Last Exam Part 14 genetics

The core promoter determines where

RNA polymerase II will bind to the DNA and begin transcription. The core promoter includes the TATA box (-25 sequence), which serves as the binding site for the general transcription factor protein TFIID and the +1 site, the first base in the template DNA strand that is transcribed by RNA polymerase II. For transcription to occur, the TATA box and the +1 site must be present. If these two sequences are the only sequences present upstream of a gene, the gene will be transcribed at a low, yet constant rate (the basal level of transcription).

Control of transcription (mediator) Mediator is a large protein complex that mediates the interaction between the regulatory transcription factors (i.e. activator and repressor proteins) and

RNA polymerase II. If mediator activates RNA polymerase II, the elongation phase of transcription begins, and RNA synthesis occurs.

Control of transcription (TFIID) We have seen that regulatory transcription factors (activator and repressor proteins) influence the ability of RNA polymerase II to transcribe a gene. However, these regulatory transcription factors do not typically bind to RNA polymerase II directly. Instead, transcription factors communicate DNA binding indirectly to RNA polymerase II through other protein complexes. Eukaryotic transcription factors influence RNA polymerase II activity through

TFIID, mediator, the enzymes involved in chromatin remodeling, and the enzymes involved in DNA methylation.

(The four different structural motifs that are found in transcription factor proteins include)Basic helix-loop-helix (bHLH).

The bHLH motif is very similar to the helix-turn-helix motif and contains a recognition helix. Instead of a turn to connect the two α helices, this type of transcription factor uses a loop to connect two α helices. bHLH transcription factors play an important role in the differentiation of cells. For example, the MyoD protein, important in the differentiation of muscle cells, is a bHLH transcription factor.

(three general ways that the activator and repressor proteins are regulated)Protein-protein interactions.

The formation of transcription factor homodimers or heterodimers influences the ability of the activator or repressor protein to bind to enhancer or silencer DNA sequences and influence transcription.

General transcription factors (GTFs).

The general transcription factor proteins include TFIID, TFIIB, TFIIF, TFIIE, and TFIIH. These proteins function to recruit RNA polymerase II to the core promoter to begin transcription. The general transcription factors are required for all transcription events. If these transcription factors are the only ones involved, the gene is transcribed at the basal level. The general transcription factors are also required for transcription rates above this basal level.

(The four different structural motifs that are found in transcription factor proteins include)Helix-turn-helix (HTH).

The helix-turn-helix motif is found in both prokaryotic and eukaryotic transcription factor proteins. The HTH motif includes two α-helices separated by a "turn" of 3-4 amino acids. One α-helix is called the recognition helix, and functions to bind to the nitrogenous bases in the major groove of the DNA. The recognition helix also includes many basic amino acids (positively charged) that bind to the DNA backbone (negatively charged). Examples of transcription factor that contain the HTH motif include sigma (σ) factor, the lac repressor protein, and the catabolite activator protein (CAP).

(The four different structural motifs that are found in transcription factor proteins include)Leucine zipper motif

The leucine zipper motif contains many leucine amino acids in a row. When the leucine-rich regions on two transcription factors recognize each other, they interact to form a protein structure called a coiled-coil that resembles a zipper. The DNA sequence is recognized by α-helices (recognition helices) that extend from the coiled-coil region. The CREB protein (see below) contains a leucine zipper motif.

(The four different structural motifs that are found in transcription factor proteins include)Zinc finger motif.

The zinc finger motif is composed of a finger-like structure composed of an α-helix (recognition helix) and two β-strands (another form of protein secondary structure). Noncovalent bonds between zinc ions (Zn2+) and amino acid side chains within the transcription factor stabilize the zinc finger motif. Steroid hormone receptors, including the glucocorticoid receptor protein (see below) contain zinc finger motifs.

_____ are proteins that influence the ability of RNA polymerase II to bind to a eukaryotic core promoter. It is estimated that 2 - 3 % of the genes in the human genome encode transcription factors.

Transcription factors

(Altering chromatin to regulate transcription) Covalent modification involves the

acetylation, methylation, and phosphorylation of histone proteins within nucleosomes. Let us consider acetylation as an example of the covalent modification of histones.

DNA methylation is thought to silence the transcription of a nearby gene in two general ways. First, methylation at a CpG island near the promoter of a gene can block an

activator protein from binding to an enhancer DNA sequence (see figure 12.11). DNA methylation inhibits activator binding because the methyl group on cytosine prevents the activator protein from binding to the major groove in the enhancer region.

(Altering chromatin to regulate transcription) Enzymes called histone acetyltransferases (HATs) add

add acetyl groups to the tails of histone proteins. Specifically, acetylation of lysine amino acids within the histone tail neutralizes the positive charge on lysine, reducing the affinity of the histone tail for the DNA backbone. The histones disassociate from the DNA, and the DNA is accessible for transcription.

Transcription factors are considered to be

trans-acting factors (i.e. can regulate genes found throughout the genome) and bind to DNA sequences called cis-acting elements (i.e. the DNA binding sites for these transcription factors tend to be near the genes they control) (see figure 12.2). However, these DNA binding sites do not need to be immediately adjacent to the core and regulatory promoters. Some transcription factor binding sites can be within the gene that they control or can be thousands of base pairs away.

Suppose an activator protein binds to an enhancer DNA sequence (see figure 12.4). This activator protein then encourages TFIID to bind to the TATA box, and TFIID then recruits the other general GTFs and RNA polymerase II to the +1 site. As a result,

transcription is up- regulated.

(comparing gene regulation in prokaryotes and eukaryotes) Regulation of a typical eukaryotic gene involves combinatorial control. For example, a single eukaryotic gene can be regulated by a combination of:

*Activator proteins binding to enhancer DNA sequences. *Repressor proteins binding to silencer DNA sequences. *Regulation of the activities of the activator and repressor proteins. This regulation involves effector molecules, covalent modification, and protein-protein interactions. *Modifying the structure of chromatin to activate or repress transcription. Modifying chromatin involves altering the structure and the arrangement of nucleosomes (see Part 2) near the core promoter of a gene. *DNA methylation to silence transcription. The methylation of cytosine bases near the core promoter region of a gene inhibits transcription.

SWI/SNF performs at least three types of chromatin remodeling:

*SWI/SNF can change the distribution of nucleosomes along the DNA. *SWI/SNF can release histones from the nucleosome core. *SWI/SNF can remove the standard histone proteins (H2A, H2B, H3, and H4) from nucleosomes and replace these standard nucleosomes with certain histone variant proteins. The presence of these histone variant proteins increases transcription. These three changes can make the core promoter more accessible to the general transcription factors (GTFs) and RNA polymerase II.

(three general ways that the activator and repressor proteins are regulated)Covalent modifications

. Phosphorylation may activate transcription factors and thus influence the transcription of a target gene. For a particular gene, one or more of the above mechanisms may be involved in regulating gene expression.

Cytosine bases within CG-rich sequences called

CpG islands are typically targets for methylation. Not surprisingly, CpG islands are located near the core promoter of a gene (see figure 12.10). Typical CpG islands are sequences that are 1,000 - 2,000 base pairs (bp) in length and contain many CpG sites (i.e. many 5'-CG-3' sequences in a row). Within CpG islands, adding methyl groups to the cytosine bases on both DNA strands is called full methylation. Full methylation inhibits transcription

islands are typically targets for methylation. Not surprisingly, CpG islands are located near the core promoter of a gene (see figure 12.10). Typical CpG islands are sequences that are 1,000 - 2,000 base pairs (bp) in length and contain many

CpG sites (i.e. many 5'-CG-3' sequences in a row). Within CpG islands, adding methyl groups to the cytosine bases on both DNA strands is called full methylation. Full methylation inhibits transcription.

Comparing gene regulation in prokaryotes and eukaryotes The lac operon provided an excellent example of how bacteria perform gene regulation in response to environmental changes. In the case of the lac operon, we learned that gene regulation involves an

activator protein (CAP) and a repressor protein (lac repressor). Effector molecules (cAMP, allolactose) regulate the ability of CAP and the lac repressor to bind to DNA sequences near the lac operon structural genes.

The functions of activator and repressor proteins are regulated in three general ways:

binding to effector molecules, protein-protein interactions, and covalent modifications.

Housekeeping genes encode proteins that are required for cell viability. The promoters of these genes are unmethylated and as a result, housekeeping genes are always transcribed. Tissue-specific genes are only expressed in

certain cell types. In cell types in which these genes are not expressed, the promoter region of the gene is fully methylated. In cell types in which the gene is expressed, the promoter region of the gene is unmethylated. As a final example, the inactive X chromosome (Barr body) in female mammals is heavily methylated.

Altering chromatin to regulate transcription The results from fibroblasts and erythroblasts discussed above suggest that nucleosomes can be altered to influence the transcription of a nearby gene. Alterations in chromatin structure to promote transcription include the

covalent modification of histone proteins and the rearrangement of nucleosomes by ATP-dependent chromatin remodeling

The β-globin gene is expressed in

erythroblasts (precursor red blood cell). When the nucleosome arrangement surrounding the β-globin gene is examined in erythroblasts, a somewhat different result is observed. Nucleosomes are displaced on the region of DNA from -500 to +200. This euchromatin area includes the core promoter. Thus, RNA polymerase II can access the core promoter and express the β-globin gene in erythroblasts.

There are two categories of transcription factors:

general transcription factors and regulatory transcription factors

Maintenance methylation also explains a phenomenon called

genomic imprinting. In oogenesis or spermatogenesis, a specific gene is methylated by de novo methylation. Following fertilization, the methylation pattern is maintained as the fertilized egg begins to divide. For example, if the maternal allele for a gene is methylated, that maternal allele remains methylated and therefore silent in the cells of the offspring.

Steroid hormones produced by endocrine glands can activate the transcription of many genes. One example is a collection of steroid hormones called

glucocorticoid hormones (GCs) produced by the adrenal glands. Glucocorticoid hormones are produced in response to fasting as well as to physical activity, leading to an increase in glucose synthesis, an increase in protein metabolism, an increase in fat metabolism, and a decrease in inflammation. Other steroid hormones, such as estrogen and testosterone, influence the development of gonads.

Suppose an activator protein binds to an enhancer DNA sequence (see figure 12.5). The activator protein activates mediator, and mediator then activates the general transcription factor TFIIH. TFIIH acts as a

helicase to separate the template and coding DNA strands. TFIIH also acts as a kinase, phosphorylating RNA polymerase II to begin the elongation step of transcription.

The four different structural motifs that are found in transcription factor proteins include:

helix-turn-helix, basic helix-loop-helix, zinc finger motif, and leucine zipper motif

In eukaryotes, the processes that regulate the expression of one gene (activators/repressor proteins bound to enhancer/silencer DNA sequences, altering chromatin structure, and DNA methylation) do not necessarily influence the regulation of an adjacent gene. DNA sequences called ______ function to define the boundaries between genes (see figure 12.13); an insulator sequence ensures that the gene regulation processes that affect one gene do not affect nearby genes.

insulators

Silencing of gene expression in many mammals and plants involves the methylation of DNA. The methyl group that is added projects

into the major groove of the DNA and inhibits the binding of activator proteins to the DNA. Cytosine bases within CG-rich sequences called CpG

In addition to the core promoter, many eukaryotic genes also include a

regulatory promoter (see figure 12.1). The components of the regulatory promoter are required for transcription levels higher than the basal level provided by the core promoter. A common regulatory promoter component that is present in most eukaryotic genes is the CAAT box. The CAAT box is located at -80 and has the sequence 5'-GGCCAATCT-3'. Another common regulatory promoter component is a GC box (5'- GGGCGG - 3') located at -100. The CAAT and GC boxes are the binding sites for certain activator proteins.

Structural motifs often contain a protein secondary structure called an

α-helix, in which certain amino acids in the polypeptide sequence interact through hydrogen bonding to produce a helical structure. An α-helix is the proper width to bind to the major groove in DNA. Thus, the α-helix is often used by transcription factors to recognize specific nucleotide sequences in the major groove.

Insulator DNA sequences can:

*Serve as the binding sites for proteins that act as physical barriers for the HATs, HDACs and SWI/SNF complexes. For example, suppose a gene is flanked by two insulator DNA sequences, and HATs modify histone tails and activate transcription of the gene. Because the proteins bound to insulators serve as physical barriers to the HATs, genes beyond the insulator sequences are not activated. *Serve as the binding sites for proteins that limit the effects of enhancer/silencer sequences. Suppose that two genes (Gene A and Gene B) have an enhancer DNA sequence and an insulator DNA sequence between them. A protein binds to the insulator DNA sequence to ensure that the enhancer only activates Gene A without up- regulating the transcription of Gene B. Insulators can limit the effects of silencer DNA sequences in a similar manner.

Arrangement of chromatin at the β-globin gene As an example of how chromatin structure can influence the transcription of a gene, consider the human β-globin gene (see figure 12.8). The β-globin gene, which encodes the β-globin protein components of hemoglobin, is not normally expressed in many cell types, including connective tissue cells called fibroblasts. When the DNA region that encompasses the β-globin gene from fibroblasts is analyzed with respect to nucleosomes, scientists discovered that nucleosomes are found in regular intervals from

-3000 to +1500. The β-globin gene in fibroblasts is in heterochromatin and is not accessible to the general transcription factors (GTFs) and RNA polymerase II. As a result, the β-globin gene is transcriptionally silent in fibroblasts.

CREB activates transcription when

1. A receptor embedded in the cell membrane on the surface of a cell binds to a peptide hormone, growth factor, or cytokine. 2. The binding of the signaling molecule to the receptor activates a G protein. 3. The G protein activates adenylyl cyclase inside the cell, which forms cAMP from ATP. 4. cAMP binds to and activates protein kinase A. 5. Protein kinase A moves into the nucleus and phosphorylates the inactive CREB protein homodimer. 6. The phosphorylated CREB protein homodimer binds to an enhancer sequence called cAMP response element (CRE). 7. CREB bound to CRE activates RNA polymerase II and transcription occurs.

Glucocorticoid hormones can increase the transcription of a gene above the basal level as follows (see figure 12.6):

1. GCs are steroid hormones, which are nonpolar in structure. As a result, these nonpolar steroid hormones diffuse through the cytoplasmic membrane of a target cell. 2. GCs act as effector molecules by binding to a transcription factor protein called glucocorticoid receptor (GR) that is found in many different cell types. Prior to glucocorticoid binding, GR is bound to a protein called HSP90. HSP90 helps maintain the proper three-dimensional shape of GR, so that GR can bind to GC when GC is produced by the adrenal glands. 3. GC binds to GR and HSP90 is released. 4. GC binding changes the conformation of GR, exposing a nuclear localization signal (NLS). The NLS is a polypeptide sequence that helps to target the GR (with bound GC) to the nucleus of the cell. 5. Two GRs (with bound GC) interact in the cytoplasm of the cell to form a GR homodimer. 6. The GR homodimer travels to the nucleus of the cell. 7. The GR homodimer binds to an enhancer sequence called a glucocorticoid response element (GRE). GREs are common enhancers found adjacent to many genes involved in metabolism. 8. GR bound to GRE activates RNA polymerase II and transcription occurs.

We learned earlier this semester that transcription in eukaryotes involves several types of

DNA sequence elements.

Enhancers and silencers Other regulatory DNA sequences assist the core promoter and regulatory promoter to regulate transcription by serving as the binding sites for regulatory transcription factor proteins. The binding of regulatory transcription factors to these DNA sequences may:

Increase the rate of transcription. Transcription can increase 10 to 1000-fold when activator proteins bind to enhancer DNA sequences (up-regulation). Activators and enhancers are generally responsible for tissue-specific expression of a gene. Decrease the rate of transcription. Transcription can decrease below the basal level when repressor proteins bind to silencer DNA sequences (down-regulation). Repressors and silencers are generally responsible for tissue-specific repression of a gene.

regulatory transcription factors

Regulatory transcription factors function to regulate transcription by either increasing the rate of transcription above the basal level or decreasing the rate of transcription below the basal level. An activator protein increases the level of transcription above the basal level; a repressor protein decreases the level of transcription below the basal level. Many transcription factors are only expressed in certain tissues or at certain times during development, thus playing a critical role in tissue-specific or time-specific gene expression.

The ATP-dependent chromatin remodeling process uses the energy in ATP to alter nucleosome structure along the DNA. One example of an ATP-dependent chromatin remodeling enzyme is a large multi-subunit complex called

SWI/SNF

(three general ways that the activator and repressor proteins are regulated)Binding to effector molecules

Similar to what we observed while studying the lac operon, small effector molecules can bind to activator or repressor proteins, change the conformation of the transcription factor, and influence the ability of the transcription factor to bind to enhancer or silencer DNA sequences. In animals, steroid hormones such as glucocorticoid, testosterone, and estrogen can serve as effectors that regulate the functions of transcription factor proteins.

Methylation blocks activator proteins and recruits HDACs DNA methylation is thought to silence the transcription of a nearby gene in two general ways. First, methylation at a CpG island near the promoter of a gene can block an activator protein from

binding to an enhancer DNA sequence (see figure 12.11). DNA methylation inhibits activator binding because the methyl group on cytosine prevents the activator protein from binding to the major groove in the enhancer region.

Many signaling molecules, such as peptide hormones, growth factors, and cytokines, which have the potential to influence transcription, are not able to diffuse through the cytoplasmic membrane. Instead, these signaling molecules bind to cell receptors on the surface of a target cell, and binding of the signaling molecule to the receptor protein is transmitted to the nucleus to activate transcription. Consider how transcription is activated by a regulatory transcription factor called

cAMP response element-binding protein (CREB)

Chromatin is a dynamic structure with a specific region of DNA alternating between

the closed and open conformation depending on the needs of the cell. When a gene is activated to be transcribed above the basal level, chromatin is converted to the open conformation. When a gene is deactivated to be transcribed below the basal level, chromatin is converted to the closed conformation.

Chromosome compaction and transcription The arrangement of nucleosomes (see Part 2) can also influence the transcription of a gene.For a gene to be transcribed, RNA polymerase II must be able to bind to the core promoter. If a gene is found in a chromosome region with tightly packed nucleosomes (heterochromatin), RNA polymerase II struggles to bind to the core promoter. As a result, the heterochromatin form of DNA is said to be in a

closed conformation and transcription is limited. Regions of the chromosome with loosely packed or absent nucleosomes are called euchromatin (open conformation). RNA polymerase II can access a core promoter located in euchromatin, and as a result, transcription occurs.

The DNA methylation pattern in the cell is established by a process called

de novo methylation (see figure 12.12). De novo methylation converts unmethylated DNA to full methylation on both DNA strands. De novo methylation is a highly regulated process that is thought to occur only in certain cell types or at a particular stage of development.

Because the DNA replication machinery does not methylate bases during replication, the daughter DNA strands produced do not contain methylated cytosines. Thus, the daughter double-stranded DNA molecules are ________, with a methylated parental strand and an unmethylated daughter DNA strand. This hemimethylated DNA is recognized by DNA methyltransferase, which subsequently methylates the cytosine bases on the daughter DNA strands, thus preserving the DNA methylation pattern established in the parental cell.

hemimethylated

(Altering chromatin to regulate transcription) When an activator protein binds to an enhancer DNA sequence, the activator can recruit HATs to the promoter. The HAT then acetylates histone tails in the promoter region. The interaction between the DNA and the histone proteins relaxes and transcription of the gene occurs. When transcription needs to be turned off, the histones can be modified using

histone deacetylase (HDAC) proteins. HDACs remove the acetyl groups from the histone tails, thus increasing the affinity of the histone tail for the DNA backbone. As a result, the chromatin is converted to the closed conformation, decreasing transcription of the gene. Repressor proteins can recruit HDACs to a promoter when the repressor protein is bound to a silencer DNA sequence.

It is important to note that all transcription factor motif structures allow transcription factors to interact. Two identical transcription factors can interact to form a transcription factor

homodimer, or two different transcription factor proteins can interact to form a heterodimer. Higher order interactions (trimers, tetramers) are also possible when transcription factors interact with each other.

Tissue-specific genes are only expressed

in certain cell types. In cell types in which these genes are not expressed, the promoter region of the gene is fully methylated. In cell types in which the gene is expressed, the promoter region of the gene is unmethylated. As a final example, the inactive X chromosome (Barr body) in female mammals is heavily methylated.

Housekeeping genes encode

proteins that are required for cell viability. The promoters of these genes are unmethylated and as a result, housekeeping genes are always transcribed.

Once the DNA methylation pattern in a cell has been established by de novo methylation, _____ ensures that the daughter cells produced by mitosis maintain the same methylation pattern as the parental cell. As an example, suppose that fully methylated DNA (both strands methylated) is replicated.

maintenance methylation

(Comparing gene regulation in prokaryotes and eukaryotes) Even though gene regulation in prokaryotes and eukaryotes is similar (involving activators, repressors, and effectors), eukaryotic gene regulation is much more complex. This complexity is because

many eukaryotic organisms are multicellular with cells in each tissue having unique phenotypes. For example, a white blood cell (leukocyte) and a muscle cell have the same genetic material; however, gene regulation ensures that a leukocyte expresses leukocyte- specific proteins, while a muscle cell expresses muscle-specific proteins. Further, many eukaryotes progress from a fertilized egg through complex developmental stages to produce the mature adult organism. Gene regulation ensures that some genes are expressed only during embryonic development, while other genes are expressed only in an adult.

Second, methylated CpG islands near promoters serve as the binding sites for a group of proteins called

methyl-CpG-binding proteins. When a methyl-CpG-binding protein binds to a methylated CpG island, the methyl-CpG-binding proteins can recruit a histone deacetylase (HDAC). HDAC then removes the acetyl groups from the histone tails, converting the promoter region of a gene into heterochromatin. Transcription of the nearby gene is therefore inhibited.

Silencing of gene expression in many mammals and plants involves the

methylation of DNA. The methyl group that is added projects into the major groove of the DNA and inhibits the binding of activator proteins to the DNA.

Structural features of transcription factors Transcription factor proteins have been identified in many organisms, including bacteria, fungi, plants, and animals. Nearly all transcription factor proteins contain conserved structural features that are important in either binding to regulatory DNA sequences, binding to effector molecules, or binding to other transcription factor proteins. These structural features are called

structural motifs.

Suppose a repressor protein binds to a silencer DNA sequence. The repressor protein inhibits

the activity of mediator. Mediator fails to activate TFIIH, and TFIIH fails to separate the template and coding DNA strands. TFIIH also fails to phosphorylate RNA polymerase II, preventing the initiation of transcription.

Suppose a repressor protein binds to a silencer DNA sequence. The repressor protein then prevents TFIID from binding to the TATA box. The absence of TFIID on the core promoter prevents

the other GTFs and RNA polymerase II from binding to the core promoter. As a result, transcription is down-regulated.

We will consider regulation of RNA polymerase II activity through TFIID first. TFIID is a general transcription factor that binds to the TATA box (the -25 sequence) within the core promoter. TFIID recruits...

the other general transcription factors (GTFs) that bring RNA polymerase II to the +1 site within the core promoter.

Recall that the mediator protein complex communicates

the signals from activator and repressor proteins to RNA polymerase II. Mediator thus serves as a link between transcription factors that bind to enhancer and silencer DNA sequences and RNA polymerase II, thereby determining the overall rate of transcription.

A particular gene can be regulated by many transcription factors bound to different combinations of enhancers and silencers. The combination of the transcription factor proteins and regulatory DNA sequences involved determines

the transcription pattern of the gene.