Glucose has an inhibitory effect on the
genes encoding these enzymes, an effect called catabolite repression. It is due to the fact that glucose lowers the concentraBon of cyclic AMP in E. coli.