28.1 The Proteins and RNAs of Gene Regulation in Chapter 28 Regulation of Gene Expression

28.1 The Proteins and RNAs of Gene Regulation

Transcription is mediated and regulated by protein-DNA interactions, especially those involving the protein components of RNA polymerase (Chapter 26). We first consider how the activity of RNA polymerase is regulated. Next, we proceed to a general description of the proteins participating in this regulation. We then examine the molecular basis for the recognition of specific DNA sequences by DNA-binding proteins. Regulatory RNAs are encountered most often in the regulation of mRNA translation in eukaryotes, but also play a role in the life of some bacterial mRNAs. We consider them briefly in this section, and in more detail in Section 28.3.

RNA Polymerase Binds to DNA at Promoters

RNA polymerases bind to DNA and initiate transcription at promoters (see Fig. 26-5), sites generally found near points at which RNA synthesis begins on the DNA template. The regulation of transcription initiation often entails changes in how RNA polymerase interacts with a promoter.

The nucleotide sequences of promoters vary considerably, affecting the binding affinity of RNA polymerases and thus the frequency of transcription initiation. Some Escherichia coli genes are transcribed once per second, others less than once per cell generation. Much of this variation is due to differences in promoter sequence. In the absence of regulatory proteins, differences in promoter sequence may affect the frequency of transcription initiation by a factor of 1,000 or more. Most E. coli promoters have a sequence close to a consensus (Fig. 28-2). Mutations that result in a shift away from the consensus sequence usually decrease the function of bacterial promoters; conversely, mutations toward consensus usually enhance promoter function.

A horizontal strand is labeled D N A 5 prime on the left end. From left to right, it has a short blue region, a purple U P element, a short blue region, a purple region containing the letters T T G A C A beneath text reading negative 35 region, a blue region labeled N subscript 17 end subscript, a purple region containing the letters T A T A A T beneath text reading negative 10 region, a blue region labeled N subscript 5 to 9 end subscript, a long yellow region, and then a short blue region. A vertical line at the boundary between the blue region labeled N subscript 5 to 9 end subscript and the yellow region joins a horizontal arrow pointing to the right and this is labeled R N A start site. A wavy green right-pointing arrow extends right from this arrow to the word m R N A. — FIGURE 28-2 Consensus sequence for many *E. coli* promoters. Most base substitutions in the $- 10$ $negative 10$ and $- 35$ $negative 35$ regions have a negative effect on promoter function. Some promoters also include the UP (upstream promoter) element.

FIGURE 28-2 Consensus sequence for many *E. coli* promoters. Most base substitutions in the $- 10$ $negative 10$ and $- 35$ $negative 35$ regions have a negative effect on promoter function. Some promoters also include the UP (upstream promoter) element.

Key convention

By convention, DNA sequences are shown as they exist in the nontemplate strand, with the $5^{'}$ $5 prime$ terminus on the left. Nucleotides are numbered from the transcription start site, with positive numbers to the right (in the direction of transcription) and negative numbers to the left. N indicates any nucleotide.

Genes for products that are required at all times, such as those for the enzymes of central metabolic pathways, are expressed continuously, with little variation in virtually every cell of a species or organism. Such genes are often referred to as housekeeping genes. Expression of a gene at approximately constant levels is called constitutive gene expression. Although housekeeping genes are expressed constitutively, the cellular concentrations of the proteins they encode vary widely. For these genes, the RNA polymerase–promoter interaction strongly influences the rate of transcription initiation. Differences in promoter sequence may be the only level of regulation for a housekeeping gene, allowing the cell to synthesize the appropriate level of each housekeeping gene product.

The basal rate of transcription initiation at the promoters of nonhousekeeping genes is also determined by the promoter sequence, but expression of these genes is further modulated by regulatory proteins. Many of these proteins work by enhancing or interfering with the interaction between RNA polymerase and the promoter.

The sequences of eukaryotic promoters are much more variable than their bacterial counterparts. The three eukaryotic RNA polymerases usually require an array of general transcription factors in order to bind to a promoter, and these can heavily influence basal transcription rates. Yet, as with bacterial gene expression, the basal level of transcription is determined in part by the effect of promoter sequences on the function of RNA polymerase and its associated transcription factors.

Transcription Initiation Is Regulated by Proteins and RNAs

At least three types of regulatory proteins regulate transcription initiation by RNA polymerase: specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters, repressors impede access of RNA polymerase to the promoter, and activators enhance the RNA polymerase–promoter interaction.

As our understanding of the roles of protein regulators slowly matures, many new roles for gene regulation by noncoding RNAs (ncRNAs) are also beginning to emerge. Among these are the long noncoding RNAs (lncRNAs), generally defined as noncoding RNAs more than 200 nucleotides long that lack an open reading frame (ORF) that encodes a protein — thus distinguishing them from the small, functional ncRNAs (miRNA, snoRNA, snRNA, etc.) described in Chapter 26. The lncRNAs are found in all types of organisms, with tens of thousands expressed in mammalian cells. Known functions of lncRNAs include regulation of nucleosome positioning and chromatin structure, control of DNA methylation and posttranscriptional histone modifications, transcriptional gene silencing, multiple roles in transcriptional activation and repression, and much more.

We introduced bacterial specificity factors in Chapter 26, although we did not refer to these proteins by that name. The σ subunit of the E. coli RNA polymerase holoenzyme is a specificity factor that mediates promoter recognition and binding. Most E. coli promoters are recognized by a single σ subunit $(M_{r} 70,000)$ $left-parenthesis upper M Subscript r Baseline 70,000 right-parenthesis$ , $σ^{70}$ $sigma Superscript 70$ (see Fig. 26-5). Under some conditions, some of the $σ^{70}$ $sigma Superscript 70$ subunits are replaced by one of six other specificity factors. One notable case arises when bacteria are subjected to heat stress, leading to the replacement of $σ^{70}$ $sigma Superscript 70$ by $σ^{32}$ $sigma Superscript 32$ $(M_{r} 32,000)$ $left-parenthesis upper M Subscript r Baseline 32,000 right-parenthesis$ . When bound to $σ^{32}$ $sigma Superscript 32$ , RNA polymerase is directed to a specialized set of promoters with a different consensus sequence (Fig. 28-3). These promoters control the expression of a set of genes that encode proteins, including some protein chaperones (p. 132), that are part of a stress-induced system called the heat shock response. Thus, through changes in the binding affinity of the polymerase that direct the enzyme to different promoters, a set of genes involved in related processes is coordinately regulated. In eukaryotic cells, some of the general transcription factors, in particular the TATA-binding protein (TBP; see Fig. 26-9), may be considered specificity factors.

A horizontal strand is labeled D N A 5 prime on the left end. From left to right, it has a short blue region, a purple region containing the letters T N T C N C C C T T G A A, a blue region labeled N subscript 13 to 15 end subscript, a purple region containing the letters C C C C A T T T A, a blue region labeled N subscript 7 end subscript, a long yellow region, and then a short blue region. A vertical line at the boundary between the blue region labeled N subscript 7 end subscript and the yellow region joins a horizontal arrow pointing to the right and this is labeled R N A start site. A wavy green right-pointing arrow extends right from this arrow to the word m R N A. — FIGURE 28-3 Consensus sequence for promoters that regulate expression of the *E. coli* heat shock genes. This system responds to temperature increases as well as some other environmental stresses, resulting in the induction of a set of proteins. Binding of RNA polymerase to heat shock promoters is mediated by a specialized σ subunit of the polymerase, $σ^{32}$ $sigma Superscript 32$ , which replaces $σ^{70}$ $sigma Superscript 70$ in the RNA polymerase initiation complex.

FIGURE 28-3 Consensus sequence for promoters that regulate expression of the *E. coli* heat shock genes. This system responds to temperature increases as well as some other environmental stresses, resulting in the induction of a set of proteins. Binding of RNA polymerase to heat shock promoters is mediated by a specialized σ subunit of the polymerase, $σ^{32}$ $sigma Superscript 32$ , which replaces $σ^{70}$ $sigma Superscript 70$ in the RNA polymerase initiation complex.

Repressors bind to specific sites on the DNA. In bacterial cells, such binding sites, called operators, are generally near a promoter. RNA polymerase binding, or its movement along the DNA after binding, is blocked when the repressor is present. Regulation by means of a repressor protein that blocks transcription is referred to as negative regulation. Repressor binding to DNA is regulated by a molecular signal, or effector, usually a small molecule or a protein that binds to the repressor and causes a conformational change. The interaction between repressor and signal molecule either increases or decreases transcription. In some cases, the conformational change results in dissociation of a DNA-bound repressor from the operator (Fig. 28-4a). Transcription initiation can then proceed unhindered. In other cases, interaction between an inactive repressor and the signal molecule causes the repressor to bind to the operator (Fig. 28-4b). In eukaryotic cells, gene regulation by a repressor is less common. Where it does occur (more often in lower eukaryotes such as yeast), the binding site for a repressor may be some distance from the promoter. Binding of these repressors to their binding sites has the same effect as in bacterial cells: inhibiting the assembly or activity of a transcription complex at the promoter.

A four-part figure shows common patterns of regulation of transcription initiation with part a showing the binding of a repressor to an operator in the absence of a molecular signal that causes dissociation of the repressor when present, part b showing the binding of a repressor in the presence of a signal with the repressor dissociating when the signal is removed, part c showing the binding of an activator in the absence of a signal that causes dissociation of the activator when present, and part d showing the binding of the activator in the presence of a signal with the activator dissociating when the signal is removed. — FIGURE 28-4 Common patterns of regulation of transcription initiation. Two types of negative regulation are illustrated. (a) Repressor binds to the operator in the absence of the molecular signal; the external signal causes dissociation of the repressor to permit transcription. (b) Repressor binds in the presence of the signal; the repressor dissociates, and transcription ensues when the signal is removed. Positive regulation is mediated by gene activators. Again, two types are shown. (c) Activator binds in the absence of the molecular signal and transcription proceeds; when the signal is added, the activator dissociates and transcription is inhibited. (d) Activator binds in the presence of the signal; it dissociates only when the signal is removed. Note that “positive” regulation and “negative” regulation refer to the type of regulatory protein involved: the bound protein either facilitates or inhibits transcription. In either case, addition of the molecular signal may increase or decrease transcription, depending on its effect on the regulatory protein.

Parts a and b show negative regulation mediated by repressors and parts c and d show positive regulation mediated by gene activators. Part a is labeled, Negative regulation, molecular signal causes dissociation of repressor from D N A, inducing transcription. A horizontal strand of D N A is shown. From left to right, it has a small blue region, a purple region labeled promoter, and a yellow region. A tan vertical oval labeled repressor is present near the right end of the strand. This oval has a triangular cutout at its top end. At the boundary between the purple and yellow regions, a vertical line extends upward to a horizontal arrow that points right to the red word O F F. An arrow points downward accompanied by a curved line showing the addition of a molecular signal shown as a green diamond. This yields a similar structure with the green diamond bound to the triangular cutout in the repressor, leaving a purple region labeled operator in the space that it vacated. From left to right, the horizontal strand now has a small blue region, a purple region labeled promoter, a short dark region labeled promoter, a short purple region, and a yellow region. At the boundary between the purple and yellow regions, a vertical line extends upward to a horizontal arrow that points right to the green word O N. An arrow points down from the yellow region to a green wavy horizontal strand labeled m R N A. Part b is labeled, Negative regulation: Molecular signal causes binding of repressor to D N A, inhibiting transcription. A horizontal strand is shown. From left to right, the horizontal strand has a small blue region, a purple region labeled promoter, a short dark region labeled promoter, a short purple region, and a yellow region. At the boundary between the purple and yellow regions, a vertical line extends upward to a horizontal arrow that points right to the green word O N. An arrow points down from the yellow region to a green wavy horizontal strand labeled m R N A. An arrow points downward accompanied by curved lines showing the addition of a molecular signal shown as a red diamond and a the addition of a tan vertical oval labeled repressor with a triangular cutout at its top end. This yields a similar strand in which the dark purple operator is covered by the vertical oval with the red diamond bound to its triangular cutout. The short vertical line extending up from the boundary between the purple and yellow regions leads to a horizontal arrow pointing right to the red word O F F. Part c is labeled, Positive regulation, Molecular signal causes dissociation of activator from D N A, inhibiting transcription. A horizontal strand is shown. From left to right, it has a light blue region, a dark blue region labeled activator binding site, a long purple region, and a yellow region. An irregular horizontal oval with rough edges labeled R N A polymerase with an oval cutout at its right side covers the left and center of the purple region. A green wavy oval labeled activator has its base against the dark blue activator binding site and leans against the left side of R N A polymerase in a position that angles it slightly toward the upper right. It has a triangular cutout at its upper left side. At the boundary between the purple and yellow regions, a vertical line extends upward to a horizontal arrow that points right to the green word O N. An arrow points down from this yellow region to a wavy horizontal green line labeled m R N A. An arrow points downward and is joined by a curved line showing the addition of a molecular signal, shown as a red diamond. This yields a similar structure in which the activator has separated from the D N A strand and is shown below with the red diamond bound to the triangular cutout in its upper left side. A short vertical line extending up from the boundary between the purple and yellow regions leads to a horizontal arrow pointing right to the red word O F F. Part d is labeled, Positive regulation, Molecular signal causes binding of activator to D N A, inducing transcription. A horizontal strand is shown. From left to right, it has a light blue region, a dark blue region, a long purple region, and a yellow region. An irregular horizontal oval with rough edges labeled R N A polymerase with an oval cutout at its right side covers the left and center of the purple region. A short vertical line extending up from the boundary between the purple and yellow regions leads to a horizontal arrow pointing right to the red word O F F. An arrow points downward accompanied by curved lines showing the addition of a molecular signal shown as a green diamond and of an activator similar to the activator in part c. This yields a similar structure in which the activator is attached to the dark blue region, has the green diamond bound to the triangular cutout in its upper left side, and leans against the left side of R N A polymerase. At the boundary between the purple and yellow regions, a vertical line extends upward to a horizontal arrow that points right to the green word O N. An arrow points down from this yellow region to a wavy horizontal green line labeled m R N A.

Activators provide a molecular counterpoint to repressors; they bind to DNA and enhance the activity of RNA polymerase at a promoter; this is positive regulation. In bacteria, activator-binding sites are often adjacent to promoters that are bound weakly or not at all by RNA polymerase alone, such that little transcription occurs in the absence of the activator. Some activators are usually bound to DNA, enhancing transcription until dissociation of the activator is triggered by the binding of a signal molecule (Fig. 28-4c). In other cases the activator binds to DNA only after interaction with a signal molecule (Fig. 28-4d). Signal molecules can therefore increase or decrease transcription, depending on how they affect the activator.

Positive regulation by activators is particularly common in eukaryotes. Many eukaryotic activators bind to DNA sites, called enhancers, that are distant from the promoter, affecting the rate of transcription at a promoter that may be located thousands of base pairs away.

The distance between a promoter and the binding site of an activator or repressor is bridged by looping out of the DNA between the two sites (Fig. 28-5). The looping is facilitated in some cases by proteins called architectural regulators that bind to intervening sites. Interaction between activators and the RNA polymerase at the promoter is often mediated by intermediary proteins called coactivators. In some instances, protein repressors may take the place of coactivators, binding to the activators and preventing the activating interaction.

A figure shows the interaction between activators/repressors and R N A polymerase in eukaryotes. — FIGURE 28-5 Interaction between activators/repressors and RNA polymerase in eukaryotes. Eukaryotic activators and repressors frequently bind sites thousands of base pairs distant from the promoters they regulate. DNA looping, often facilitated by architectural regulators, brings the sites together. The interaction between activators and RNA polymerase may be mediated by coactivators, as shown. Repression is sometimes mediated by repressors (described later) that bind to activators, thereby preventing the activating interaction with RNA polymerase.

A horizontal strand has its 5 prime end on the left and its 3 prime end on the right. From left to right, it has a short blue region, an orange region with two overlapping spheres bound to its upper side and labeled activator, a long blue region, a brown region labeled architectural regulator binding site, a blue region, a long purple region labeled promoter, and a yellow region. An arrow points downward accompanied by a curved line showing the addition of a yellow oval labeled architectural regulator. This yields a similar structure in which the yellow oval is horizontally bound to the brown architectural regulator binding site and the strand has bent upward to the left to this site. An arrow labeled recruitment points downward accompanied by a curved line showing the addition of tan P o l Roman numeral 2 and a curved line below showing the addition of a green coactivator. P o l Roman numeral 2 is roughly oval with a bottom half that is horizontal across the bottom and curves up to the left and a shorter, elongated oval at the upper left that extends to a thin protrusion to the upper left of the overall structure. A small triangle extends down from the top part into the open region between the top and bottom parts, which is wide to the right and has a narrow opening to the upper left. The coactivator is roughly oval with a wider left side than right side and with the right side bending upward into a rounded protrusion. This yields a similar structure in which the purple region of the strand runs through the middle of P o l Roman numeral 2 and the coactivator has its rounded end against the lower left of P o l Roman numeral 2 and extends horizontally upward. The left side of the horizontal strand of D N A is bent diagonally to the upper right to position the two spheres bound to the orange region so that they are adjacent to the upper left of the green coactivator. A vertical line extends up from the boundary between the purple and yellow regions to meet a short horizontal arrow pointing right to the green word O N.

Many Bacterial Genes Are Clustered and Regulated in Operons

Bacteria have a simple general mechanism for coordinating the regulation of multiple genes: these genes are clustered on the chromosome and are transcribed together. Many bacterial mRNAs are polycistronic — multiple genes on a single transcript — and the single promoter that initiates transcription of the cluster is the site of regulation for expression of all the genes in the cluster. The gene cluster and promoter, plus additional sequences that function together in regulation, are called an operon (Fig. 28-6). Operons that include two to six genes transcribed as a unit are common; some operons contain 20 or more genes. The identity and order of the genes in an operon are not random. In many cases, genes in the same operon encode subunits of a larger protein complex, and cotranslation directly enables assembly of the complex. Some operons organize genes involved in related processes that require coordinated regulation. In other cases, the genes may seem to be unrelated, but they encode products required by the cell under similar conditions.

A figure shows the structure of a representative bacterial operon with three genes. — FIGURE 28-6 Representative bacterial operon. Genes A, B, and C are transcribed on one polycistronic mRNA. Typical regulatory sequences include binding sites for proteins that either activate or repress transcription from the promoter.

Many of the principles of bacterial gene expression were first defined by studies of lactose metabolism in E. coli, which can use lactose as its sole carbon source. In 1960, François Jacob and Jacques Monod published a short paper in the Proceedings of the French Academy of Sciences that described how two adjacent genes involved in lactose metabolism were coordinately regulated by a genetic element located at one end of the gene cluster. The genes were those for β-galactosidase, which cleaves lactose to galactose and glucose, and for galactoside permease, which transports lactose into the cell (Fig. 28-7). The terms “operon” and “operator” were first introduced in this paper. With the operon model, gene regulation could, for the first time, be considered in molecular terms.

A figure shows the steps of lactose metabolism in italicized E. coli end italics. — FIGURE 28-7 Lactose metabolism in *E. coli*. Uptake and metabolism of lactose require the activities of galactoside (lactose) permease and β-galactosidase. Conversion of lactose to allolactose by transglycosylation is a minor reaction also catalyzed by β-galactosidase.

A slightly curved horizontal plasma membrane extends across the top part of the illustration, separating the extracellular space above from the cytoplasm below. A vertical purple channel with crescents forming the left and right sides, with the ends extending outward above and below, is labeled galactoside (lactose) permease. The word lactose above this channel has an arrow pointing downward to the word lactose in the cytoplasm, indicating that lactose passes through the channel to enter the cell. Lactose is shown as a disaccharide. The left-hand ring has O substituted for C at its upper right vertex, an upper left vertex bonded to C H 2 O H above and H below, a left side vertex bonded to O H above and H below, a lower left vertex bonded to O H above and H below, a lower right vertex bonded to H above and O H below, and a right side vertex bonded to H below and to O above that joins it to the left side vertex of the right-hand ring above. The right-hand ring has a left side vertex bonded to H above and to O below further bonded to the left-hand ring, an upper left vertex bonded to C H 2 O H above and H below, O substituted for C at the upper right vertex, a right side vertex bonded to O H above and H below, a lower right vertex bonded to H above and O H below, and a lower left vertex bonded to O H above and H below. An arrow labeled blue highlighted galactosidase end highlight points downward and curves to the left and right. The left-hand arrow is thicker and shows a curved line indicating the addition of H 2 O above the word hydrolysis. This yields galactose plus glucose. The thinner right-hand arrow is labeled isomerization and points to allolactose. The structures are as follows. Galactose: A six-membered ring with O at the upper right vertex, the right side vertex bonded to O H above and H below, the lower right vertex bonded to H above and O H below, the lower left vertex bonded to H above and H below, the left side vertex bonded to O H above and H below, and the upper left vertex bonded to H below and C H 2 O H above. Glucose: Similar to galactose except that the left side vertex is bonded to H above and O H below. Allolactose: The left-hand ring is the same as the left-hand ring of lactose, but its right side vertex bonds to O above that bonds to C H 2 to the left that bonds to the lower left vertex of the right-hand ring. The right-hand ring has a lower left vertex bonded to H above and to C H 2 further bonded to O bonded to the left-hand ring below, a left side vertex bonded to O H above and H below, an upper left vertex bonded to H above and O H below, an upper right vertex bonded to O H above and H below, a right side vertex bonded to H above and O H below, and O substituted for C at the lower right vertex.

The lac Operon Is Subject to Negative Regulation

The lactose (lac) operon (Fig. 28-8a) includes the genes for β-galactosidase (Z), galactoside permease (Y), and thiogalactoside transacetylase (A). The last of these enzymes seems to modify toxic galactosides to facilitate their removal from the cell. Each of the three genes is preceded by a ribosome-binding site (not shown in Fig. 28-8) that independently directs the translation of that gene (Chapter 27). Regulation of the lac operon by the lac repressor protein (Lac) follows the pattern outlined in Figure 28-4a.

A three-part figure shows the structure of the lac operon by showing the horizontal strand of D N A in part a, the binding of the repressor in part b, and a surface contour view of the repressor bound to discontinuous segments of D N A in part c. — FIGURE 28-8 The *lac* operon. (a) In the *lac* operon, the *lacI* gene encodes the Lac repressor. The *lac Z*, Y, and A genes encode β-galactosidase, galactoside permease, and thiogalactoside transacetylase, respectively. P is the promoter for the *lac* genes, and $P_{I}$ $upper P Subscript upper I$ is the promoter for the I gene. $O_{1}$ $upper O Subscript 1$ is the main operator for the *lac* operon; $O_{2}$ $upper O Subscript 2$ and $O_{3}$ $upper O Subscript 3$ are secondary operator sites of lesser affinity for the Lac repressor. The inverted repeat to which the Lac repressor binds in $O_{1}$ $upper O Subscript 1$ is shown. (b) The Lac repressor binds to the main operator and $O_{2}$ $upper O Subscript 2$ or $O_{3}$ $upper O Subscript 3$ , and it seems to form a loop in the DNA. (c) Lac repressor (shades of red) is shown bound to short, discontinuous segments of DNA (blue and orange). [(c) Data from PDB ID 2PE5, R. Daber et al., *J. Mol. Biol.* 370:609, 2007.]

FIGURE 28-8 The *lac* operon. (a) In the *lac* operon, the *lacI* gene encodes the Lac repressor. The *lac Z*, Y, and A genes encode β-galactosidase, galactoside permease, and thiogalactoside transacetylase, respectively. P is the promoter for the *lac* genes, and $P_{I}$ $upper P Subscript upper I$ is the promoter for the I gene. $O_{1}$ $upper O Subscript 1$ is the main operator for the *lac* operon; $O_{2}$ $upper O Subscript 2$ and $O_{3}$ $upper O Subscript 3$ are secondary operator sites of lesser affinity for the Lac repressor. The inverted repeat to which the Lac repressor binds in $O_{1}$ $upper O Subscript 1$ is shown. (b) The Lac repressor binds to the main operator and $O_{2}$ $upper O Subscript 2$ or $O_{3}$ $upper O Subscript 3$ , and it seems to form a loop in the DNA. (c) Lac repressor (shades of red) is shown bound to short, discontinuous segments of DNA (blue and orange). [(c) Data from PDB ID 2PE5, R. Daber et al., *J. Mol. Biol.* 370:609, 2007.]

Part a shows a horizontal strand that runs from its 5 prime end on the left to its 3 prime end on the right. From left to right, it has a short blue piece, a purple piece labeled P subscript I end subscript, a light red piece labeled italicized lac end italics I, an orange piece labeled O subscript 3 end subscript, a short blue piece, a purple piece labeled P, an orange piece labeled O subscript 1 end subscript, a long tan piece labeled italicized lac Z end italics, an orange piece labeled O subscript 2 end subscript, a tan piece, a tan piece labeled italicized lac Y end italics, a yellow piece labeled lac A end italics, and a short blue piece. At the boundary between purple P subscript I end subscript and light red italicized lac I end italics and at the boundary between orange O 1 and tan italicized lac Z end italics, there are short vertical lines that meet horizontal arrows pointing to the right. A close-up of the orange region labeled O subscript 1 end subscript shows the base pairs of this double-stranded piece of D N A. The top strand begins at the 5 prime end, then has a light orange region containing A A T T G T, then has an unshaded region containing G A G C G G A T A, then has a dark orange region containing A C A A T T before ending at the 3 prime end. The bottom strand begins at the 3 prime end, then has a dark orange region containing T T A A C A, then has an unshaded region containing C T C G C C T A T before a light orange region containing T G T T A A before ending at the 5 prime end. Part b shows a similar structure except that the orange region labeled O subscript 3 end subscript curves upward, the blue region to its right curves further to the right, and the purple region labeled P forms a loop above before meeting the left side of the orange region labeled O subscript 1 end subscript, which curves back down to horizontal. A red repressor below binds to O subscript 3 end subscript and O subscript 1 end subscript. The left front of the repressor is a light red oval that angles slightly toward the upper left to meet a smaller, almost horizontal oval that runs against the lower right of O subscript 3 end subscript. Behind it, a more vertical oval has a smaller oval that extends to the upper right to bind to the other side of O subscript 3 end subscript and the lower left of the blue region. In the right front, an almost vertical oval joins to the left-hand front oval and extends up until the small oval at its top angles up to the left along the lower left of O subscript 1 end subscript. Behind it, another oval extends slightly to its right and has a smaller oval that runs horizontally to meet the lower right of O subscript 1 end subscript. Beneath, the horizontal strand is shown again but the repressor is now beneath O subscript 1 end subscript and O subscript 2 end subscript with italicized lac Z end italics forming the loop above. The shape of the repressor is the same. Part c shows a close-up of the structure of the repressor. It has four subunits. Each bottom subunit is roughly oval and joins to a smaller, roughly oval part above. The left front piece is pink, the left rear piece is dark red, the right rear piece is bright red, and the right front piece is purplish red. The small ovals at the top of the left two halves bind to the bottom of one short horizontal piece of D N A and the two small ovals at the top of the right two halves bind to the bottom of a second short horizontal piece of D N A.

The study of lac operon mutants has revealed some details of the workings of the operon’s regulatory system. In the absence of lactose, the lac operon genes are repressed. Mutations in the operator or in another gene, the I gene, result in constitutive synthesis of the gene products. When the I gene is defective, repression can be restored by introducing a functional I gene into the cell on another DNA molecule, demonstrating that the I gene encodes a diffusible molecule that causes gene repression. This molecule proved to be a protein, now called the Lac repressor, a tetramer of identical monomers. The operator to which it binds most tightly $(O_{1})$ $left-parenthesis upper O Subscript 1 Baseline right-parenthesis$ abuts the transcription start site (Fig. 28-8a). The I gene is transcribed from its own promoter $(P_{I})$ $left-parenthesis upper P Subscript upper I Baseline right-parenthesis$ independent of the lac operon genes. The lac operon has two secondary binding sites for the Lac repressor: $O_{2}$ $upper O Subscript 2$ and $O_{3}$ $upper O Subscript 3$ . $O_{2}$ $upper O Subscript 2$ is centered near position $+ 410$ $plus 410$ , within the gene encoding β-galactosidase (Z); $O_{3}$ $upper O Subscript 3$ is near position $- 90$ $negative 90$ , within the I gene. To repress the operon, the Lac repressor seems to bind to both the main operator and one of the two secondary sites, with the intervening DNA looped out (Fig. 28-8b, c). Either binding arrangement blocks transcription initiation.

Despite this elaborate binding complex, repression is not absolute. Binding of the Lac repressor reduces the rate of transcription initiation by a factor of $10^{3}$ $10 cubed$ . If the $O_{2}$ $upper O Subscript 2$ and $O_{3}$ $upper O Subscript 3$ sites are eliminated by deletion or mutation, the binding of repressor to $O_{1}$ $upper O Subscript 1$ alone reduces transcription by a factor of about $10^{2}$ $10 squared$ . Even in the repressed state, each cell has a few molecules of β-galactosidase and galactoside permease, presumably synthesized on the rare occasions when the repressor transiently dissociates from the operators. This basal level of transcription is essential to operon regulation.

When cells are provided with lactose, the lac operon is induced. An inducer (signal) molecule binds to a specific site on the Lac repressor, causing a conformational change that results in dissociation of the repressor from the operator. The inducer in the lac operon system is not lactose itself but allolactose, an isomer of lactose (Fig. 28-7). After entry into the E. coli cell (via the few preexisting molecules of lactose permease), lactose is converted to allolactose by one of the few preexisting β-galactosidase molecules. Release of the operator by Lac repressor, triggered as the repressor binds to allolactose, allows expression of the lac operon genes and leads to a $10^{3}$ $10 cubed$ -fold increase in the concentration of β-galactosidase.

Several β-galactosides structurally related to allolactose are inducers of the lac operon but are not substrates for β-galactosidase; others are substrates but not inducers. One particularly effective and nonmetabolizable inducer of the lac operon that is often used experimentally is isopropylthiogalactoside (IPTG).

Isopropyl-beta-D-thiogalactoside (I P T G) is a six-membered ring with O at its upper right vertex; its right side vertex bonded to H below and to S above further bonded to C bonded to C H 3 above and below and to H to the right; the lower right vertex bonded to H above and O H below; the lower left vertex bonded to O H above and H below; the left side vertex bonded to OH above and H below; and the upper left vertex bonded to C H 2 O H above and H below.

An inducer that cannot be metabolized allows researchers to explore the physiological function of lactose as a carbon source for growth, separate from its function in the regulation of gene expression.

In addition to the multitude of operons now known in bacteria, a few polycistronic operons have been found in the cells of lower eukaryotes. In the cells of higher eukaryotes, however, almost all protein-coding genes are transcribed separately.

The mechanisms by which operons are regulated can vary significantly from the simple model presented in Figure 28-8. Even the lac operon is more complex than indicated here, with an activator also contributing to the overall scheme, as we shall see in Section 28.2. Before any further discussion of the layers of regulation of gene expression, however, we examine the critical molecular interactions between DNA-binding proteins (such as repressors and activators) and the DNA sequences to which they bind.

Regulatory Proteins Have Discrete DNA-Binding Domains

Regulatory proteins generally bind to specific DNA sequences. Their affinity for these target sequences is roughly $10^{4}$ $10 Superscript 4$ to $10^{6}$ $10 Superscript 6$ times higher than their affinity for any other DNA sequence. Most regulatory proteins have discrete DNA-binding domains containing substructures that interact closely and specifically with the DNA. These binding domains usually include one or more of a relatively small group of recognizable and characteristic structural motifs.

To bind specifically to DNA sequences, regulatory proteins must recognize surface features on the DNA (see Fig. 8-13). Most of the chemical groups that differ among the four bases and thus permit discrimination between base pairs are hydrogen-bond donor and acceptor groups exposed in the major groove of DNA (Fig. 28-9), and most of the protein-DNA contacts that impart specificity are hydrogen bonds. A notable exception is the nonpolar surface near C-5 of pyrimidines, where thymine is readily distinguished from cytosine by its protruding methyl group. Protein-DNA contacts are also possible in the minor groove of the DNA, but the hydrogen-bonding patterns there generally do not allow ready discrimination between base pairs.

A two-part figure shows the groups in D N A that are available for protein binding with part a showing the functional groups on all four base pairs in the major and minor grooves of D N A and part b showing recognition patterns for each base pair. — FIGURE 28-9 Groups in DNA available for protein binding. (a) Shown here are functional groups on all four base pairs that are displayed in the major and minor grooves of DNA. Hydrogen-bond acceptor (A) and donor (D) atoms are marked by blue and red disks, respectively. Other hydrogen atoms (H) are marked with purple disks, and methyl groups (M) with yellow disks. (b) Recognition patterns for each base pair (from left to right). The much greater variation in the patterns for the major groove gives rise to a much greater discriminatory power in the major groove relative to the minor groove. [Information from J. L. Huret, *Atlas Genet. Cytogenet. Oncol. Haematol.* 2006, http://atlasgeneticsoncology.org/Educ/DNAEngID30001ES.html.]

Part a shows four structures representing adenine bound to thymine with the major groove on top, guanine bound to cytosine with the major groove on top, thymine bound to adenine with the major groove on top, and cytosine bound to guanine with the major groove on top. The left-hand structure shows blue adenine on the left and yellow thymine on the right. A double-headed arrow across the top indicates the minor groove and a similar arrow at the bottom indicates the minor groove. Similar arrows indicating the major groove on top and minor groove below are shown for all four structures. Adenine is a five-membered ring with a double bond at its upper left side, a red N substituted for C at its upper left vertex beneath a blue circle labeled A, N at its lower left vertex connected by three dots to the lower left, and a double bond at its lower right side shared with the left side of a six-membered ring to its right that has red N at its bottom vertex next to a blue circle labeled A, a double bond at its lower right side, its lower right vertex bonded to red H next to a purple circle labeled H, N at the upper right vertex connected by a set of three short blue lines representing a hydrogen bond to N at the lower left vertex of T to the right, a double bond at the upper right side, and a top vertex bonded to N bonded to H to the upper right that is hydrogen bonded to O of T and bonded to red H to the upper left next to a red circle labeled D. T is a six-membered ring with N at the lower right vertex connected by three dots to the lower right, a bottom vertex double bonded to red O next to a blue circle labeled A, N at the lower left vertex bonded to H that is hydrogen bonded to N at the upper right vertex of the six-membered ring of A, an upper left vertex double bonded to red O beneath a red circle labeled A and hydrogen bonded to H bonded to N bonded to the top vertex of the six-membered ring of A, a top vertex bonded to red C H 3 beneath a yellow circle labeled M, and a double bond at the upper right side. The second structure shows green guanine on the left and red cytosine on the right. Guanine is a five-membered ring with a double bond at its upper left side, a red N substituted for C at its upper left vertex beneath a blue circle labeled A, N at its lower left vertex connected by three dots to the lower left, and a double bond at its lower right side shared with the left side of a six-membered ring to its right that has red N at its bottom vertex next to a blue circle labeled A, a double bond at its lower right side, its lower right vertex bonded to N bonded to red H below next to a red circle labeled D and bonded to H to the upper right hydrogen bonded to red O bonded to the bottom vertex of C, N at the upper right vertex bonded to H further hydrogen bonded to N at the lower left vertex of C, and a top vertex double bonded to red O next to a blue circle labeled A and hydrogen bonded to H bonded to N bonded to the upper left vertex of C. C is a six-membered ring with its bottom vertex double bonded to red O next to a blue circle labeled A and hydrogen bonded to H bonded to N bonded to the lower right vertex of G, N at the lower left side hydrogen bonded to H bonded to N at the upper right vertex of G, a double bond at its left side, an upper left vertex bonded to N bonded to red H above next to a red circle labeled D and bonded to N hydrogen bonded to O double bonded to the top vertex of G, a top vertex bonded to red H next to a purple circle labeled H, a double bond at it supper right side, and a lower left side with N connected by three dots to the lower right. The third structure shows yellow T on the left and blue A on the right. T is a six-membered ring with N at the lower left vertex connected by three dots to the lower left, a double bond at the upper left side, an upper left vertex bonded to red C H 3 next to a yellow circle labeled M, an upper right vertex double bonded to red O next to a blue circle labeled A and hydrogen bonded to H bonded to N bonded to the upper left vertex of the six-membered ring of A to the right, N at the right side vertex bonded to H further hydrogen bonded to N at the left side vertex of A to the right, and a lower right vertex double bonded to red O next to a blue circle labeled A. Adenine has a five-membered ring on the right with N at the lower left vertex connected by three dots to the lower right, a double bond at the upper right side, red N at the upper right with a blue circle labeled A above, and a double bond at the left side shared with the upper left side of an adjacent six-membered ring. The six-membered ring has red N at its lower right vertex next to a blue circle labeled A, a double bond at its bottom side, a lower left vertex bonded to red H next to a purple circle labeled H, N at its left side vertex hydrogen bonded to H bonded to N at the right side vertex of T, a double bond at the upper left side, and an upper left vertex bonded to N bonded to red H to the upper right next to a red circle labeled D and bonded to H to the left further hydrogen bonded to red O double bonded to the upper right vertex of T. The fourth structure has red C on the left and green G on the right. C is a six-membered structure with N at the lower left vertex connected by three dots to the lower left, a double bond at the upper left side, an upper left vertex bonded to red H next to a purple circle labeled H, an upper right side bonded to N bonded to red H to the upper left next to a red circle labeled D and bonded to H tot eh right hydrogen bonded to red O double bonded to the upper left vertex of G, a double bond at the upper right side, N at the right side vertex hydrogen bonded to N at the left side vertex of G, and a lower right vertex double bonded to red O next to a blue circle labeled A and hydrogen bonded to H bonded to N bonded to the lower left vertex of G. G has a five-membered ring with N at the lower right vertex connected by three dots to the lower right, a double bond at the upper right side, red N at the upper right vertex next to a blue circle labeled A above, and a double bond at the left side shared with the upper right side of the adjacent six-membered ring. This six-membered ring has red N at the lower right vertex next to a blue circle labeled A, a double bond at the bottom side, a lower left side bonded to N bonded to red H to the lower left next to a red circle labeled D and bonded to H to the upper left hydrogen bonded to red O double bonded to the lower right vertex of C, N at the left side vertex bonded to H hydrogen bonded to N at the right side vertex of C, and an upper left vertex double bonded to red O next to a blue circle labeled A and hydrogen bonded to H bonded to N bonded to the upper right vertex of C. Part b shows a key that indicates the following: a blue circle labeled A represents an H-bond acceptor, a red circle labeled D represents an H-bond donor, a purple circle labeled H represents other H, and a yellow circle labeled M represents a methyl group. Under the heading major groove, there are four rows. Each row has a base on the left and right with four circles in between. Row 1: base A blue circle A red circle D blue circle A yellow circle M base T; Row 2: base G blue circle A blue circle A red circle D purple circle H base C; Row 3: base T yellow circle M blue circle A red circle D blue circle A base A; Row 4: base C purple circle H red circle D blue circle A blue circle A base G. Under the heading minor groove, there are four rows. Each row has a base on the left and right with three circles in between. Row 1: base A blue circle A purple circle H blue circle A base T; Row 2: base G blue circle A red circle D blue circle A; Row 3: base T blue circle A purple circle H blue circle A base A; Row 4: base C blue circle A red circle D blue circle A base G.

Within regulatory proteins, the amino acid side chains most often hydrogen-bonding to bases in the DNA are those of Asn, Gln, Glu, Lys, and Arg residues. Is there a simple recognition code in which a particular amino acid always pairs with a particular base? The two hydrogen bonds that can form between Gln or Asn and the $N^{6}$ $upper N Superscript 6$ and N-7 positions of adenine cannot form with any other base. And an Arg residue can form two hydrogen bonds with N-7 and $O^{6}$ $upper O Superscript 6$ of guanine (Fig. 28-10). Examination of the structure of many DNA-binding proteins, however, has shown that a protein can recognize each base pair in more than one way, leading to the conclusion that there is no simple amino acid–base code. For some proteins, the Gln-adenine interaction can specify $A ═ T$ $upper A box drawings double horizontal upper T$ base pairs, but in others a van der Waals pocket for the methyl group of thymine can recognize $A ═ T$ $upper A box drawings double horizontal upper T$ base pairs. Researchers cannot yet examine the structure of a DNA-binding protein and infer the DNA sequence to which it binds.

A figure shows two examples of specific amino acid residue-base pair interactions. — FIGURE 28-10 Specific amino acid residue–base pair interactions. The two examples shown have been observed in DNA-protein binding.

On the left, thymine is shown bonded to adenine that is bonded to red-highlighted glutamine (or asparagine). It is labeled thymine double bond symbol adenine. Thymine is a six-membered ring with N substituted for C at position 1 at the lower left vertex and further bonded, the lower right vertex at position 2 double bonded to O, N substituted for C at position 3 at the right side vertex and bonded to H hydrogen bonded to N at position 1 in adenine, C at position 4 double bonded to O further hydrogen bonded to H bonded to N boned to position 6 of adenine, C at position 5 bonded to C H 3, and a double bond between C 5 and C 6. Adenine is shown as a six-membered ring on the left with N substituted for C at position 1 and hydrogen bonded to H bonded to N at position 3 of thymine, a double bond at the bottom side between positions 2 and 3, a double bond on the upper right side between positions 4 and 5 shared with the left side of the adjacent five-membered ring, and C at position 6 at the upper left side bonded to N bonded to 2 H. One of these H is hydrogen bonded to O double bonded to the upper right vertex at position 4 of thymine and the other H is hydrogen bonded to red highlighted O of glutamine (or asparagine). The five-membered ring of adenine has N substituted for C at position 9 and further bonded, a double bond between positions 7 and 8, and N substitute for C at position 7 that is hydrogen bonded to H of glutamine (or asparagine). Hydrogens 6 and 7 are both numbered in adenine. Glutamine (or asparagine) is entirely highlighted in red. It has N at the lower right bonded to H to the lower right, to H to the lower left that is hydrogen bonded to N at position 7 of adenine, and to C to the upper left that is double bonded to O to the lower left that is hydrogen bonded to N bonded to C at position 6 of adenine and bonded to C H 2 above further bonded to C H 2 bonded to C bonded to H above, to N to the left further bonded to R to the left and to H above, and to C to the right double bonded to O above and bonded to R prime to the right. On the right, cytosine is shown bonded to guanine that is bonded to red-highlighted arginine. It is labeled cytosine triple bond symbol guanine. Cytosine is a six-membered ring with N substituted for C at the lower left vertex at position 1 and further bonded, with C 2 at the lower right vertex double bonded to O further hydrogen bonded to H bonded to N bonded to position 2 of guanine, N substituted for C at the right side vertex at position 3 hydrogen bonded to H bonded to N at position 1 of guanine, a double bond at the upper right side between positions 3 and 4, C at the upper right vertex at position 4 bonded to N bonded to H to the upper left and to H to the lower right hydrogen bonded to O double bonded to C at position 6 of guanine, and a double bond between positions 5 and 6. Guanine has a six-membered ring on the left with N substituted for C at position 3 at the lower left vertex, a double bond at the bottom side between C 2 and C 3, C 2 at the lower left vertex bonded to N bonded to H to the lower left and to H to the upper left hydrogen bonded to O double bonded to C at position 2 of cytosine, N substitute for C at position 1 at the left side vertex bonded to H further hydrogen bonded to N at position 3 of cytosine, C at position 6 double bonded to O hydrogen bonded to H bonded to N bonded to position 4 of cytosine and also hydrogen bonded to H of arginine above, and a double bond at the upper right side between positions 4 and 5 shared with the five-membered ring to the right. The five-membered ring has N substituted for C at position 9 at the lower right vertex and further bonded to the lower right, a double bond at the upper right side between positions 7 and 8, and N substituted for C at position 7 at the top vertex hydrogen bonded to H of arginine above. Carbons 6 and 7 are numbered in guanine. Arginine has N at the lower right bonded to H to the upper right, bonded to H below that is hydrogen bonded to N at position 7 of guanine, and bonded to C to the upper right that is further bonded above and bonded to N to the lower left bonded to H to the left and bonded to H below that is hydrogen bonded to O double bonded to position 6 of guanine. There is a dashed curved line between the two N atoms on either side of the central C and there is a positive charge on C. This central C is bonded to N H above further bonded to a chain of three C H 2 further bonded to C above that is bonded to H above, to N to the left bonded to R to the left and H below, and to C to the right double bonded to O above and bonded to R prime to the right.

To interact with bases in the major groove of DNA, a protein requires a relatively small substructure that can stably protrude from the protein surface. The DNA-binding domains of regulatory proteins tend to be small (60 to 90 amino acid residues), and the structural motifs within these domains that are actually in contact with the DNA are smaller still. Many small proteins are unstable because of their limited capacity to form layers of structure to bury hydrophobic groups. The DNA-binding motifs provide either a very compact stable structure or a way of allowing a segment of protein to protrude from the protein surface.

The DNA-binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple (usually two) subunits of a regulatory protein bind cooperatively. The Lac repressor is unusual in that it functions as a tetramer, with two dimers tethered together at the end distant from the DNA-binding sites (Fig. 28-8b). An E. coli cell usually contains about 20 tetramers of the Lac repressor. Each of the tethered dimers separately binds to a palindromic operator sequence, in contact with 17 bp of a 22 bp region in the lac operon. And each of the tethered dimers can independently bind to an operator sequence, with one generally binding to $O_{1}$ $upper O Subscript 1$ and the other to $O_{2}$ $upper O Subscript 2$ or $O_{3}$ $upper O Subscript 3$ (as in Fig. 28-8b). The symmetry of the $O_{1}$ $upper O Subscript 1$ operator sequence corresponds to the twofold axis of symmetry of two paired Lac repressor subunits. The tetrameric Lac repressor binds to its operator sequences in vivo with an estimated dissociation constant of $10^{- 10} M$ $10 Superscript negative 10 Baseline upper M$ . The repressor discriminates between the operators and other sequences by a factor of about $10^{6}$ $10 Superscript 6$ , so binding to these few base pairs among the 4.6 million or so of the E. coli chromosome is highly specific.

Several DNA-binding motifs have been described, but here we focus on two that play prominent roles in the binding of DNA by regulatory proteins from all domains of life: the helix-turn-helix and the zinc finger. We also consider two other types of such motifs: the homeodomain and the RNA recognition motif, which, as its name implies, also binds RNA; both motifs play prominent roles in some eukaryotic regulatory proteins.

Helix-Turn-Helix

The helix-turn-helix motif is crucial to the interaction of many regulatory proteins with DNA in bacteria, and similar motifs occur in some eukaryotic regulatory proteins. The helix-turn-helix comprises about 20 amino acid residues in two short α-helical segments, each 7 to 9 residues long, separated by a β turn (Fig. 28-11). This structure generally is not stable by itself; it is simply the interactive portion of a somewhat larger DNA-binding domain. One of the two α-helical segments is called the recognition helix, because it usually contains many of the amino acids that interact with DNA in a sequence-specific way. This α helix is stacked on other segments of the protein structure so that it protrudes from the protein surface. When bound to DNA, the recognition helix is positioned in or nearly in the major groove. The Lac repressor has this DNA-binding motif.

Part a shows a horizontal double-stranded piece of D N A. Beneath, a dark blue helix angles down slightly to the right to meet the center of a light blue helix that angles from the lower left to upper right. A light purple thread bends back away from the lower right end of the dark blue helix at a position labeled turn and the strand joins to a purple helix labeled recognition helix that angles away from the viewer, then ends with a light blue strand connecting its far end to the upper right side of the light blue helix. Part b shows a larger structure with a box around a piece to the upper left center that is shown in close-up in part a. Within the box, two almost parallel helices are shown with a diagonal helix running from the center of the upper helix to the lower part of the lower helix. The lower helix has a strand connecting it to a yellow helix below that is adjacent to another yellow helix to its left that connects to several light blue helices to the upper right. Below, the roughly rectangular vertical left-hand part of the overall structure angles down to the right and consist of dark red helices in the back and pink helices behind that connect with two almost horizontal green helices above two green helices extending away from the viewer at the bottom. These bottom two green helices are labeled alpha helices involved in tetramer formation. The roughly rectangular right half that angles up to the right and includes a bright red rear portion and a purplish red front portion. It has two yellow helices at the upper right like those on the left and these are labeled linker helices. A set of three light blue helices are to the left and right of the linker helices. The upper left set of light blue helices, which extend to the upper right center, are labeled D N A-binding domain.

Zinc Finger

In a zinc finger, about 30 amino acid residues form an elongated loop held together at the base by a single ${Zn}^{2 +}$ $Zn Superscript 2 plus$ ion, which is coordinated to 4 of the residues (4 Cys, or 2 Cys and 2 His). The zinc does not itself interact with DNA; rather, the coordination of zinc with the amino acid residues stabilizes this small structural motif. Several hydrophobic side chains in the core of the structure also lend stability. Figure 28-12 shows the interaction between DNA and three zinc fingers of a single polypeptide from the mouse regulatory protein Zif268.

A piece of blue D N A runs from the lower left to upper right. A zing finger motif is shown at the lower left. It has a dark red helix that runs from lower left to upper right to the lower left of the D N A. A dark red wireframe model at the bottom of the helix runs strand below and extends to a ring above with two blue patches. Just below halfway up the helix, another wireframe model has a red base that bends to a similar ring with two blue patches that almost touches a purple sphere to its left and above the ring below. This sphere is labeled Z n 2 plus. A strand from the upper right of the helix coils back to the zinc ion, including a thicker beta strand, and wireframe protrusions show two yellow pieces adjacent to zinc. The strand curves away upward to the upper left of a light red helix in the top zinc finger motif. The top motif has a bright red helix that runs from upper right to lower left, ending near the top center of the D N A. It has a similar bent wireframe model with a ring on the end with two blue patches near a purple sphere labeled Z n s plus. It also has a loop that bends back toward the zinc ion. To the lower right, a third zinc finger motif is similar but light pink. The helix extends away from the viewer near the top and is connected by a black strand to the red zinc finger at the top. The wireframe rings are labeled H I s. The yellow pieces of the wireframe structure are labeled C y s. The wireframe wrings extend down from the helix near the purple sphere representing the zing ion below. A beta strand is visible to the left of the sphere and a strand loops to the lower right. Two yellow wireframe pieces are visible near the zinc ion.

Many eukaryotic DNA-binding proteins contain zinc fingers. The interaction of a single zinc finger with DNA is typically weak, and many DNA-binding proteins, like Zif268, have multiple zinc fingers that substantially enhance binding by interacting simultaneously with the DNA. One DNA-binding protein of the frog Xenopus has 37 zinc fingers. There are few known examples of the zinc finger motif in bacterial proteins.

The precise manner in which proteins with zinc fingers bind to DNA differs from one protein to the next. Some zinc fingers contain the amino acid residues that are important in sequence discrimination, whereas others seem to bind DNA nonspecifically (the amino acids required for specificity are located elsewhere in the protein). Zinc fingers can also function as RNA-binding motifs, such as in certain proteins that bind eukaryotic mRNAs and act as translational repressors. We discuss this role later (Section 28.3).

Homeodomain

Another type of DNA-binding domain has been identified in some proteins that function as transcriptional regulators, especially during eukaryotic development. This domain of 60 amino acid residues — called the homeodomain, because it was discovered in homeotic genes (genes that regulate the development of body patterns) — is highly conserved and has now been identified in proteins from a wide variety of organisms, including humans (Fig. 28-13). The DNA-binding segment of the domain is related to the helix-turn-helix motif. The DNA sequence that encodes this domain is known as the homeobox.

A vertical blue piece of D N A is shown with helices on each side. A wavy orange strand runs horizontally across the bottom to meet an almost vertical white helix on the left that runs up behind an overlapping almost vertical blue helix. A yellow strand labeled turn extends from thee bottom of the blue strand to a purple helix extending away from the viewer labeled recognition helix. Across the top of the D N A, a similar wavy orange strand runs almost horizontally to join the upper left of a whit helix that angles down to the left. A purple helix runs from the upper left into the venter of the D N A and crosses behind the white helix just above its center. A short blue helix runs up along the D N A to end near the end of the purple helix and a yellow strand connects the top of the blue helix with the left end of the purple helix.

RNA Recognition Motif

New classes of proteins with RNA-binding domains continue to be identified. RNA recognition motifs (RRMs) are found in some eukaryotic gene activators, where they may do double duty in binding DNA and RNA. When bound to specific binding sites in DNA, these activators induce transcription. The same activators are sometimes regulated in part by specific lncRNAs that compete with DNA binding and decrease gene transcription. Other proteins with RRM motifs bind to mRNA, rRNA, or any of a range of other smaller, noncoding RNAs. The RRM consists of 90 to 100 amino acid residues, arranged in a four-strand antiparallel β sheet sandwiched against two α helices, with a $β_{1} - α_{1} - β_{2} - β_{3} - α_{2} - β_{4}$ $beta 1 zero width space hyphen alpha Subscript 1 Baseline zero width space hyphen beta 2 zero width space hyphen beta 3 zero width space hyphen alpha 2 zero width space hyphen beta 4$ topology (Fig. 28-14a). This motif may be present as part of DNA-binding regulatory proteins that also have other DNA-binding motifs or may occur in proteins that bind uniquely to RNA. The RRM is just one of many diverse protein motifs known to interact with RNA (Fig. 28-14b).

FIGURE 28-14 RNA recognition motifs (RRMs). (a) An RRM from the p50 subunit of regulatory protein $NF- κ B$ $NF hyphen kappa upper B$ is shown, bound to (left) DNA and (right) RNA. Black lines indicate hydrogen-bonding interactions between particular amino acid residues and bases in the DNA or RNA. $NF- κ B$ $NF hyphen kappa upper B$ is the name of a family of structurally related eukaryotic transcription factors that regulate processes ranging from immune and inflammatory responses to cell growth and apoptosis. (b) Three additional RRM motifs from widespread RNA-binding protein families. [(a) Data from (left) PDB ID 1OOA, D. B. Huang et al., *Proc. Natl. Acad. Sci. USA* 100:9268, 2003; (right) PDB ID 1VKX, F. E. Chen et al., *Nature* 391:410, 1998. (b) DEAD box RIG1: PDB ID 3LRR, C. Lu et al., *Structure* 18:1032, 2010; ROQ domain: PDB ID 4QIK, D. Tan et al., *Nat. Struct. Mol. Biol.* 21:679, 2014; Pumilio-Nos-hunchback: PDB ID 5KL1, C. A. Weidmann et al., *eLife* 5, 2016.]

Part a shows a roughly rounded white structure on the upper right with helices in the upper right and beta strands in the venter and left. It fits against a horizontal strand of D N A below. At least six hydrogen bonds are visible between D N A and wireframe structures along the bottom of the white structure, which has blue and red segments visible. A similar white structure is shown to the right with double-stranded R N A below that forms a loop to the lower right. At least six hydrogen bonds are visible between the R N A and wireframe structures along the bottom of the white structure. Part b shows three different R R Ms. D E A D box R I G 1 shows double-stranded D N A across the bottom with beta strands to the left, right, and above. The R O Q domain shows horizontal double-stranded D N A beneath a structure that consists of many helices, including a clump of helices to the upper right. Pumilio-Nos-hunchback shows a single-stranded R N A molecule beneath a crescent-shaped structure consisting of many helices. There are many parallel helices that extend down toward the R N A and a line of helices forming a rounded outer surface. Strands extend down across the R N A to the right.

Regulatory Proteins Also Have Protein-Protein Interaction Domains

Regulatory proteins contain domains not only for DNA binding but also for protein-protein interactions — with RNA polymerase, other regulatory proteins, or other subunits of the same regulatory protein. Examples include many eukaryotic transcription factors that function as gene activators, which often bind as dimers to the DNA through DNA-binding domains that contain zinc fingers. Some structural domains are devoted to the interactions required for dimer formation, which is generally a prerequisite for DNA binding. Like DNA-binding motifs, the structural motifs that mediate protein-protein interactions tend to fall within one of a few common categories. Two important examples are the leucine zipper and the basic helix-loop-helix. Structural motifs such as these are the basis for classifying some regulatory proteins into structural families.

Leucine Zipper

The leucine zipper is an amphipathic α helix with a series of hydrophobic amino acid residues concentrated on one side (Fig. 28-15), with the hydrophobic surface forming the area of contact between the two polypeptides of a dimer. A striking feature of these α helices is the occurrence of Leu residues at every seventh position, forming a straight line along the hydrophobic surface. Although researchers initially thought the Leu residues interdigitated (hence the name “zipper”), we now know that they line up, side by side, as the interacting α helices coil around each other (forming a coiled coil; Fig. 28-15b). Regulatory proteins with leucine zippers often have a separate DNA-binding domain with a high concentration of basic (Lys or Arg) residues that can interact with the negatively charged phosphates of the DNA backbone. Leucine zippers have been found in many eukaryotic proteins and a few bacterial proteins.

Amino acid sequences are shown for three mammal regulatory proteins and one yeast regulatory protein. Each horizontal line shows the source (mammal or yeast), the name of the regulatory protein, and the amino acid sequence. The amino acid sequence is divided into a left-hand D N A-binding sequence, a connector (6 amino acids), and a leucine zipper. A consensus sequence is shown below. Row 1: source mammal, regulatory protein C/E B P, D N A-binding region D K N S N E Y R V yellow R yellow R E yellow R light red N N I A V R yellow K S yellow R D yellow K A; connector K Q R N V E; leucine zipper T Q Q K V L E light red L T S D N D R light red L R K R V E Q light red L S R E L D T light red L R G dash. Row 2: source mammal, regulatory protein J u n, D N A-binding region S Q E R I K A E R yellow K yellow R M yellow R light red N R I A A S yellow K C yellow R K yellow R yellow K, connector sequence L E R I A R, leucine zipper light red L E E K V K T light red L K A Q N S E light red L A S T A N M light red L T E Q V A Q light red L K Q dash. Row 3: source mammal, regulatory protein F o s, D N A-binding region E E R R R I R R I yellow R yellow R E yellow R light red N K M A A A yellow K C yellow R N yellow R yellow R, connector sequence R E L T D T, leucine zipper light red L Q A E T D Q light red L E D K K S A light red L Q T E I A N light red L L K E K E K light red L E F dash. Row 4: source yeast, regulatory protein G C N 4, D N A-binding region P E S S D P A A L yellow K yellow R A yellow R light red N T E A A R yellow R S yellow R A yellow R yellow K, connector sequence L Q R M K Q, leucine zipper light red L E D K V E E light red L L S K N Y H light red L E N E V A R light red L K K L V G E R space space no dash. Consensus sequence: 9 dashes, then yellow R over K; yellow R over K; dash; yellow R over K; light red N; five dashes, yellow R over K, dash, yellow R; dash; yellow R over K; yellow R over K; six dashes; light red L; six dashes; light red L; six dashes; light red L; six dashes; light red L; six dashes; light red L; space space no dash. The first light red N of the consensus sequence is labeled invariant A s n and a vertical line extends down to point to a light red box in part b. A vertical piece of D N A in shown in part b. Two helices extend across it. One helix extends from the upper left to just below the center left and the other extends from the lower left to just above the center right. The D N A-binding region is shown as the region that crosses the D N A. It contains multiple yellow pieces with yellow wireframe models out that contain pieces of blue. Two light red pieces are present, one on each strand. The top one is near the center of the D N A and is labeled invariant A s n. The second is to its lower left. After the two strands cross, the leucine zipper region begins. This includes several light red pieces. Wireframe structures extend down from the light red region in the upper strand to connect with similar structures in the lower strand.

Basic Helix-Loop-Helix

Another common structural motif, the basic helix-loop-helix, occurs in some eukaryotic regulatory proteins implicated in the control of gene expression during development of multicellular organisms. These proteins share a conserved region of about 50 amino acid residues important in both DNA binding and protein dimerization. This region can form two short, amphipathic α helices linked by a loop of variable length, the helix-loop-helix (distinct from the helix-turn-helix motif associated with DNA binding). The helix-loop-helix motifs of two polypeptides interact to form dimers (Fig. 28-16). In these proteins, DNA binding is mediated by an adjacent short amino acid sequence rich in basic residues, similar to the separate DNA-binding region in proteins containing leucine zippers.

A figure shows a helix-loop-helix structure. — FIGURE 28-16 Helix-loop-helix. The human transcription factor Max, bound to its DNA target site. The protein is dimeric; one subunit is colored. The recognition helix is linked via the loop to the dimer-forming helix, which merges with the carboxyl-terminal end of the subunit. Interaction of the carboxyl-terminal helices of the two subunits describes a coiled coil very similar to that of a leucine zipper (see Fig. 28-15b), but with only one pair of interacting Leu residues (side chains at the right) in this example. The overall structure is sometimes called a helix-loop-helix/leucine zipper motif. [Data from PDB ID 1HLO, P. Brownlie et al., *Structure* 5:509, 1997.]

A vertical blue double helix of D N A is shown on the left. A pink helix extends from the lower left across the D N A to end at just above the venter right. A white helix begins just above the center of the D N A and extends to the lower right. Where they cross, these helices are labeled recognition helix. A yellow strand connects the pink helix with the left end of a horizontal purple helix that runs behind it and ends to its left. This yellow strand is labeled loop. A white helix overlaps the lower right end of the first white helix and extends up above the purple helix to end just above the right end of the purple helix. Red wireframe models are shown connecting the right ends of the white and purple helices. Where the white and purple helices cross, they are labeled, dimer-forming helix.

Protein-Protein Interactions in Eukaryotic Regulatory Proteins

In eukaryotes, most genes are regulated by activators, and most genes are monocistronic. If a different activator were required for each gene, the number of activators (and genes encoding them) would need to be equivalent to the number of regulated genes. However, in yeast, about 300 transcription factors (many of them activators) are responsible for the regulation of many thousands of genes. Many of the transcription factors regulate the induction of multiple genes, but most genes are subject to regulation by multiple transcription factors. Appropriate regulation of different genes is accomplished by different combinations of a limited repertoire of transcription factors at each gene, a mechanism referred to as combinatorial control.

Combinatorial control is accomplished in part by mixing and matching the variants within a regulatory protein family to form a series of different active protein dimers. Several families of eukaryotic transcription factors have been defined on the basis of close structural similarities. Within each family, dimers can sometimes form between two identical proteins (a homodimer) or between two different members of the family (a heterodimer). A hypothetical family of four different leucine-zipper proteins could thus form up to 10 different dimeric species. In many cases, the different combinations have distinct regulatory and functional properties and regulate different genes. As we shall see, multiple regulatory proteins of this kind function in the regulation of most eukaryotic genes, further contributing to combinatorial control.

In addition to having structural domains devoted to DNA binding and protein dimerization, which direct a particular protein dimer to a particular gene, many regulatory proteins have domains that interact with RNA polymerase, with regulatory RNAs, with unrelated regulatory proteins, or with some combination of the three. At least three types of additional domains for protein-protein interaction have been characterized (primarily in eukaryotes): glutamine-rich, proline-rich, and acidic domains, the names reflecting the amino acid residues that are especially abundant.

Protein-DNA and protein-RNA binding interactions are the basis of the intricate regulatory circuits fundamental to gene function. We now turn to a closer examination of these gene regulatory schemes, first in bacteria, then in eukaryotes.

SUMMARY 28.1 The Proteins and RNAs of Gene Regulation

Transcription is initiated when an RNA polymerase interacts with a site called a promoter. In bacteria, the frequency of transcription initiation is dictated in part by sequence changes within the promoter. For gene products required all the time at a defined level — the products of housekeeping genes — the promoter sequence may be the sole element of regulation. For genes encoding products that are not always needed, regulation is imposed by additional proteins and RNAs.
Regulation of gene transcription is imposed primarily by three types of proteins: specificity factors, repressors, and activators. Regulatory RNAs also play an important role in regulating the expression of many genes.
In bacteria, genes that encode products with interdependent functions are often clustered in an operon, a single transcriptional unit. Transcription of the genes is generally blocked by binding of a specific repressor protein at a DNA site called an operator. Dissociation of the repressor from the operator is mediated by a specific small molecule, an inducer.
Many principles of gene regulation in bacteria were first elucidated in studies of the lactose (lac) operon. The Lac repressor dissociates from the lac operator when the repressor binds to its inducer, allolactose.
Regulatory proteins are DNA-binding proteins that recognize specific DNA sequences; most have distinct DNA-binding domains. Within these domains, common structural motifs that bind DNA (and/or RNA) are the helix-turn-helix, zinc finger, homeodomain, and RNA recognition motif.
Regulatory proteins often contain domains such as the leucine zipper and helix-loop-helix, required for dimerization or other protein-protein interactions, and other motifs required for activation of transcription. Mixing and matching of protein family variants in dimeric transcription factors provides for more efficient and responsive regulation through combinatorial control.