Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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Sources: Data for pancreas from V. E. Velculescu, L. Zhang, B. Vogelstein, and K. W. Kinzler, Serial analysis of gene

expression, Science 270(1995):484

487. Data for liver from T. Yamashita, S. Hashimoto, S. Kaneko, S. Nagai, N. Toyoda, T.

Suzuki, K. Kobayashi, and K. Matsushima, Comprehensive gene expression profile of a normal human liver, Biochem. Biophys.

Res. Commun. 269(2000):110

116.

III. Synthesizing the Molecules of Life 31. The Control of Gene Expression

Programming Gene Expression. Complex biological processes often involve coordinated control of the expression of

many genes. The maturation of a tadpole into a frog is largely controlled by thyroid hormone. This hormone regulates

gene expression by binding to a protein, the thyroid hormone receptor, shown at the right. In response to hormone

binding, this protein binds to specific DNA sites in the genome and modulates the expression of nearby genes. [(Left)

Shanon Cummings/Dembinsky Photo Associates.]

III. Synthesizing the Molecules of Life 31. The Control of Gene Expression

31.1. Prokaryotic DNA-Binding Proteins Bind Specifically to Regulatory Sites in

Operons

Bacteria such as E. coli usually rely on glucose as their source of carbon and energy. 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 essential enzyme in the metabolism of lactose is β-galactosidase, which hydrolyzes lactose into galactose

and glucose. These products are then metabolized by pathways discussed in Chapter 16.

This reaction can be conveniently followed in the laboratory through the use of alternative galactoside substrates that

form colored products such as X-Gal (Figure 31.1) 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 31.2). The presence of lactose in the culture medium induces a

large increase in the amount of β-galactosidase by eliciting the synthesis of new enzyme molecules rather than by

activating a preexisting but inactive precursor.

A crucial clue to the mechanism of gene regulation was the observation 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. The transacetylase is not essential for lactose metabolism but

appears to play a role in the detoxification of compounds that also may be transported by the permease. Thus, the

expression levels of a set of enzymes that all contribute to the adaptation to a given change in the environment change

together. Such a coordinated unit of gene expression is called an operon.

31.1.1. An Operon Consists of Regulatory Elements and Protein-Encoding Genes

The parallel regulation of β-galactosidase, the permease, and the transacetylase suggested that the expression of genes

encoding these enzymes is controlled by a common mechanism. Francois Jacob and Jacques Monod proposed the operon

model to account for this parallel regulation as well as the results of other genetic experiments (Figure 31.3). The genetic

elements of the model are a regulator gene, a regulatory DNA sequence called an operator site, and a set of structural

genes.

The regulator gene encodes a repressor protein that binds to the operator site. The binding of the repressor to the

operator prevents transcription of the structural genes. The operator and its associated structural genes constitute 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, respectively. The operon also contains a

promoter site (denoted by p), which directs the RNA polymerase to the correct transcription initiation site. The z, y, and

a genes are transcribed to give a single mRNA molecule that encodes all three proteins. An mRNA molecule encoding

more than one protein is known as a polygenic or polycistronic transcript.

31.1.2. The lac Operator Has a Symmetric Base Sequence

The operator site of the lac operon has been extensively studied (Figure 31.4). The nucleotide sequence of the operator

site shows a nearly perfect inverted repeat, indicating that the DNA in this region has an approximate twofold axis of

symmetry. Recall that cleavage sites for restriction enzymes such as EcoRV have similar symmetry properties (Section

9.3.3). Symmetry in the operator site usually corresponds to symmetry in the repressor protein that binds the operator

site. Symmetry matching is a recurring theme in protein-DNA interactions.

31.1.3. The lac Repressor Protein in the Absence of Lactose Binds to the Operator and

Blocks Transcription

How does the lac repressor inhibit the expression of the lac operon? The lac repressor can exist as a dimer of 37-kd

subunits, and two dimers often come together to form a tetramer. In the absence of lactose, the repressor binds very

tightly and rapidly to the operator. When the lac repressor is bound to DNA, it prevents bound RNA polymerase from

locally unwinding the DNA to expose the bases that will act as the template for the synthesis of the RNA strand (Section

28.1.3).

How does the lac repressor locate the operator site in the E. coli chromosome? The lac repressor binds 4 × 10

times as

strongly to operator DNA as it does to random sites in the genome. This high degree of selectivity allows the repressor to

find the operator efficiently even with a large excess (4.6 ×10

) of other sites within the E. coli genome. The dissociation

constant for the repressor-operator complex is approximately 0.1 pM (10

-13

M). The rate constant for association ( 10

-1

) is strikingly high, indicating that the repressor finds the operator by diffusing along a DNA molecule (a one-

dimensional search) rather than encountering it from the aqueous medium (a three-dimensional search).

Inspection of the complete E. coli genome sequence reveals two sites within 500 bp of the primary operator site that

approximate the sequence of the operator. Other lac repressor dimers can bind to these sites, particularly when aided by

cooperative interactions with the lac repressor dimer at the primary operator site. No other sites that closely match

sequence of the lac operator site are present in the rest of the E. coli genome sequence. Thus, the DNA-binding

specificity of the lac repressor is sufficient to specify a nearly unique site within the E. coli genome.

The three-dimensional structure of the lac repressor has been determined in various forms. Each monomer consists of a

small amino-terminal domain that binds DNA and a larger domain that mediates the formation of the dimer and the

tetramer (Figure 31.5). A pair of the amino-terminal domains come together to form the functional DNA-binding unit.

Complexes between the lac repressor and oligonucleotides that contain the lac operator sequence have been structurally

characterized. The lac repressor binds DNA by inserting an α helix into the major groove of DNA and making a series of

contacts with the edges of the base pairs as well as with the phosphodiester backbone (Figure 31.6). For example, an

arginine residue in the α helix forms a pair of hydrogen bonds with a guanine residue within the operator. Other bases

are not directly contacted but may still be important for binding by virtue of their effects on local DNA structure. As

expected, the twofold axis of the operator coincides with a twofold axis that relates the two DNA-binding domains.

31.1.4. Ligand Binding Can Induce Structural Changes in Regulatory Proteins

How does the presence of lactose trigger expression from the lac operon? Interestingly, lactose itself does not have this

effect; rather, allolactose, a combination of galactose and glucose with an α-1,6 rather than an α-1,4 linkage, does.

Allolactose is thus referred to as the inducer of the lac operon. Allolactose is a side product of the β-galactosidase

reaction produced at low levels by the few molecules of β-galactosidase that are present before induction. 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.

How does the presence of the inducer modulate gene expression? When the lac repressor is bound to the inducer, the

repressor's affinity for operator DNA is greatly reduced. The inducer binds in the center of the large domain within each

monomer. This binding leads to local conformational changes that are transmitted to the interface with the DNA-binding

domains (Figure 31.7). The relation between the two small DNA-binding domains is modified so that they cannot easily

contact DNA simultaneously, leading to a dramatic reduction in DNA-binding affinity.

Let us recapitulate the processes that regulate gene expression in the lactose operon (Figure 31.8). 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 little β-galactosidase, permease, or transacetylase are produced. The addition of lactose to the

environment leads to the formation of allolactose. This inducer binds to the lac repressor, leading to conformational

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 utilization of lactose.

The structure of the large domain of the lac repressor is similar to those of a large class of proteins that are present

in E. coli and other bacteria. This family of homologous proteins binds ligands such as sugars and amino acids at

their centers. Remarkably, domains of this family are utilized by eukaryotes in taste proteins and in neurotransmitter

receptors, as will be discussed in Chapter 32.

31.1.5. The Operon Is a Common Regulatory Unit in Prokaryotes

Many other gene-regulatory networks function in ways analogous to those of 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% identical 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 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

boldface type are particularly important. Examination of the E. coli genome sequence reveals the presence of more than

20 such sites, regulating 19 operons including more than 25 genes (Figure 31.9).

Because the DNA binding sites for these regulatory proteins are relatively short, it is likely that they evolved

independently from one another 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 may evolve adjacent to additional genes, allowing them

to become regulated in a physiologically appropriate manner. Binding sites for the pur repressor have evolved in the

regulatory regions of a wide range of genes taking part in nucleotide biosynthesis. All such genes can then be regulated

in a concerted manner.

31.1.6. Transcription Can Be Stimulated by Proteins That Contact RNA Polymerase

All the DNA-binding proteins discussed thus far function by inhibiting transcription until some environmental condition,

such as the presence of lactose, is met. There are also DNA-binding proteins that stimulate transcription. One

particularly well studied example is the catabolite activator protein (CAP), which is also known as the cAMP response

protein (CRP). When bound to cAMP, CAP, which also is a sequence-specific DNA-binding protein, stimulates the

transcription of lactose- and arabinose-catabolizing genes. Within the lac operon, CAP binds to an inverted repeat that is

centered near position -61 relative to the start site for transcription (Figure 31.10). CAP functions as a dimer of identical

subunits.

The CAP-cAMP complex stimulates the initiation of transcription by approximately a factor of 50. A major factor in this

stimulation is the recruitment of RNA polymerase to promoters to which CAP is bound. Studies have been undertaken to

localize the surfaces on CAP and on the α subunit of RNA polymerase that participate in these interactions (Figure

31.11). These energetically favorable protein-protein contacts increase the likelihood that transcription will be initiated at

sites to which the CAP-cAMP complex is bound. Thus, in regard to the lac operon, gene expression is maximal when the

binding of allolactose relieves the inhibition by the lac repressor, and the CAP-cAMP complex stimulates the binding of

RNA polymerase.

The E. coli genome contains many CAP-binding sites in positions appropriate for interactions with RNA polymerase.

Thus, an increase in the cAMP level inside an E. coli bacterium results in the formation of CAP-cAMP complexes that

bind to many promoters and stimulate the transcription of genes encoding a variety of catabolic enzymes.When grown

on glucose, E. coli have a very low level of catabolic enzymes such as β-galactosidase. Clearly, it would be wasteful to

synthesize these enzymes when glucose is abundant. The inhibitory effect of glucose, called catabolite repression, is due

to the ability of glucose to lower the intracellular concentration of cyclic AMP.

31.1.7. The Helix-Turn-Helix Motif Is Common to Many Prokaryotic DNA-Binding

Proteins

The structures of many prokaryotic DNA-binding proteins have now been determined, and amino acid sequences are

known for many more. Strikingly, the DNA-binding surfaces of many (but not all) of these proteins consist of a pair of α

helices separated by a tight turn (Figure 31.12). This helix-turn-helix motif is present in the lac repressor family, CAP,

and many other gene-regulatory proteins. In complexes with DNA, the second of these two helices (often called the

recognition helix) lies in the major groove, where amino acid side chains make contact with the edges of base pairs,

whereas residues of the first helix participate primarily in contacts with the DNA backbone. Helix-turn-helix motifs are

very frequently present on proteins that bind DNA as dimers, and thus two of the units will be present, one on each

monomer. In this case, the two helix-turn-helix units are related by twofold symmetry along the DNA double helix.

Although the helix-turn-helix motif 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

31.13). This protein binds DNA through the insertion of a pair of β strands into the major groove. We shall shortly

encounter a variety of other DNA-binding motifs found in eukaryotic cells.