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5D: Binding and the Control of Gene Transcription - Biology


Learning Objectives

  • describe general mechanisms of how a gene for a given protein might be negatively and positively regulated at the level of gene transcription;
  • describe the structure/function/role of promoters, response elements, RNA polymerase, transcription factors, nucleosomes, histone proteins, epigenetic modifications of DNA in gene transcription;
  • explain the differences (structural, Kds) between specific and nonspecific binding of a ligand to a macromolecule, at the structural level;
  • describe the structural features of both proteins and DNA that result in specific and nonspecific binding;
  • describe and give examples of how post-translational modifications of proteins and epigenetic modifications of DNA can alter gene expression;
  • explain how the apparent Kd for a protein binding to DNA can be altered by the presence of another protein bound to DNA at a proximal site
  • describe the basis of RNA interference in gene expression

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contain the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cells can change (differentiate) into different types of cells by altering the expression of genes. The central dogma of biology describes how genes are first transcribed to messenger RNA (mRNA), and then the mRNA is translated into a corresponding protein sequence.


D1. Introduction to Transcription

Imagine you have been given a string approximately 2 meters long, which represents an unwound, deproteinated, human chromosome. In actuality, such naked DNA does not exist in the cell nucleus. Rather, it is wound around a series of positively charged histone proteins. In the electron microscope it resembles beads on a string. This structure is wound into a cylindrical "solenoid" structure which is further packaged to fit into the nucleus along with the rest of the chromosomes. There just happens to be a small dot in the dead center of the string you have been given. It represents the gene for a particular protein called metallothionein. This gene is expressed and the protein metallothionein is made when cells are exposed to heavy metals like Cd. How might the cell not express the gene, or express it to a small, constitutive level, in the absence of Cd exposure? How might the gene might be activated - through transcription of the gene and translation of the resulting mRNA to form the metallothionein protein upon exposure to Cd. (Hint: Cd doe not DIRECTLY bind to DNA.)?

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contains the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cell can change (differentiate) into different types of cells by altering the expression of genes. The Central Dogma of Biology describes how genes are first transcribed to messenger RNA (mRNA), and then the mRNA is translated into a corresponding protein sequence. The links below should be reviewed by those who have little background on the Central Dogma of Biology and of the nature of a gene.

Updated Simple DNA Tutorial Jmol14 (Java) | JSMol (HTML5)

Once made, p roteins can then be post-translationally modified, localized to certain sites within the cells, and ultimately degraded. If functional proteins are considered the end-product of gene expression, the control of gene expression could theoretically occur at any of these steps in the process.

Mostly, however, gene expression appears to be controlled at the level of transcription. This makes great biological sense, since it would be less energetically wasteful to induce or inhibit the ultimate expression of a functional protein at a step early in the process. How can gene expression be regulated at the transcriptional level? Many examples have been documented. The main control is typically exerted at the level of RNA polymerase binding just upstream (5') of a site for transcriptional initiation. Other factors, called transcription factors (which are usually proteins), bind to the same region and promote the binding of RNA polymerase at its binding site, called the promoter . Proteins can also bind to sites on DNA (operator in prokaryotes) and inhibit the assembly of the transcription complex and hence transcription. Regulation of gene transcription then becomes a matter of binding the appropriate transcription factors and RNA polymerase to the appropriate region at the start site for gene transcription. Regulation of gene expression by proteins can be either positive or negative. Regulation in prokaryotes is usually negative while it is positive in eukaryotes.


5D: Binding and the Control of Gene Transcription - Biology

One of the central questions of modern biology is what controls gene expression. As we have previously described, genes must be "turned on" at the right time, in the right cell. To a first approximation, all the cells in an organism contains the same DNA (with the exception of germ cells and immune cells). Cell type is determined by what genes are expressed at a given time. Likewise, cell can change ( differentiate) into different types of cells by altering the expression of genes. The central dogma of biology describes how genes are first transcribed to RNA, and then the mRNA is translated into a corresponding protein sequence. Proteins can then be post-translationally modified, localized to certain locales within the cells, and ultimately degraded. If functional proteins are considered the end-product of gene expression, the control of gene expression could theoretically occur at any of these steps in the process.

Mostly, however, gene expression is controlled at the level of transcription. This makes great biological sense, since it would be less energetically wasteful to induce or inhibit the ultimate expression of a functional protein at a step early in the process. How can gene expression be regulated at the transcriptional level? Many examples have been documented. The main control is typically exerted at the level of RNA polymerase binding just upstream (5') of a a site for transcriptional initiation. Other factors, called transcription factors (which are usually proteins), bind to the same region and promote the binding of RNA polymerase at its binding site, called the promoter. Proteins can also bind to sites on DNA (operator in prokaryotes) and inhibit the assembly of the transcription complex and hence transcription. Regulation of gene transcription then becomes a matter of binding the appropriate transcription factors and RNA polymerase to the appropriate region at the start site for gene transcription. Regulation of gene expression by proteins can be either positive or negative. Regulation in prokaryotes is usually negative while it is positive in eukaryotes.

CONTROL OF GENE TRANSCRIPTION IN PROKARYOTES: THE E. COLI LAC OPERON

The regulation of the genes involved in lactose utilization won Jacob and Monod (of MWC fame) the Nobel Prize. Lactose can be used as the sole source of carbon by E. Coli. Three genes are required for lactose utilization, beta-galactosidase (lac Z, cleaves lactose to gal and glu), galactoside permease (lac Y, transports lac into the cell) and thiogalactoside transacetylase (lac A, function unknown). These genes follow one another on the DNA, and have 1 promoter region. On transcription and translation, one long poly-protein is made, which is cleaved post-translationally to form the individual proteins.

In addition, another gene, the gal repressor, is found just upstream of the gal utilization genes. It has its own promoter (P I ) The gene cluster, including promoter and any regulatory DNA sequences is called an operon, for example, the Lac operon. In this case, the operon is induced in response to a molecular signal - i.e. the presence of lactose, or allolactose. This binds to the repressor protein, which is bound to the operator DNA, inhibiting transcription. When allolactose or another beta-galactosides, such as isopropylthiogalactoside (IPTG), bind to the repressor protein, a conformation change occurs in the repressor, resulting in a higher Kd for the operator DNA, and subsequent dissociation of the repressor-galactoside complex. Transcription ensues.

IPTG is an inducer of the lac operon but is not a substrate for the enzymes produced. We will use IPTG to induce expression of human adipocyte acid phosphatase b (HAAP- b) in lab 4B. The plasmid containing the gene for HAAP- b has been engineered to contain the lac promoter just before the start site for gene transcription. By adding IPTG to the growing cells, the cells can be induced to synthesize the protein, HAAP- b .

Many analogous but distinct methods are used to control gene transcription in prokaryotes. The control of lac operon transcription is but one example.

CONTROL OF GENE TRANSCRIPTION IN EUKARYOTES

  • multiple changes occur in the structure of chromatin at the site of transcription
  • positive mechanisms regulate transcriptions much more often than negative ones.
  • transcription and translation occur at spatially and temporally distinct sites and times.

The genomes of eukaryotes are much larger than prokaryotes. This poses some problems with respect to binding. Remember, DNA binding proteins demonstrate both nonspecific and specific binding. Nonspecific binding may help a protein find a specific site in the genome, but as the size of the genome increases, the chance of finding multiple specific sites randomly distributed increases. This problem can be avoided if multiple proteins are required to generate an active transcription complex. The chance of finding two or more specific sites for different proteins in proximity at sites other than required for gene transcription are very low. Multiple negative regulators would not be needed since just the binding of one regulator would probably be sufficient. Most eukaryotic genes have about 5 regulatory sites for binding transcription factors and RNA polymerase. Examples of these transcription factors are show in the figure below.

Light can even regulate gene expression. A great new example by Quail shows how light can change and activate an inactive transcription factor in plants. "The basic helix-loop-helix transcription factor PIF3 binds to a G-box motif in the promoter region of light-responsive genes. Upon absorbing red light, a phytochrome photoreceptor is converted from the inactive Pr form to the active Pfr form, which moves to the nucleus.

Here, Pfr is recruited to the promoter region of target genes by binding to PIF3 and then activates the expression of genes encoding MYB class transcription factors (CCA1, LHY). The transcription factors in turn activate the expression of secondary genes. Far-red light shuts down this signaling pathway by converting Pfr back to Pr, promoting its release from the PIF3 complex."

STRUCTURAL FEATURES OF SPECIFIC DNA BINDING SITES

Since RNA polymerase must interact at the promoter site of all genes, you might expect that all genes would have a similar nucleotide sequence in the promoter region. This is found to be true for both prokaryotic and eukaryotic genes. You would expect, however, that all transcription factors would not have identical DNA binding sequences.

Prokaryotic Promoter Sequences

Spacer -10 Region Spacer RNA start trp operon TTGACA N17 TTAACT N7 A tRNAtyr TTACA N16 TATGAT N7 A l P2 TTGACA N17 GATACT N6 G lac operon TTTACA N17 TATGTT N6 A rec A TTGATA N16 TATAAT N7 A lex A TTCCAA N17 TATACT N6 A T7A3 TTGACA N17 TACGAT N7 A consensus TTGACA TATAAT

Eukaryotic Response Elements (RE)s

Consensus DNA bound Factor Size (daltons) Heat Shock HSE CNNGAANNTCCNNG 27 bp HSTF 93,000 Glucocorticoid GRE TGGTACAAATGTTCT 20 bp Receptor 94,000 Cadmium MRE CGNCCCGGNCNC . ? . Phorbol Ester TRE TGACTCA 22 bp AP1 39,000 Serum SRE CCATATTAGG 20 bp SRF 52,000

Proteins can interact specifically with DNA through electrostatic, H-bond, and hydrophobic interactions. AT and GC base pairs have available H bond donors and acceptors which are exposed in the major and minor grove of the ds DNA helix, allowing specific protein-DNA interactions.

Gene Transcription and Control of Membrane Lipids

An interesting example of transcriptional control occurs to maintain the balance of lipids in biological membranes. The phospholipids and spingolipids in membranes are extremely heterogeneous, owing to the diversity of head groups and acyl chain composition. Given this great diversity, it is remarkable the different cells are able to maintain the specificity of lipid types in different cells, in different membranes within cells, and within a given leaflet of a membrane (remember our discussion of lipid rafts). How can the cell regulate the type of lipids that it synthesizes? What controls the transcription of genes for lipid synthesis?

Regulation of transcription of these genes appears to be controlled by proteins that bind to sterol response elements in the DNA. The proteins, called sterol response element binding proteins (SREBPs) are activated to become transcription factors and migrate to the nucleus when they are released from Golgi membrane by proteolysis by Golgi proteases. The SREBP in the Golgi is in complex with another protein, SCAP, which facilitates movement of the SREBP to the Golgi from its site of synthesis in the endoplasmic reticulum. Lipid regulation occurs when fatty acids, cholesterol, or PL derivatives like phosphoethanolamine (from ceramide) inhibits proteolytic activation of the SREBP. Regulation depends on whether or not SCAP "ferries" SREBP to the Golgi.

STRUCTURAL FEATURES OF DNA-BINDING PROTEINS

The figures shows two such proteins, the cro repressor form bacteriophage 434 and the lambda repressor from the bacteriophage lambda. (Bacteriophages are viruses that infect bacteia.) Notice how specificity is achieved, in part, by the formation of specific H-bonds between the protein and the major grove of the operator DNA.

Figure: Lambda Repressor/DNA Complex

Figure: H Bond interactions between l repressor and DNA

  • zinc finger: (eukaryotes) These proteins have a common sequence motif of
    X 3 - Cys-X 2-4 - Cys-X 12 - His-X 3-4 - His-X 4- in which X is any amino acid. Zn 2+ is tetrahedrally coordinated with the Cys and His side chains, which are on one of two antiparallel beta strands, and an alpha helix, respectively. The zinc finger, stabilized by the zinc, binds to the major groove of DNA. ]
  • steroid hormone receptors: (eukaryotes) In contrast to most hormones, which bind to cell surface receptors, steroid hormones (derivatives of cholesterol) pass through the cell membrane and bind to cytoplasmic receptors through a hormone binding domain. This changes the shape of the receptor which then binds to a specific site on the DNA (hormone response element) though a DNA binding domain. In a structure analogous to the zinc finger, Zn 2+ is tetrahedrally coordinated to 4 Cys, in a globular-like structure which binds as a dimer to two identical, but reversed sequences of DNA (palindrome) within the major grove. (An example of a palindrome: Able was I ere I saw Elba.)
  • leucine zippers: (eukaryotes) These proteins contain stretches of 35 amino acids in which Leu is found repeatedly at intervals of 7 amino acids. These regions of the protein form amphiphilic helices, with Leu on one face. Two of these proteins can form a dimer, stabilized by the binding of these amphiphilic helices to one another, forming a coiled-coil, much as in the muscle protein myosin. Hence the leucine zipper represents the protein binding domain of the protein. The DNA binding domain is found in the first 30 N-terminal amino acids, which are basic and form an alpha helix when the protein binds to DNA. The leucine zipper then functions to bring two DNA binding proteins together, allowing the N-terminal bases helices to interact with the major grove of DNA in a base-specific fashion.

PHOSPHORYLATION AND CONTROL OF GENE EXPRESSION

A common way to control gene expression is by controlling the post-translational phosphorylation of transcription factors by ATP. This modification might activate or inhibit the transcription factor in turning on gene expression. The added phosphate groups might be necessary for direct binding interactions leading to gene transcription or they might lead to a conformational change in the transcription factor, which could activate or inhibit gene transcription. A recent example of this later case is the control of the activity of the transcription factor p53. p53 has many activities in the cell, a primary one as a suppressor of tumor cell growth. If a cell is subjected to stress that results in genetic damage (an evident which could lead a cell to transform into a tumor cell), this protein becomes an active transcription factor, leading to the expression of many genes, including those involved in programmed cell death and cell cycle regulation. Both of these effects could clearly inhibit cell proliferation. Hence p53 is a tumor suppressor gene. p53 is usually bound to the protein HDM2 which down regulates its activity by leading to its degradation. Stress signals lead to the activation of protein kinases in the cell (such as p38, JNK, and cdc2), causing phosphorylation of Ser 33 and 315 and Thr 81 in p53. This leads to the binding of Pin 1, a peptidyl-prolyl isomerase , which catalyzes the trans<=>cis conformational changes of X-Pro bounds. Pin 1 appears to bind only when p53 is phosphorylated. The ensuing change in p53 conformation presumably leads to its activation as a transcription factor.

POSITIVE TRANSCRIPTION FACTORS - A NEW CLASIFICATION

As inferred from above, transcription factors can be classified based on their protein structure. A newer classification scheme, based on the function/activity of the transcription factors, has been proposed by Brivan, Lou and Darnell (Science, 295, pg 813, 2002), as illustrated in the flowchart below, along with specific examples. The classes of transcription factors include those that are:

constitutively active : are always active in the nucleus of the cell and probably activate transcription of genes that must always be turned on

The rest must be activated by some means, which include those that are:

developmental or cell type-specific whose genes must be transcribed (probably in a regulated fashion) to form the transcription factor which then enters the nucleus

signal dependent transcription factors , which are activated through a signaling event.

There are classes of signal-dependent transcription factors that are activated by:

steroids , which are cholesterol derivatives that can pass through the cell membrane and bind steroid-specific transcription factors which turn on specific sets of genes most of these transcription factors are present in the nucleus and are activated there by steroid hormones. One exception is the glucocorticoid receptor (GR) which is found in the cytoplasm

internal signal s derived from the cell, such as internally made lipid signals.

cell surface receptor-ligand interactions

There are two types of receptor-ligand interactions that lead to transcription factor initiation.

small ligand molecules (like epinephrine) bind transmembrane receptors leads to formation of second messengers or signals inside the cell, which ultimately activate Ser-phosphorylation activity. Nuclear transcription factors can become phosphorylated and activated.

small ligands bind transmembrane receptors which then bind to and activate latent transcription factors in the cytoplasm, which then migrate to the nucleus.

COOPERATIVE BINDING OF PROTEINS TO DNA

We have just spend much time studying the cooperative binding of oxygen to hemoglobin. Cooperativity seemed to be require conformational changes in a multimeric protein. Is it possible to get cooperative binding of ligands without conformational changes? In a recent book by Ptashne and Gann (Genes and Signals, Cold Spring Harbor Press, 2002), it is argued that you can and through a very simple mechanism.

It must be clear that to activate gene transcription, several transcription factor proteins must assembly at the promoter before RNA polymerase can transcribe a gene. There are multiple DNA-protein and protein-protein contacts. To simplify this discussion, consider the case of two proteins, A and B, that must bind to the DNA and to each other for transcription to occur.

The binding of each protein alone is characterized by a characteristic Kd, k on , and k off. What happens to k on and k off for protein B, for example, when A is already bound? You can imagine that k on doesn't change much, but what about k off after the protein is interacting both with its DNA site and with protein A? If B did dissociate from its DNA site, it would still be held in close approximation to that site because of its interaction with the bound protein A. Its effective concentration goes up and you should readily image that it would rebind very quickly to its DNA site. The net effect would be that it's apparent k off would decrease, which would increase its apparent binding affinity and decrease its apparent Kd (remember that Kd = k off /k on ). Hence prior binding of A would lead to cooperative binding of protein B.

EPIGENTIC CONTROL OF DNA TRANSCRIPTION - METHYLATION OF DNA

The fertilized egg is a totipotent cell. That is, through a series of divisions, its progeny cells can eventually become any of about 200 histologically different cell types. With subsequent cell division in the developing embryo, cells find themselves in different topological environments and have different cell-cell contact. Through signal transduction through the cell membrane, these cells start to become different, or differentiate, into other cells types. They do so by activating and inhibiting the expression of a different set of genes to form a different set of proteins in the cells. Most cells become terminally differentiated and eventually (after maybe a hundred cell divisions) lose the ability to divide and hence begin to die. However, a few types of cells, called stem cells, retain the ability to differentiate into other cells types in a regenerative process. These cells are pluripotent in that they can differentiate into other cell types.

How do dividing cells know what types of genes to actively transcribe? How can they have "memory" of the cells type they were before division? This appears to happen without alteration of the nucleotide sequence of the DNA in these cells. The main mechanism appears to be a inheritable but modifiable pattern of chemical modifications to the DNA (not unlike co- or post-translational modification of proteins) involving methylation/demethylation (by a methylase and demethylase) of cytosine in CpG dinucleotide repeats in the DNA. Also proteins can bind to DNA and methylated DNA to modify the course of gene expression in daughter cells. Such chemical modifications to the DNA which modify gene transcription are examples of epigenetic mechanisms controlling gene expression.

You are all familiar with the cloning of animals from the DNA of adult cells (from Jurassic Park to the actual cloning of the sheep Dolly). In this process, the nucleus from an adult somatic cell (like a check epithelial cell) is removed and placed in an egg from which the nucleus has been removed (enucleated). The egg now has a full complement of DNA, just like an adult cell, but it didn't get the full set by normal means - i.e. by receiving half from a sperm to complement its own normal half. There is another potentially big problem. The DNA in the egg has the methylation pattern of a terminally differentiated adult cell. It must be reprogrammed by undergoing extensive demethylation and remethylation to the "correct" epigenetic methylation state if the egg has a chance to form a normal embryo, fetus, and adult. Obviously this can happen, as evidence by Dolly and success in cloning cows, cats, pigs, and mice. However, it is very difficult to achieve and probably accounts for the low success rate of cloning.

Methylation patterns can account for gene silencing (in which one gene in a pair of identical chromosomes is not expressed) and inactivation of one entire X chromosome in a female (who has 2 X chromosomes). In general, transcription from genes that are methylated is inhibited.

CHROMATIN REMODELING AND GENE EXPRESSION

Control of DNA transcription in eukaryotes was thought to involve the assembly of many proteins at the promoter into a pre-initiation complex (PIC). Once assembled, RNA polymerase could bind and transcription would be initiated. But wait a minutes! Isn't DNA packaged in the nucleus into chromatin in which 147 BP of DNA is wound around a core of 4 pairs of positively charged histone proteins - including H2A, 2B, 3, and 4 - to form a nucleosome , seen under a microscope as beads on a string?

Isn't this chromatin further wound into fibers which result in the classic picture of sister chromatids ready to separate at cell division. How could the transcription factors and RNA polymerase recognize target sites on DNA given this degree of "folding" and condensation of the DNA?

Clearly the complex compacted state of DNA and its interaction with the histone proteins must be "remodeled" to allow interactions of the transcription factors and RNA polymerase (which is about the same size as a nucleosome). The regulation of this chromatin remodeling clearly affects gene transcription, and is another example of epigenetic changes that can affects phenotype. The state of chromatin structure is regulated by enzymes that affect histone structure and function by chemically modifying the histone proteins (through acetylation, methylation, and phosphorylation) . Likewise, the DNA at the promoter region is changed by enzymes that remodel the DNA through an ATP dependent series of modifications. For example when histones are modified by histone acetyltransferase (HAT's), other modeling factors ( SWI/SNF) are recruited to the chromatin. Chromatin remodeling would also be affected by that cell cycle stage of the cell. For example, chromatin condensed in sister chromatids ready for cells division would have different remodeling requirements for gene transcription than might chromatin in the form of bead on a string. Likewise remodeling efforts would also be gene-specific.

The figure below shows how remodeling is coupled to formation of the pre-initiation complex for three genes:

yeast HO gene: Swi5p activator binding results in the interaction of the SWI/SNF ATP-dependent remodeling enzyme, which leads to the binding of histone acetyltransferase (HAT). These facilitate formation of the pre-initiation complex.

human interferon- b gene: gene sequences known as activators, 5' to the promote, bind HATs. When histones are acetylated, SWI/SNF interacts to remodel the chromatin and facilitate PIC formation.

human a -1 antitrypsin gene: the PIC is preformed and recruits HAT and SWI/SNF, which leads to gene transcription.

Alternations in chromatin remodeling could lead to changes in gene expression, in some cases causing cancer. SNF5 is a component of the SWI/SNF complex and in its normal form acts to suppress tumors (i.e. its gene is a tumor suppressor gene). Mutations in SNF5 are associated with rare and aggressive childhood tumors. Stuart Orkin has developed a technique to alter the gene in some mouse cells to produce an inverted gene which produces no functional SNF5. Cells with this mutation become tumor cells almost immediately.

DNA winds around the histone core to form the nucleosome. However, histone tails not associated with DNA binding protrude from the nucleosome, and the function of these tails is just being unraveled. The amino acids in these tails are clearly sites for posttranslational modifications, including methylation, acetylation, and phosphorylation. When modified, these tails would provide additional binding sites for protein which could regulate transcription and chromatin modeling, thus modifying the "genetic code". Understanding the "histone code" and how it affects gene transcription becomes important. For example, the methylation of Lys 9 on histone 3 leads to binding of heterochromatin-associated protein, leading to inhibition of gene transcription (an example of epigentic silencing). Acetylation of the tails generally leads to activation of gene transcription at that site.

Epigenetic changes (through methylation of DNA or acetylation, methylation, and phosphorylation of histone proteins causing chromatin remodeling may change phenotype (characteristics of the individual) as evidenced by the fact that identical twins can eventually diverge in ways that effect their propensities to disease. Differences in diet and lifestyle, which can alter disease propensity, might exert their effects through epigenetic changes in gene expression. The Human Epigenome Consortium is developing a catalog of methylation pattern differences in the human genome which might be correlated with disease risk.

CONTROL OF GENE EXPRESSION BY RNA MOLECULES

What accounts for the increased complexity of organisms like humans? As was discussed in the DNA chapter, it is not the number of chromosomes or even the number of possible genes in an organisms. One big difference between bacterial and human cells, for example, is the percentage of DNA coding for proteins. In bacteria, most of the DNA codes for proteins, but in human eukaryotic cells, most of the DNA (up to 98%) is "junk" in that it does not code for proteins. The DNA consists of intervening sequences within DNA coding for a given protein, and sequences between genes. Up to 98 % of the RNA transcribed in human cells is derived from this "junk" DNA. What function does this RNA serve? New evidence shows that this RNA binds to other RNA molecules like mRNA (to inhibit its translation), to DNA (to control gene transcription) or to proteins (to alter gene transcription as well). These process are called RNA interference (RNAi)

MicroRNAs (miRNAs) are formed when an enzyme called dicer cleaves a host RNA gene transcript that in animal cells contains a stem-loop hairpin structure. Dicer recognizes the double-stranded regions of the stem. The cleaved RNA might then bind a host mRNA, inhibiting its translation. If the complementarity of the mRNA and miRNA is less than ideal, then binding of miRNA to the mRNA may only attenuate the translation of the mRNA..

Another class of RNA, s hort i nterfering or si lencing RNAs ( siRNAs) can also infer with mRNA translation. siRNAs are formed when dicer cleaves viral double-stranded RNA in infected cells. (Viruses often produced dsRNA during their life cycle). These differ from miRNA in that siRNA are not derived by transcription from discrete host gene. Dicer works by cleaving the dsRNA to form a small dsRNA between 20 and 25 nucleotide pairs long called siRNAs. These can then bind to a protein complex called RISC ( RNA- induced silencing complex), which promotes unraveling of the siRNA. A complex of RISC and one of the short RNA strands from the siRNA then can bind to complementary stretches in mRNA for a specific gene (viral or host) and inhibit translation of the mRNA into protein. Inhibition occurs when RISC complex cleaves the RISC-siRNA- mRNA complex. (i.e one of the components in the RISC complex is an RNA nuclease.) This mechanism might be linked to defense mechanisms of virally-infected cells by inhibition of viral mRNA translation.

siRNAs can also bind to host RNA targets by binding to host mRNA of complementary sequence to form a dsRNA complex, inhibiting its translation. This technique has recently been used to ascertain the functions of gene products in the nematode worm, C. elegans . This organism has about 20,000 genes which code for proteins. Kamathk et. al. have fed these worms E. Coli transformed with plasmid DNA designed to produced dsRNA upon transcription, one strand of which was complementary to mRNA sequences in the worm. Plasmids containing almost 17,000 different dsRNA encoding genes were constructed and used to "knock out" gene expression (by forming dsRNA complexes from the mRNA) by RNAi. Phenotypic changes in the organism were studied. About 1700 of the dsRNA experiments led to observable (phenotypical) changes in the organism. Genes whose inactivation was lethal (and hence were essential for survival) were generally those that had counterparts in all other organism, while those associated with nonlethal changes were more likely to be homologous to higher organisms and more recently evolved. They also selectively looked at which genes influenced lipid metabolism by incorporating a fluorescent type which bound to lipid deposits in the organism. Around 300 genes were found to influence fluorescence and hence regulate fat deposition in the organism.

Recently, a new mechanism in control of gene expression has been offered which involves regulation of translation of a mRNA. mRNA must have a sequence, the Shine-Delgarno sequence, which allows it to bind to ribosomes. If a ligand binds to this site, mRNA could not bind to the ribosome and translation would be inhibited. Such is the case in the mRNA encoding proteins involved in the transport and synthesis of vitamins B1 (thiamine) and B12 (adenosyl cobalamin). Thiamin and thyamine pyrophosphate were shown to bind to the leader sequence of an E. Coli mRNA involved in thiamine biosynthesis and inhibit the translation of the mRNA. This allosteric mechanism for inhibition makes physiological sense since the presence of high levels of cellular B1 would obviate the need for its synthesis or transport.

EUKARYOTIC SPECIES COMPLEXITY AND CONTROL OF GENE TRANSCRIPTON

The increasing complexity of eukaryotic organisms was thought to arise from an increasing number of genes. This simplistic assumptions has not been validated from the results of sequencing and annotating the genomes of many eukaryotic organisms. Compare these statistics: the number of putative genes in the simple nematode round worm C. Elegans, the fruit fly drosophila, and the human are approximately 20,000, 14,000, and about 36,000. There seems to be little correlation of species complexity with number of genes. Other possible mechanisms for increasing complexity from a given genome size include producing different proteins from the same genes through differential splicing of RNA transcripts and rearranging DNA as occurs in immune cells to produce the huge repertoire of possible antibody molecules necessary for recognition of nonself molecules (such as viruses and bacteria). These mechanisms can not account for the incredible complexity of the human species. Levine and Tjian have proposed two other mechanisms that could account for increasing complexity. Complexity would arise from the number of gene expression patterns and involve the involvement of nonprotein-coding regions of the genome, which in humans accounts for up to 98% of the genome. One mechanism requires the present of greater number and complexity of DNA regulatory sequences (enhancers, silencers, promoters) in more complex organisms. Since these sequences are in the DNA (the molecule that is transcribed), they are called cis-regulatory sequences. The second mechanism involves an increase in the elaboration and complexity of proteins (trans-regulatory elements) that regulate gene expression in more complex organisms.. These proteins could include transcription factors, proteins interacting with enhancer sequences, and proteins involved in chromatin remodeling (described above). They estimate that up to a third of the human genome (1 billion base pairs) might be involved in the regulation of gene transcription. In addition, 5-10% of all proteins expressed from genes appear to regulate gene transcription. There appears to be about 300, 1000, and 3000 transcription factor in yeast, drosophila and C. elegans, and humans, respectively. There is about one transcription factor for every gene in yeast, but one for every ten in humans.

In simple eukaryotes, cis regulatory elements would include the promoter (TATA box region), and upstream regulatory sequences (enhancer) and silencers about 100-200 base pairs from the promoter. In more complex eukaryotic species like humans, the promoter is more complex, containing the TATA box, initiator sequences (INR) and downstream promoter elements (DPE). Upstream cis regulatory elements (as far as 10 kb from the promoter) include multiple enhancers, silencers, and insulators. Most promoters have TATA boxes, where TATA Binding Protein (TBP) binds. Upstreams elements in turn regulate the binding of TBP.

Comparative Genomics - Gene Expression Differences Between Humans and Chimpanzees

Our closest biological relative is the chimpanzee, who branched off from a common ancestor of both of us about six million years ago. Our DNA sequence appears to be 98.6 % identical (not just homologous). If we are so close in our genetic blue print, how can we be so different. They are many possible conjectures that may soon be answered when the chimp genome is sequenced and compared to that of the human. Our genes are presumably very similar. People suspect that there are two major kinds of differences that make our species different:

  • our genes are very similar but are transcribed differently in the two species. Recent evidence show that the types of RNA transcribed by human and chimp livers are very similar, but many more genes are transcribed in human brains compared to chimp brains.
  • humans may have lost genes (or their function) that are required for chimp survival in the jungle. The observations that chimps are resistant to many of the disease pathogens that affect humans (immunodeficiency viruses like HIV, influenza A virus, hepatitis B/C, malarial parasite) could be explained by the loss of "protective" genes in humans. In addition, cardiovascular disease and certain types of cancer are rarer in chimps. Humans have apparently "lost" genes involved in body hair, strength, and early maturation, traits that would adapt the chimp to life in the jungle.

We previously discussed an example of a loss of gene function in humans. We have lost a hydroxylase gene involved in formation of certain types of sialic acids, specifically N -glycolylneuraminic acid , found on cell surface glycogroteins of mammals other than humans. Chimps have a lectin receptor for this sialic acid. Recent work has shown that human lack a critical Arg in our version of the lectin that would recognize N -glycolylneuraminic acid , making it unable to bind this ligand. Hence both pairs of genes involved in these type of interactions (cell:cell) are missing. Since sialic acid molecules are often involved in pathogen:host binding, these difference in humans compared to chimps might account for the difference in disease susceptibility as mentioned above.

With respect to gene transcription in the brain, Lai et al. have found a mutation in the human gene FOXP2, a transcription factor, in a family that has significant difficulty in controlling muscles required for articulation of words. This mutation also causes problems in language processing and grammar construction. Comparsion of the normal human gene with other primate genes shows distinct differences in the human gene which may have conferred on human the ability to use speech.


Structure of Transcriptional Activators

Many transcriptional activators are essentially modular in structure in that the DNA-binding domain and the transactivation (or activation) domain can almost be thought of as two distinct proteins that are physically linked. The DNA-binding domain is the part of the molecule that contacts DNA at the promoter site. The transactivation domain is the part that recruits other factors to the promoter such that the rate of transcription of the gene increases. Although transcription factor DNA-binding domains vary in amino acid sequence, many can be placed into structural categories based on their three-dimensional structures. Among these are the zinc finger, helix-loop-helix,


Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.

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Figures

Characterization of the FRDA fibroblast…

Characterization of the FRDA fibroblast lines. ( A ) Determination of the number…

Transcriptional silencing induced by expanded…

Transcriptional silencing induced by expanded GAA repeats is restricted to the FXN gene.…

Epigenetic changes induced by expanded…

Epigenetic changes induced by expanded GAAs in FRDA cells are restricted to the…

Expanded GAA repeats impede transcription…

Expanded GAA repeats impede transcription elongation rate at intron 1 of the FXN…

Histone H4K20me1 is decreased downstream…

Histone H4K20me1 is decreased downstream of the expanded GAA repeats in FRDA cells.…


Expression of Genes

For a cell to function properly, necessary proteins must be synthesized at the proper time. All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed. For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.

The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required. In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.

The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer.

Gene regulation makes cells different

Gene regulation is how a cell controls which genes, out of the many genes in its genome, are “turned on” (expressed). Thanks to gene regulation, each cell type in your body has a different set of active genes—despite the fact that almost all the cells of your body contain the exact same DNA. These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job.

For example, one of the jobs of the liver is to remove toxic substances like alcohol from the bloodstream. To do this, liver cells express genes encoding subunits (pieces) of an enzyme called alcohol dehydrogenase. This enzyme breaks alcohol down into a non-toxic molecule. The neurons in a person’s brain don’t remove toxins from the body, so they keep these genes unexpressed, or “turned off.” Similarly, the cells of the liver don’t send signals using neurotransmitters, so they keep neurotransmitter genes turned off (Figure 1).

Figure 1. Different cells have different genes “turned on.”

There are many other genes that are expressed differently between liver cells and neurons (or any two cell types in a multicellular organism like yourself).

How do cells “decide” which genes to turn on?

Now there’s a tricky question! Many factors that can affect which genes a cell expresses. Different cell types express different sets of genes, as we saw above. However, two different cells of the same type may also have different gene expression patterns depending on their environment and internal state.

Broadly speaking, we can say that a cell’s gene expression pattern is determined by information from both inside and outside the cell.

  • Examples of information from inside the cell: the proteins it inherited from its mother cell, whether its DNA is damaged, and how much ATP it has.
  • Examples of information from outside the cell: chemical signals from other cells, mechanical signals from the extracellular matrix, and nutrient levels.

How do these cues help a cell “decide” what genes to express? Cells don’t make decisions in the sense that you or I would. Instead, they have molecular pathways that convert information—such as the binding of a chemical signal to its receptor—into a change in gene expression.

As an example, let’s consider how cells respond to growth factors. A growth factor is a chemical signal from a neighboring cell that instructs a target cell to grow and divide. We could say that the cell “notices” the growth factor and “decides” to divide, but how do these processes actually occur?

Figure 2. Growth factor prompting cell division

  • The cell detects the growth factor through physical binding of the growth factor to a receptor protein on the cell surface.
  • Binding of the growth factor causes the receptor to change shape, triggering a series of chemical events in the cell that activate proteins called transcription factors.
  • The transcription factors bind to certain sequences of DNA in the nucleus and cause transcription of cell division-related genes.
  • The products of these genes are various types of proteins that make the cell divide (drive cell growth and/or push the cell forward in the cell cycle).

This is just one example of how a cell can convert a source of information into a change in gene expression. There are many others, and understanding the logic of gene regulation is an area of ongoing research in biology today.

Growth factor signaling is complex and involves the activation of a variety of targets, including both transcription factors and non-transcription factor proteins.

In Summary: Expression of Genes

  • Gene regulation is the process of controlling which genes in a cell’s DNA are expressed (used to make a functional product such as a protein).
  • Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA.
  • The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties.
  • In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription.

Alcohol dehydrogenase. (2016, January 6). Retrieved April 26, 2016 from Wikipedia: https://en.wikipedia.org/wiki/Alcohol_dehydrogenase.

Cooper, G. M. (2000). Regulation of transcription in eukaryotes. In The cell: A molecular approach. Sunderland, MA: Sinauer Associates. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK9904/.

Kimball, John W. (2014, April 19). The human and chimpanzee genomes. In Kimball’s biology pages. Retrieved from http://www.biology-pages.info/H/HominoidClade.html.

OpenStax College, Biology. (2016, March 23). Eukaryotic transcription gene regulation. In _OpenStax CNX. Retrieved from http://cnx.org/contents/[email protected]:[email protected]/Eukaryotic-Transcription-Gene-.

OpenStax College, Biology. (2016, March 23). Regulation of gene expression. In _OpenStax CNX. Retrieved from http://cnx.org/contents/[email protected]:[email protected]/Regulation-of-Gene-Expression

Phillips, T. (2008). Regulation of transcription and gene expression in eukaryotes. Nature Education, 1(1), 199. Retrieved from http://www.nature.com/scitable/topicpage/regulation-of-transcription-and-gene-expression-in-1086.

Purves, W. K., Sadava, D. E., Orians, G. H., and Heller, H.C. (2003). Transcriptional regulation of gene expression. In Life: The science of biology (7th ed., pp. 290-296). Sunderland, MA: Sinauer Associates.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). Eukaryotic gene expression is regulated at many stages. In Campbell Biology (10th ed., pp. 365-373). San Francisco, CA: Pearson.


17.2.2 The lac Operon: An Inducer Operon

The lac operon in E. coli has more complex regulation, involving both a repressor and an activator. E. coli uses glucose for food, but is able to use other sugars, such as lactose, when glucose concentrations are low. Three proteins are needed to break down lactose they are encoded by the three genes of the lac operon.

When lactose is not present, the proteins to digest lactose are not needed. Therefore, a repressor binds to the operator and prevents RNA polymerase from transcribing the operon.

When lactose is present, lactose binds to the repressor and removes it from the operator. RNA polymerase is now free to transcribe the genes necessary to digest lactose (Figure 17.4)

Figure 17.4 Transcription of the lac operon only occurs when lactose is present. Lactose binds to the repressor and removes it from the operator.

However, the story is more complex than this. Since E. coli prefers to use glucose for food, the lac operon is only expressed at low levels even when the repressor is removed. But what happens when ONLY lactose is present? Now the bacterium needs to ramp up production of the lactose-digesting proteins. It does so by using an activator protein called catabolite activator protein (CAP).

When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. cAMP binds to CAP and the complex binds to the lac operon promoter (Figure 17.5). This increases the binding ability of RNA polymerase to the promoter and ramps up transcription of the genes.

Figure 17.5 When there is no glucose, the CAP activator increases transcription of the lac operon. However, if no lactose is present, the operon is not activated.

In summary, for the lac operon to be fully activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed maximally. This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available (Table 17.2).

Table 17.2 Summary of signals that induce or repress transcription of the lac operon.


Biology 171

By the end of this section, you will be able to do the following:

  • Explain how chromatin remodeling controls transcriptional access
  • Describe how access to DNA is controlled by histone modification
  • Describe how DNA methylation is related to epigenetic gene changes

Eukaryotic gene expression is more complex than prokaryotic gene expression because the processes of transcription and translation are physically separated. Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels. Epigenetic changes are inheritable changes in gene expression that do not result from changes in the DNA sequence. Eukaryotic gene expression begins with control of access to the DNA. Transcriptional access to the DNA can be controlled in two general ways: chromatin remodeling and DNA methylation. Chromatin remodeling changes the way that DNA is associated with chromosomal histones. DNA methylation is associated with developmental changes and gene silencing.

Epigenetic Control: Regulating Access to Genes within the Chromosome

The human genome encodes over 20,000 genes, with hundreds to thousands of genes on each of the 23 human chromosomes. The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments can be accessed as needed by a specific cell type.

The first level of organization, or packing , is the winding of DNA strands around histone proteins. Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions ((Figure)a). Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string ((Figure)b).


These beads (histone proteins) can move along the string (DNA) to expose different sections of the molecule. If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription ((Figure)).


In females, one of the two X chromosomes is inactivated during embryonic development because of epigenetic changes to the chromatin. What impact do you think these changes would have on nucleosome packing?

How closely the histone proteins associate with the DNA is regulated by signals found on both the histone proteins and on the DNA. These signals are functional groups added to histone proteins or to DNA and determine whether a chromosomal region should be open or closed ((Figure) depicts modifications to histone proteins and DNA). These tags are not permanent, but may be added or removed as needed. Some chemical groups (phosphate, methyl, or acetyl groups) are attached to specific amino acids in histone “tails” at the N-terminus of the protein. These groups do not alter the DNA base sequence, but they do alter how tightly wound the DNA is around the histone proteins. DNA is a negatively charged molecule and unmodified histones are positively charged therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. By adding chemical modifications like acetyl groups, the charge becomes less positive, and the binding of DNA to the histones is relaxed. Altering the location of nucleosomes and the tightness of histone binding opens some regions of chromatin to transcription and closes others.

The DNA molecule itself can also be modified by methylation. DNA methylation occurs within very specific regions called CpG islands. These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes. The cytosine member of the CG pair can be methylated (a methyl group is added). Methylated genes are usually silenced, although methylation may have other regulatory effects. In some cases, genes that are silenced during the development of the gametes of one parent are transmitted in their silenced condition to the offspring. Such genes are said to be imprinted. Parental diet or other environmental conditions may also affect the methylation patterns of genes, which in turn modifies gene expression. Changes in chromatin organization interact with DNA methylation. DNA methyltransferases appear to be attracted to chromatin regions with specific histone modifications. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.


Epigenetic changes are not permanent, although they often persist through multiple rounds of cell division and may even cross generational lines. Chromatin remodeling alters the chromosomal structure (open or closed) as needed. If a gene is to be transcribed, the histone proteins and DNA in the chromosomal region encoding that gene are modified in a way that opens the promoter region to allow RNA polymerase and other proteins, called transcription factors , to bind and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur ((Figure)).

View Chromatin, Histones and Modifications (video) which describes how epigenetic regulation controls gene expression.

Section Summary

In eukaryotic cells, the first stage of gene-expression control occurs at the epigenetic level. Epigenetic mechanisms control access to the chromosomal region to allow genes to be turned on or off. Chromatin remodeling controls how DNA is packed into the nucleus by regulating how tightly the DNA is wound around histone proteins. The DNA itself may be methylated to selectively silence genes. The addition or removal of chemical modifications (or flags) to histone proteins or DNA signals the cell to open or close a chromosomal region. Therefore, eukaryotic cells can control whether a gene is expressed by controlling accessibility to the binding of RNA polymerase and its transcription factors.

Art Connections

(Figure) In females, one of the two X chromosomes is inactivated during embryonic development because of epigenetic changes to the chromatin. What impact do you think these changes would have on nucleosome packing?

(Figure) The nucleosomes would pack more tightly together.

Free Response

In cancer cells, alteration to epigenetic modifications turns off genes that are normally expressed. Hypothetically, how could you reverse this process to turn these genes back on?

You can create medications that reverse the epigenetic processes (to add histone acetylation marks or to remove DNA methylation) and create an open chromosomal configuration.

A scientific study demonstrated that rat mothering behavior impacts the stress response in their pups. Rats that were born and grew up with attentive mothers showed low activation of stress-response genes later in life, while rats with inattentive mothers had high activation of stress-response genes in the same situation. An additional study that swapped the pups at birth (i.e., rats born to inattentive mothers grew up with attentive mothers and vice versa) showed the same positive effect of attentive mothering. How do genetics and/or epigenetics explain the results of this study?

Swapping the pups at birth indicates that the genes inherited from the attentive or inattentive mothers do not explain the rats’ stress-responses later in life. Instead, researchers found that the attentive mothering caused the methylation of genes that control the expression of stress receptors in the brain. Thus, rats that received attentive maternal care exhibited epigenetic changes that limited the expression of stress-response genes, and that the effect was durable over their lifespans.

Some autoimmune diseases show a positive correlation with dramatically decreased expression of histone deacetylase 9 (HDAC9, an enzyme that removes acetyl groups from histones). Why would the decreased expression of HDAC9 cause immune cells to produce inflammatory genes at inappropriate times?

Histone acetylation reduces the positive charge of histone proteins, loosening the DNA wrapped around the histones. This looser DNA can then interact with transcription factors to express genes found in that region. Normally, once the gene is no longer needed, histone deacetylase enzymes remove the acetyl groups from histones so that the DNA becomes tightly wound and inaccessible again. However, when there is a defect in HDAC9, the deacetylation may not occur. In an immune cell, this would mean that inflammatory genes that were made accessible during an infection are not tightly rewound around the histones.

Glossary


Watch the video: Gene Expression (January 2022).