Genetics - Chapter 17

Small interfering RNAs and microRNAs regulate gene expression through at least four distinct mechanisms:

(1) cleavage of mRNA, (2) inhibition of translation, (3) transcriptional silencing, or (4) degradation of mRNA.

Within the regulatory promoter are typically several different consensus sequences to which different transcriptional activators can bind.

Among different promoters, activator-binding sites are mixed and matched in different combinations (Figure 17.6), and so each promoter is regulated by a unique combination of transcriptional activator proteins.

The tails of histone proteins are often modified by the addition or removal of phosphate groups, methyl groups, or acetyl groups.

Another modification of histones is ubiquitination, in which small molecules called ubiquitin are added or removed from the histones.

In bacteria, transcription and translation take place simultaneously. In eukaryotes, transcription takes place in the nucleus and the pre-mRNAs are then processed before moving to the cytoplasm for translation, allowing opportunities for gene control after transcription.

Consequently, posttranscriptional gene regulation assumes an important role in eukaryotic cells. A common level of gene regulation in eukaryotes is RNA processing and degradation.

CpG island:

DNA region that contains many copies of a cytosine base followed by a guanine base; often found near transcription start sites in eukaryotic DNA. The cytosine bases in CpG islands are commonly methylated when genes are inactive but are demethylated before the initiation of transcription.

One type of gene control in eukaryotic cells is accomplished through the modification of chromatin structure.

In the nucleus, histone proteins associate to form octamers, around which helical DNA tightly coils to create chromatin (see Figure 11.4). In a general sense, this chromatin structure represses gene expression. For a gene to be transcribed, transcription factors, other regulator proteins, and RNA polymerase must bind to the DNA. How can these events take place with DNA wrapped tightly around histone proteins? The answer is that, before transcription, chromatin structure changes and the DNA becomes more accessible to the transcriptional machinery.

In male embryos, which have a single X chromosome (see Figure 17.11), the promoter that transcribes the Sxl gene in females is inactive, so no Sxl protein is produced. In the absence of Sxl protein, tra pre-mRNA is spliced at a different 3′ splice site to produce a nonfunctional form of Tra protein (Figure 17.12).

In turn, the presence of this nonfunctional Tra in males causes dsx pre-mRNAs to be spliced differently from that in females, and a male-specific Dsx protein is produced (see Figure 17.11). This event causes the development of male-specific traits.

Ribosomes, aminoacyl tRNAs, initiation factors, and elongation factors are all required for the translation of mRNA molecules. The availability of these components affects the rate of translation and therefore influences gene expression. For example, the activation of T lymphocytes (T cells) is critical to the development of immune responses to viruses (see Chapter 22). T cells are normally in the G0 stage of the cell cycle and not actively dividing.

On exposure to viral antigens, however, specific T cells become activated and undergo rapid proliferation (Figure 17.14). Activation includes a 7- to 10-fold increase in protein synthesis that causes cells to enter the cell cycle and proliferate. This burst of protein synthesis does not require an increase in mRNA synthesis. Instead, a global increase in protein synthesis is due to the increased availability of initiation factors taking part in translation—initiation factors that allow ribosomes to bind to mRNA and begin translation. This increase in initiation factors leads to more translation from the existing mRNA molecules, increasing the overall amount of protein synthesized. Similarly, insulin stimulates the initiation of overall protein synthesis by increasing the availability of initiation factors. Initiation factors exist in inactive forms and, in response to various cell signals, can be activated by chemical modifications of their structure, such as phosphorylation.

Another type of histone modification that affects chromatin structure is acetylation, the addition of acetyl groups (CH3CO) to histone.

The acetylation of histones usually stimulates transcription.

The basic idea of ChIP is that a particular protein and the DNA to which it is bound are isolated, the protein and DNA are then separated, and the DNA sequence to which the protein was formally bound is identified.

The technique has provided a powerful means of determining the genome-wide locations of modified histones and the binding sites for transcription factors and other proteins that affect transcription.

Other enzymes, called histone demethylases,

remove methyl groups from histones.

Histones in the octamer core of the nucleosome have two domains:

(1) a globular domain that associates with other histones and the DNA and (2) a positively charged tail domain that interacts with the negatively charged phosphate groups on the backbone of DNA.

This relaxation of the chromatin structure allows regulatory proteins access to binding sites on the DNA. Indeed, many DNase I hypersensitive sites correspond to known binding sites for regulatory proteins. At least three different processes affect gene regulation by altering chromatin structure:

(1) chromatin remodeling; (2) the modification of histone proteins; and (3) DNA methylation. Each of these mechanisms will be discussed in the sections that follow.

Although most eukaryotic cells do not possess operons, several eukaryotic genes may be activated by the same stimulus. Groups of bacterial genes are often coordinately expressed (turned on and off together) because they are physically clustered as an operon and have the same promoter, but coordinately expressed genes in eukaryotic cells are not clustered. How, then, is the transcription of eukaryotic genes coordinately controlled if they are not organized into an operon?

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In summary, the Tra, Tra-2, and Sxl proteins regulate alternative splicing that produces male and female phenotypes in Drosophila. Exactly how these proteins regulate alternative splicing is not yet known, but the Sxl protein (produced only in females) possibly blocks the upstream splice site on the tra pre-mRNA. This blockage would force the spliceosome to use the downstream 3′ splice site, which causes the production of Tra protein and eventually results in female traits (see Figure 17.12)

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In general, acetyl groups destabilize chromatin structure, allowing transcription to take place (Figure 17.2).

Acetyl groups are added to histone proteins by acetyltransferase enzymes; other enzymes called deacetylases strip acetyl groups from histones and restore chromatin structure, which represses transcription. Certain transcription factors (see Chapter 13) and other proteins that regulate transcription either have acetyltransferase activity or attract acetyltransferases to DNA.

Stalling was formerly thought to take place at only a small number of genes, but research now indicates that stalling is widespread throughout eukaryotic genomes. For example, stalled RNA polymerases were found at hundreds of genes in Drosophila. Several factors that promote stalling have been identified; one of these is a protein called negative elongation factor (NELF), which binds to RNA polymerase and causes it to stall after initiation.

Another protein called positive transcription elongation factor b (P-TEFb) relieves stalling and promotes elongation by phosphorylating NELF and RNA polymerase, perhaps by causing NELF to dissociate from the polymerase.

Several types of changes are observed in chromatin structure when genes become transcriptionally active.

As genes become transcriptionally active, regions around the genes become highly sensitive to the action of DNase I These regions, called DNase I hypersensitive sites, frequently develop about 1000 nucleotides upstream of the start site of transcription, suggesting that the chromatin in these regions adopts a more open configuration during transcription.

ChIP analysis has been used to determine the locations of modified histones that activate or repress transcription.

As mentioned earlier, the H3K4me3 histone mark is associated with promoters of active genes. nChIP analysis has successfully identified locations of this modified histone in the human genome, helping to identify active promoters in different tissues.

Other response elements found upstream of the metallothionein gene contribute to increasing its rate of transcription. For example, several copies of a metal response element (MRE) are upstream of the metallothionein gene (Figure 17.9). Heavy metals stimulate the binding of activator proteins to MREs, which elevates the rate of transcription of the metallothionein gene.

Because there are multiple copies of the MRE, high rates of transcription are induced by metals. Two enhancers also are located in the upstream region of the metallothionein gene; one enhancer contains a response element known as TRE, which stimulates transcription in the presence of an activated protein called AP1. A third response element called GRE is located approximately 250 nucleotides upstream of the metallothionein gene and stimulates transcription in response to certain hormones.

Evidence indicates that an association exists between DNA methylation and the deacetylation of histones, both of which repress transcription.

Certain proteins that bind tightly to methylated CpG sequences form complexes with other proteins that act as histone deacetylases. In other words, methylation appears to attract deacetylases, which remove acetyl groups from the histone tails, stabilizing the nucleosome structure and repressing transcription. The demethylation of DNA allows acetyltransferases to add acetyl groups, disrupting nucleosome structure and permitting transcription.

DNase I hypersensitive site:

Chromatin region that becomes sensitive to digestion by the enzyme DNase I.

insulator:

DNA sequence that blocks or insulates the effect of an enhancer; must be located between the enhancer and the promoter to have blocking activity; also may limit the spread of changes in chromatin structure.

Changes in chromatin structure that affect gene expression, such as the mechanisms just described (chromatin remodeling, histone modification, and DNA methylation), are examples of the phenomenon of epigenetics.

Epigenetics is defined as alterations to DNA and chromatin structure that affect traits and are passed on to other cells or generations but are not caused by changes in the DNA base sequences.

The amount of a protein that is synthesized depends on the amount of corresponding mRNA available for translation. The amount of available mRNA, in turn, depends on both the rate of mRNA synthesis and the rate of mRNA degradation.

Eukaryotic mRNAs are generally more stable than bacterial mRNAs, which typically last only a few minutes before being degraded. Nonetheless, there is great variability in the stability of eukaryotic mRNA: some mRNAs persist for only a few minutes; others last for hours, days, or even months. These variations can produce large differences in the amount of protein that is synthesized.

Evidence suggests at least two mechanisms by which remodeling complexes reposition nucleosomes.

First, some remodeling complexes cause the nucleosome to slide along the DNA, allowing DNA that was wrapped around the nucleosome to occupy a position in between nucleosomes, where it is more accessible to proteins affecting gene expression (see Figure 17.1). Second, some complexes cause conformational changes in the DNA, in nucleosomes, or in both so that DNA that is bound to the nucleosome assumes a more exposed configuration.

Transcriptional activator proteins have two distinct functions (see Figure 17.5).

First, they are capable of binding DNA at a specific base sequence, usually a consensus sequence in a regulatory promoter or enhancer; for this function, most transcriptional activator proteins contain one or more DNA-binding motifs, such as the helix-turn-helix, zinc finger, and leucine zipper (see Chapter 16). A second function is the ability to interact with other components of the transcriptional apparatus and influence the rate of transcription.

Genes that are coordinately expressed in eukaryotic cells are able to respond to the same stimulus because they have short regulatory sequences in common in their promoters or enhancers.

For example, different eukaryotic heat-shock genes possess a common regulatory element upstream of their start sites. Such DNA regulatory sequences are called response elements; they are short sequences that typically contain consensus sequences (Table 17.1) at varying distances from the gene being regulated. The response elements are binding sites for transcriptional activators. A transcriptional activator protein binds to the response element and elevates transcription. The same response element may be present in different genes, allowing multiple genes to be activated by the same stimulus.

Much of development in multicellular eukaryotes is through gene regulation: different genes are turned on and off at specific times (see Chapter 22). In fact, when miRNAs were first discovered, researchers noticed that a mutation in an miRNA in C. elegans caused a developmental defect. Research now demonstrates that miRNA molecules are key factors in controlling development in plants, animals, and humans.

For example, the vertebrate heart develops through the programmed differentiation and proliferation of cardiomyocytes, which are controlled by a specific miRNA termed miR-1-1.

UASG exhibits the properties of an enhancer—a regulatory sequence that may be some distance from the regulated gene and is independent of the gene in position and orientation (see Chapter 13). When bound to UASG, GAL4 activates the transcription of yeast genes needed for metabolizing galactose.

GAL4 and a number of other transcriptional activator proteins contain multiple amino acids with negative charges that form an acidic activation domain. These acidic activators stimulate transcription by enhancing the assembly of the basal transcription apparatus.

The basal transcription apparatus—consisting of RNA polymerase, transcription factors, and other proteins—assembles at the core promoter. When the initiation of transcription has taken place, RNA polymerase moves downstream, transcribing the structural gene and producing an RNA product. At some genes, RNA polymerase initiates transcription and transcribes from 24 to 50 nucleotides of RNA but then pauses or stalls. For example, stalling is observed at genes that encode heat-shock proteins in Drosophila—proteins that help to prevent damage from stressing agents such as extreme heat.

Heat-shock proteins are produced by a large number of different genes. During times of environmental stress, the transcription of all the heat-shock genes is greatly elevated. RNA polymerase initiates transcription at heat-shock genes in Drosophila but, in the absence of stress, stalls downstream of the transcription initiation site. Stalled polymerases are released when stress is encountered, allowing rapid transcription of the genes and the production of heat-shock proteins that facilitate adaptation to the stressful environment.

Recent research demonstrates that many enhancers are themselves transcribed into short RNA molecules called enhancer RNAs (eRNAs). Evidence suggests that transcription of an enhancer is often associated with transcription at the promoters that the enhancers affect.

How transcription at the enhancer might affect transcription occurring at a distant promoter is not clear. Enhancers might recruit RNA polymerase, which is then transferred to the promoter when the enhancer interacts with the promoter. Alternatively, transcription of the enhancer might allow the chromatin to adopt a more open configuration, which then facilitates transcription at nearby promoters.

many features of gene regulation are common to both bacterial and eukaryotic cells. For example, in both types of cells, DNA-binding proteins influence the ability of RNA polymerase to initiate transcription.

However, there are also some differences. First, many prokaryotic genes are organized into operons and are transcribed into a single RNA molecule. Although some operon-like gene clusters have been found in worms and even some primitive chordates, most eukaryotic genes have their own promoters and are transcribed separately. Second, chromatin structure affects gene expression in eukaryotic cells; DNA must unwind from the histone proteins before transcription can take place. Third, the presence of the nuclear membrane in eukaryotic cells separates transcription and translation in time and space. Therefore, the regulation of gene expression in eukaryotic cells is characterized by a greater diversity of mechanisms that act at different points in the transfer of information from DNA to protein.

Most enhancers are capable of stimulating any promoter in their vicinities. Their effects are limited, however, by insulators (also called boundary elements), which are DNA sequences that block or insulate the effect of enhancers in a position-dependent manner.

If the insulator lies between the enhancer and the promoter, it blocks the action of the enhancer; but, if the insulator lies outside the region between the two, it has no effect (Figure 17.8). Specific proteins bind to insulators and play a role in their blocking activity. Some insulators also limit the spread of changes in chromatin structure that affect transcription. Some enhancer-like elements are found in prokaryotes.

Changes in a relatively small number of regulatory sequences help produce the large phenotypic differences between humans and chimpanzees.

Illustrated here is a network of interacting genes for transcription factors that are differentially expressed in the brains of humans and chimpanzees and control the expression of other genes. Red circles represent transcription factors that are more highly expressed in the human brain; green circles represent transcription factors that are more highly expressed in the chimpanzee brain.

Cellular RNA is degraded by ribonucleases, enzymes that specifically break down RNA. Most eukaryotic cells contain 10 or more types of ribonucleases, and there are several different pathways of mRNA degradation.

In one pathway, the 5′ cap is first removed, followed by 5′→3′ removal of nucleotides. A second pathway begins at the 3′ end of the mRNA and removes nucleotides in the 3′→5′ direction. In a third pathway, the mRNA is cleaved at internal sites.

A version of this technique, called native ChIP (nChIP) does not utilize crosslinking. It is often used for finding the locations of modified histone proteins.

In this case, crosslinking is not required because the DNA and histone proteins are naturally linked by the nucleosome structure. The chromatin is isolated from the cell and fragmented, and antibodies to a particular protein (usually a specific modified histone) are used to precipitate the protein-DNA complexes. The protein and DNA are separated, the protein digested, and the DNA fragments to which the modified histones were attached are sequenced or otherwise identified.

One version of ChIP, called crosslinked ChIP (XChIP) is used for identifying the binding sites of transcription factors and other proteins that bind to DNA.

In this procedure (Figure 17.4), the protein and associated DNA are temporarily crosslinked, which means that they are treated with formaldehyde or UV light to create covalent bonds between the DNA and protein. The crosslinking holds the DNA and protein together so that the DNA to which the protein is bound will separate along with the protein. After crosslinking, the cell is lysed and the chromatin is broken into pieces by digestion with an enzyme or by mechanical shearing. Antibodies specific for a particular protein—such as a specific transcription factor—are then applied. The antibodies attach to the protein-DNA complexes and cause them to precipitate. The crosslink is then reversed, separating the DNA and protein. The protein is removed by an enzyme that digests protein but not DNA, leaving fragments of the DNA to which the protein was bound. These fragments can then be sequenced or identified with other methods. The result is the information about the genomic locations of binding sites for the specific protein.

Alternative splicing allows a pre-mRNA to be spliced in multiple ways, generating different proteins in different tissues or at different times in development (see Chapter 14).

Many eukaryotic genes undergo alternative splicing, and the regulation of splicing is an important means of controlling gene expression in eukaryotic cells.

Mechanisms also exist for the regulation of translation of specific mRNAs. The initiation of translation in some mRNAs is regulated by proteins that bind to an mRNA's 5′ UTR and inhibit the binding of ribosomes, similar to the way in which repressor proteins bind to operators and prevent the transcription of structural genes. The translation of some mRNAs is affected by the binding of proteins to sequences in the 3′ UTR.

Many eukaryotic proteins are extensively modified after translation by the selective cleavage and trimming of amino acids from the ends, by acetylation, or by the addition of phosphate groups, carboxyl groups, methyl groups, or carbohydrates to the protein. These modifications affect the transport, function, and activity of the proteins.

histone code

Modification of histone proteins, such as the addition or removal of phosphate groups, methyl groups, or acetyl groups, that encode information affecting how genes are expressed. The histone code affects gene expression by altering chromatin structure directly or, in some cases, by serving as recognition sites for proteins that bind to DNA and that then regulate transcription.

This example illustrates a common feature of eukaryotic transcriptional control: a single gene may be activated by several different response elements found in both promoters and enhancers.

Multiple response elements allow the same gene to be activated by different stimuli. At the same time, the presence of the same response element in different genes allows a single stimulus to activate multiple genes. In this way, response elements allow complex biochemical responses in eukaryotic cells.

Recent studies demonstrate that, through their effects on gene expression, miRNAs play important roles in many diseases and disorders. For example, a genetic form of hearing loss has been associated with a mutation in the gene that encodes an miRNA. Other miRNAs are associated with heart disease.

One miRNA called miR-1-2 is highly expressed in heart muscle. Mice genetically engineered to express only 50% of the normal amount of miR-1-2 frequently have holes in the wall that separates their left and right ventricles, a common congenital heart defect seen in newborn humans. Overexpression of another miRNA called miR-1 in the hearts of adult mice causes cardiac arrhythmia—irregular electrical activity of the heart that can be life-threatening in humans. Changes in the expression of other miRNAs have been associated with cancer.

Transcriptional activator proteins bind to the consensus sequences in the regulatory promoter and affect the assembly or stability of the basal transcription apparatus at the core promoter.

One of the components of the basal transcription apparatus is a complex of proteins called the mediator (see Figure 17.6). Transcriptional activator proteins binding to sequences in the regulatory promoter (or enhancer, see next section) make contact with the mediator and affect the rate at which transcription is initiated. Some regulatory promoters also contain sequences that are bound by proteins that lower the rate of transcription through inhibitory interactions with the mediator.

In spite of the large phenotypic gulf between humans and chimpanzees, sequencing of their genomes reveals that their DNA is remarkably similar.

Only about 1% of individual base pairs differ between the two species, along with a 3% difference in insertions and deletions. Thus, 96% of the DNA of humans and chimpanzees is identical. But clearly humans are not chimpanzees. How then, did humans and chimpanzees come to be so different? Where are the genes that make us human?

The expression of a number of eukaryotic genes is controlled through RNA interference, also known as RNA silencing and posttranscriptional gene silencing (see Chapter 14). Research suggests that as much as 30% of human genes are regulated by RNA interference.

RNA interference is widespread in eukaryotes, existing in fungi, plants, and animals. This mechanism is also widely used as a powerful technique for artificially regulating gene expression in genetically engineered organisms (see Chapter 19).

Transcription in eukaryotes is often regulated through factors that affect the initiation of transcription, including changes in chromatin structure, transcription factors, and transcriptional regulatory proteins.

Research indicates that transcription may also be controlled through factors that affect stalling and elongation of RNA polymerase after transcription has been initiated.

A final mechanism by which miRNAs regulate gene expression is by triggering the decay of mRNA in a process that does not require Slicer activity. For example, a short-lived mRNA with an AU-rich element in its 3′ UTR is degraded by an RNA-silencing mechanism.

Researchers have identified an miRNA with a sequence that is complementary to the consensus sequence in the AU-rich element. This miRNA binds to the AU-rich element and, in a way that is not yet fully understood, brings about the degradation of the mRNA in a process that requires Dicer and RISC.

Many of the enzymes and proteins that modify histones, such as methyltransferases and demethylases, do not bind to specific DNA sequences, and must be recruited to specific chromatin sites.

Sequence-specific binding proteins, pre-existing histone modifications, and RNA molecules serve to recruit histone-modifying enzymes to specific sites.

Enhancers are capable of affecting transcription at distant promoters. For example, an enhancer that regulates the gene encoding the alpha chain of the T-cell receptor is located 69,000 bp downstream of the gene's promoter. Furthermore, the exact position and orientation of an enhancer relative to the promoter can vary. How can an enhancer affect the initiation of transcription taking place at a promoter that is tens of thousands of base pairs away? In many cases, regulator proteins bind to the enhancer and cause the DNA between the enhancer and the promoter to loop out, bringing the promoter and enhancer close to each other, and so the transcriptional regulator proteins are able to directly interact with the basal transcription apparatus at the core promoter (see Figure 17.5).

Some enhancers may be attracted to promoters by proteins that bind to sequences in the regulatory promoter and "tether" the enhancer close to the core promoter. Enhancers may also affect transcription by undergoing modifications that alter chromatin structure. A typical enhancer is about 500 bp in length and contains 10 binding sites for proteins that regulate transcription.

A protein called splicing factor 2 (SF2) enhances the production of mRNA encoding the small t antigen (see Figure 17.10). Splicing factor 2 has two binding domains: one domain is an RNA-binding region and the other has alternating serine and arginine amino acids. These two domains are typical of SR (serine- and arginine-rich) proteins, which often play a role in regulating splicing.

Splicing factor 2 stimulates the binding of small nuclear ribonucleoproteins (snRNPs) to the 5′ splice site, one of the earliest steps in RNA splicing (see Chapter 14). The precise mechanism by which SR proteins influence the choice of splice sites is poorly understood. One model suggests that SR proteins bind to specific splice sites on mRNA and stimulate the attachment of snRNPs, which then commit the site to splicing.

A common modification is the addition of three methyl groups to lysine 4 in the tail of the H3 histone protein, abbreviated H3K4me3 (K is the abbreviation for lysine). Histones containing the H3K4me3 modification are frequently found near promoters of transcriptionally active genes in eukaryotes

Studies have identified proteins that recognize and bind to H3K4me3, including nucleosome remodeling factor (NURF). NURF and other proteins that recognize H3K4me3 have a common protein-binding domain that binds to the H3 histone tail and then alters chromatin packing, allowing transcription to take place. Research has also demonstrated that some transcription factors, which are necessary for the initiation of transcription (see Chapter 13 and Section 17.3), directly bind to H3K4me3.

Among the many genes that control flowering in Arabidopsis is flowering locus C (FLC), which plays an important role in suppressing flowering until after an extended period of coldness (a process called vernalization).

The FLC gene encodes a regulator protein that represses the activity of other genes that affect flowering (Figure 17.3). As long as FLC is active, flowering remains suppressed. The activity of FLC is controlled by another locus called flowering locus D (FLD), the key role of which is to stimulate flowering by repressing the action of FLC. In essense, flowering is stimulated because FLD represses the repressor. How does FLD repress FLC?

RNA interference is triggered by microRNAs (miRNAs) and small interfering RNAs (siRNAs), depending on their origin and mode of action (see Chapter 14). An enzyme called Dicer cleaves and processes double-stranded RNA to produce single-stranded siRNAs or miRNAs that are from 21 to 25 nucleotides in length (Figure 17.13) and pair with proteins to form an RNA-induced silencing complex (RISC).

The RNA component of RISC then pairs with complementary base sequences of specific mRNA molecules, most often with sequences in the 3′ UTR of the mRNA. Small interfering RNAs tend to base pair perfectly with the mRNAs, whereas miRNAs often form less-than-perfect pairings.

Transcriptional activator proteins stimulate and stabilize the basal transcription apparatus at the core promoter.

The activators may interact directly with the basal transcription apparatus or indirectly through protein coactivators. Some activators and coactivators, as well as the general transcription factors, also have acetyltransferase activity and so further stimulate transcription by altering chromatin structure.

Other siRNAs silence transcription by altering chromatin structure. These siRNAs combine with proteins to form a complex called RITS (for RNA transcriptional silencing; see Figure 17.13c), which is analogous to RISC. The siRNA component of RITS then binds to its complementary sequence in DNA or an RNA molecule in the process of being transcribed and represses transcription by attracting enzymes that methylate the tails of histone proteins.

The addition of methyl groups to the histones causes them to bind DNA more tightly, restricting the access of proteins and enzymes necessary to carry out transcription (see earlier section on histone modification). Some miRNAs bind to complementary sequences in DNA and attract enzymes that methylate the DNA directly, which also leads to the suppression of transcription (see earlier section on DNA methylation).

A single eukaryotic gene may be regulated by several different response elements. For example, the metallothionein gene protects cells from the toxicity of heavy metals by encoding a protein that binds to heavy metals and removes them from cells.

The basal transcription apparatus assembles around the TATA box, just upstream of the transcription start site for the metallothionein gene, but the apparatus alone is capable of only low rates of transcription.

Some miRNAs regulate genes by inhibiting the translation of their complementary mRNAs (see Figure 17.13b). For example, an important gene in flower development in Arabidopsis thaliana is APETALA2. The expression of this gene is regulated by an miRNA that base pairs with nucleotides in the coding region of APETALA2 mRNA and inhibits its translation.

The exact mechanism by which miRNAs repress translation is not known, but some research suggests that it can inhibit both the initiation step of translation and steps after translation initiation such as those causing premature termination. Many mRNAs have multiple miRNA-binding sites, and translation is most efficiently inhibited when multiple miRNAs are bound to the mRNA.

FLD encodes a deacetylase enzyme, which removes acetyl groups from histone proteins in the chromatin surrounding FLC (see Figure 17.3). The removal of acetyl groups from histones alters chromatin structure and inhibits transcription.

The inhibition of transcription prevents FLC from being transcribed and removes its repression on flowering. In short, FLD stimulates flowering in Arabidopsis by deacetylating the chromatin that surrounds FLC, thereby removing its inhibitory effect on flowering.

Another change in chromatin structure associated with transcription is the methylation of cytosine bases, which yields 5-methylcytosine

The methylation of cytosine in DNA is distinct from the methylation of histone proteins mentioned earlier. Heavily methylated DNA is associated with the repression of transcription in vertebrates and plants, whereas transcriptionally active DNA is usually unmethylated in these organisms. Abnormal patterns of methylation are also associated with some types of cancer.

Messenger RNA degradation from the 5′ end is most common and begins with the removal of the 5′ cap. This pathway is usually preceded by the shortening of the poly(A) tail. Poly(A)-binding proteins (PABPs) normally bind to the poly(A) tail and contribute to its stability-enhancing effect.

The presence of these proteins at the 3′ end of the mRNA protects the 5′ cap. When the poly(A) tail has been shortened below a critical limit, the 5′ cap is removed, and nucleases then degrade the mRNA by removing nucleotides from the 5′ end. These observations suggest that the 5′ cap and the 3′ poly(A) tail of eukaryotic mRNA physically interact with each other, most likely by the poly(A) tail bending around so that the PABPs make contact with the 5′ cap (see Figure 15.18).

Another example of alternative mRNA splicing that regulates gene expression is the determination of whether a fruit fly develops as male or female. Sex differentiation in Drosophila arises from a cascade of gene regulation. When two X-chromosomes are present, a female-specific promoter is activated early in development and stimulates the transcription of the sex-lethal (Sxl) gene (Figure 17.11).

The protein encoded by Sxl regulates the splicing of the pre-mRNA transcribed from another gene called transformer (tra). The splicing of tra pre-mRNA results in the production of the Tra protein (see Figure 17.11). Together with another protein (Tra-2), Tra stimulates the female-specific splicing of pre-mRNA from yet another gene called doublesex (dsx). This event produces a female-specific Dsx protein, which causes the embryo to develop female characteristics.

The importance of histone acetylation in gene regulation is demonstrated by the control of flowering in Arabidopsis thaliana, a plant with a number of characteristics that make it an excellent genetic model for plant systems

The time at which flowering takes place is critical to the life of a plant; if flowering is initiated at the wrong time of year, pollinators may not be available to fertilize the flowers or environmental conditions may be unsuitable for the survival and germination of the seeds. Consequently, flowering time in most plants is carefully regulated in response to multiple internal and external cues, such as plant size, photoperiod, and temperature.

Chromatin-remodeling complexes are targeted to specific DNA sequences by transcriptional activators or repressors that attach to a remodeling complex and then bind to the promoters of specific genes.

There is also evidence that chromatin-remodeling complexes work together with enzymes that alter histones, such as acetyltransferase enzymes (see next section), to change chromatin structure and expose DNA for transcription.

One type of histone modification is the addition of methyl groups to the tails of histone proteins.

These modifications can bring about either the activation or the repression of transcription, depending on which particular amino acids in the histone tail are methylated.

Some transcription factors and other regulatory proteins alter chromatin structure without altering the chemical structure of the histones directly.

These proteins are called chromatin-remodeling complexes. They bind directly to particular sites on DNA and reposition the nucleosomes, allowing transcription factors and RNA polymerase to bind to promoters and initiate transcription (Figure 17.1).

RISCs that contain an siRNA (and some that contain an miRNA) pair with mRNA molecules and cleave the mRNA near the middle of the bound siRNA (see Figure 17.13a).

This cleavage is carried out by a protein that is sometimes referred to as "Slicer." After cleavage, the mRNA is further degraded. Thus, the presence of siRNAs and miRNAs increase the rate at which mRNAs are broken down and decrease the amount of protein produced.

One of the best-studied examples of a chromatin-remodeling complex is SWI-SNF, which is found in yeast, humans, Drosophila, and other organisms.

This complex utilizes energy derived from the hydrolysis of ATP to reposition nucleosomes, exposing promoters in the DNA to the action of other regulatory proteins and RNA polymerase.

Much of RNA degradation takes place in specialized complexes called P bodies. However, P bodies appear to be more than simply destruction sites for RNA. Evidence suggests that P bodies can temporarily store mRNA molecules, which may later be released and translated.

Thus, P bodies help control the expression of genes by regulating which RNA molecules are degraded and which are sequestered for later release. RNA degradation facilitated by small interfering RNAs (siRNAs) also may take place within P bodies (see next section).

Some regulatory proteins in eukaryotic cells act as repressors, inhibiting transcription. These repressors bind to sequences in the regulatory promoter or to distant sequences called silencers, which, like enhancers, are position and orientation independent.

Unlike repressors in bacteria, most eukaryotic repressors do not directly block RNA polymerase. These repressors may compete with activators for DNA binding sites: when a site is occupied by an activator, transcription is activated, but, if a repressor occupies that site, there is no activation. Alternatively, a repressor may bind to sites near an activator site and prevent the activator from contacting the basal transcription apparatus. A third possible mechanism of repressor action is direct interference with the assembly of the basal transcription apparatus, thereby blocking the initiation of transcription.

We just considered one level at which gene expression is controlled—the alteration of chromatin and DNA structure.

We now turn to another important level of control—control through the binding of proteins to DNA sequences that affect transcription.

A particular region of GAL4 binds another protein called GAL80, which regulates the activity of GAL4 in the presence of galactose.

When galactose is absent, GAL80 binds to GAL4, preventing GAL4 from activating transcription (Figure 17.7). When galactose is present, however, it binds to another protein called GAL3, which interacts with GAL80, causing a conformational change in GAL80 so that it can no longer bind GAL4. The GAL4 protein is then free to activate the transcription of the genes, whose products metabolize galactose.

DNA regions with many CpG sequences are called CpG islands and are commonly found near transcription start sites.

While genes are not being transcribed, these CpG islands are often methylated, but the methyl groups are removed before the initiation of transcription. CpG methylation is also associated with long-term gene repression, such as on the inactivated X chromosome of female mammals

Fossil evidence indicates that humans and chimpanzees diverged genetically only 5 to 7 million years ago—a mere blink of the eye in evolutionary time.

Yet humans and chimps differ in a large number of anatomical, physiological, behavioral, and cognitive traits. For example, there are numerous differences in the structure of the backbone, pelvis, skull, jaw, teeth, hands, and feet of humans and chimpanzees. The size of the human brain is more than twice that of the chimpanzee; and humans exhibit complex language and cultural characteristics not seen in chimpanzees. Indeed, the degree of phenotypic difference between chimpanzees and humans is so large that scientists place them into entirely different primate families (humans in the family Hominidae and chimpanzees in the family Pongidae).

All of these modifications have sometimes been called the histone code,

because they encode information that affects how genes are expressed.

For example, the addition of a single acetyl group to lysine 16 in the tail of the H4 histone prevents the formation of the 30-nm chromatin fiber (see Figure 11.4),

causing the chromatin to be in an open configuration and available for transcription.

Our understanding of how changes in chromatin structure are associated with gene expression, as well as how DNA binding proteins affect transcription, have been greatly advanced by the use of a technique called

chromatin immunoprecipitation (ChIP). This technique allows researchers to determine the specific locations within the genome where proteins interact with DNA. Those proteins might be histones that have undergone modifications, transcription factors, or other proteins that bind to promoters and enhancers (sequences that affect the transcription of distant genes

Transcription is an important level of control in eukaryotic cells, and this control requires a number of different types of proteins and regulatory elements.

general transcription factors and RNA polymerase assemble into a basal transcription apparatus, the complex of RNA polymerase, transcription factors, and other proteins that carry out transcription. The basal transcription apparatus binds to a core promoter located immediately upstream of a gene and is capable of minimal levels of transcription; transcriptional regulator proteins are required to bring about normal levels of transcription. These proteins bind to a regulatory promoter, which is located upstream of the core promoter (Figure 17.5), and to enhancers, which may be located some distance from the gene. Some transcriptional regulator proteins are activators, stimulating transcription. Others are represssors, inhibiting transcription.

Eukaryotic gene regulation is less well understood than bacterial regulation, partly owing to the

larger genomes in eukaryotes, their greater sequence complexity, and the difficulty of isolating and manipulating mutations that can be used in the study of gene regulation. Nevertheless, great advances in our understanding of the regulation of eukaryotic genes have been made in recent years.

Enzymes called histone methyltransferases add

methyl groups to specific amino acids (usually lysine or arginine) of histone proteins.

Other parts of eukaryotic mRNA, including sequences in the 5′ untranslated region (5′ UTR), the coding region, and the 3′ UTR, also affect mRNA stability. Some short-lived eukaryotic mRNAs have one or more copies of a consensus sequence consisting of 5′-AUUUAUAA-3′,

referred to as the AU-rich element, in the 3′ UTR. Messenger RNAs containing AU-rich elements are degraded by a mechanism in which microRNAs take part (see next section).

genetic variations that make us human are concentrated in regulatory sequences—

those parts of the genome that control the expression of other genes. In this way, small genetic changes might influence the expression of numerous other genes and affect the phenotypes of many traits simultaneously. Unfortunately, there were no techniques available at the time to examine regulatory sequences and to test their hypothesis.

An example of a transcriptional activator protein is GAL4, which regulates the transcription of several yeast genes whose products metabolize galactose. Like the genes in the lac operon, the genes that control galactose metabolism are inducible:

when galactose is absent, these genes are not transcribed and the proteins that break down galactose are not produced; when galactose is present, the genes are transcribed and the enzymes are synthesized. GAL4 contains several zinc fingers and binds to a DNA sequence called UASG (upstream activating sequence for GAL4).