Information

Why is the gene regulation in eukaryotic cells needs multiple level of control than in prokaryotic cells?


That "eukaryotic cells are more complex" and "compartmentalized" are used to justify the need of more level of control of gene expression. I get the basic idea but can't convince myself why complexity or compartmentalization leads to more complex control (i.e. multiple levels) of gene regulation!

In control theory, we often use multiple level of control to minimize the variance (there are few other benefits as well from design perspective); probably that is what eukaryotic cell would also do. Moreover, simple combinatorics suggest that two level of control with $n_1$ and $n_2$ molecules involved at each level respectively would give me $n_1n_2$ possibilities of output which is higher than $n_1 + n_2$ distinct output I might get if I were to use these molecules with 1 level of control/regulation.

I was wondering if such simple reasoning are correct and can be used in the context of evolution? Are there other reasons as well?


I think you need to consider the overall amount of information that is needed to implement a proper control system in living organisms.

In prokaryotic cells, in most of the cases, you will find that one gene code for one enzyme, so if a simple organisms need 500 enzymes to stay alive you can expect 500 genes that code for that enzymes. A more complex organism may need 100 times more enzymes to stay alive and so theoretically 100 times more genes. Now, evolution wise, to have a linear increase of DNA vs complexity is not a good option for very complex organisms. To keep everything compartmentalised in one single membrane is also not a good idea if you need to run millions of chemical reactions using thousands of enzymes that can cross react and need different optimal conditions to work properly, so multi-compartimentalization is a must. Linked with it arises the problem of sending the right enzyme in the right place and this has been solved by adding tags, transporter enzymes, etc… in the pot… So, lots of more information is needed to make everything run smoothly.

It turned out that an efficient way to code for all that enzymes, tags, promoters and other regulators without increment the size of the DNA too much is by multi level control of the expression of a gene i.e. the same gene can be used to produce different enzymes (splicing etc… ). The tradeoff to pay to have a more compact genome is to increase the complexity of its control system.

Lastly, consider that differently from normal electronic controllers, the biological one do affect each other. A biological controller system may produce an output molecule that interact with the system under control but also with other reactions going on all over… that is another burden that require an increase of complexity in the control system to make it work.


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


16.3 Eukaryotic Epigenetic Gene Regulation

In this section, you will explore the following question:

Connection for AP ® Courses

One reason that eukaryotic gene expression is more complex than prokaryotic gene expression is because the processes of transcription and translation are physically separated within the eukaryotic cell. Eukaryotic cells also package their genomes in a more sophisticated way compared with prokaryotic cells. Consequently, eukaryotic cells can regulate gene expression at multiple levels, beginning with control of access to DNA. Because genomic DNA is folded around histone proteins to create nucleosome complexes, nucleosomes physically regulate the access of proteins, such as transcription factors and enzymes, to the underlying DNA. Methylation of DNA and histones causes nucleosomes to pack tightly together, preventing transcription factors from binding to the DNA. Methylated nucleosomes contain DNA that is not expressed. On the other hand, histone acetylation results in loose packing of nucleosomes, allowing transcription factors to bind to DNA. Acetylated nucleosomes contain DNA that may be expressed.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.B Expression of genetic information involves cellular and molecular mechanisms.
Essential Knowledge 3.B.1 Gene regulation results in differential gene expression, leading to cell specialization.
Science Practice 7.1 The student can connect phenomena and models across spatial and temporal scales
Learning Objective 3.19 The student is able to describe the connection between the regulation of gene expression and observed differences between individuals in a population

Epigenetic Control: Regulating Access to Genes within the Chromosome

As stated earlier, one reason why eukaryotic gene expression is more complex than prokaryotic gene expression is because the processes of transcription and translation are physically separated. Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels. Eukaryotic gene expression begins with control of access to the DNA. This form of regulation, called epigenetic regulation, occurs even before transcription is initiated.

Teacher Support

Introduce epigenetics and have students work on an epigenetics activity found on the University of Utah’s website.

The human genome encodes over 20,000 genes each of the 23 pairs of human chromosomes encodes thousands of genes. 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 16.6a). Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string (Figure 16.6b). These beads (histone proteins) can move along the string (DNA) and change the structure 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 16.7). Nucleosomes can move to open the chromosome structure to expose a segment of DNA, but do so in a very controlled manner.

Visual Connection

  1. Methylation of DNA and hypo-acetylation of histones causes the nucleosomes to pack tightly together, inactivating one of the X chromosomes at random in each cell.
  2. Methylation of DNA and hypo-acetylation of histones causes the nucleosomes to pack tightly together, inactivating the top half of the paternal chromosome and the bottom half of the maternal chromosome.
  3. Acetylation of DNA and hyper-methylation of histones causes the nucleosomes to unwind, inactivating one of the X chromosomes at random in each cell.
  4. Acetylation of DNA and hyper-methylation of histones causes the nucleosomes to unwind, inactivating only the paternal chromosome.

How the histone proteins move is dependent on signals found on both the histone proteins and on the DNA. These signals are tags added to histone proteins and DNA that tell the histones if a chromosomal region should be open or closed (Figure 16.8 depicts modifications to histone proteins and DNA). These tags are not permanent, but may be added or removed as needed. They are chemical modifications (phosphate, methyl, or acetyl groups) that are attached to specific amino acids in the protein or to the nucleotides of the DNA. The tags 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 therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a large positive charge by adding chemical modifications like acetyl groups, the charge becomes less positive.

The DNA molecule itself can also be modified. This 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. When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added). This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region. Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.

This type of gene regulation is called epigenetic regulation. Epigenetic means “around genetics.” The changes that occur to the histone proteins and DNA do not alter the nucleotide sequence and are not permanent. Instead, these changes are temporary (although they often persist through multiple rounds of cell division) and alter the chromosomal structure (open or closed) as needed. A gene can be turned on or off depending upon the location and modifications to the histone proteins and DNA. If a gene is to be transcribed, the histone proteins and DNA are modified surrounding the chromosomal region encoding that gene. This opens the chromosomal region to allow access for RNA polymerase and other proteins, called transcription factors , to bind to the promoter region, located just upstream of the gene, 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 16.7).

Science Practice Connection for AP® Courses

Think About It

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 will have on nucleosome packaging and, consequently, gene expression?

Teacher Support

The question is an application of Learning Objective 3.19 and Science Practice 7.1 because students are asked to describe how epigenetic changes to chromatin during development can result in differential gene expression and, consequently, differences among cells and organisms.

Answer:

Link to Learning

View this video that describes how epigenetic regulation controls gene expression.

  1. Epigenetics would allow new body parts to be synthesized that could replace those damaged by cancer.
  2. Epigenetics could change the genetic code of all cells in the body to prevent them from becoming cancerous.
  3. New therapies could be made that changes the genetic code of harmful cancer genes.
  4. New therapies could be made that do not require altering the cancer cell’s DNA.

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    Gene Regulation in Eukaryotes

    Let us make an in-depth study of the gene regulation in eukaryotes. After reading this article you will learn about: 1. Chromatin Modification 2. Control of Transcription by Hormones 3. Regulation of Processing of mRNA 4. Control of Life Span of mRNA 5. Gene Amplification 6. Post Translation Regulation and 7. Post Transcription Gene Silencing.

    Introduction to Gene Regulation:

    The expression of genes can be regulated in eukaryotes by all the principles as those of prokaryotes. But there are many additional mechanisms of control of gene expression in eukaryotes as genome is much bigger. The genes are present in the nucleus where mRNA is synthesized. The mRNA is then exported to cytoplasm where translation takes place.

    In eukaryotes, the organization is multicellular and specialized into tissues and organs. The cells are differentiated and cells of a tissue generally produce a specific protein involving a particular set of genes. All other genes become permanently shut off and are never transcribed.

    Structural features of eukaryotes that influence the gene expression are the presence of nucleosomes in chromatin, heterochromatin and the presence of the split genes in chromosomes.

    As compared to prokaryotic genes, the eukaryotic genes have many more regulatory binding sites and they are controlled by many more regulatory proteins. Regulatory sequences can be present thousands of nucleotides away from the promoter, may lie upstream and downstream. These regulatory sequences act from a distance. The intervening DNA loops out, so that the regulatory sequence and promoter come to lie near each other.

    Most of the regulation of gene control occurs at the initiation of transcription level. Initiation of translation also influences gene regulation immensely.

    Chromatin Modification:

    The genome of eukaryotes is wrapped in histone proteins to form nucleosomes. This condition leads to partial concealment of genes and reduces the expression of genes.

    The packing of DNA with histone octomers is not permanent. Any portion of DNA can be released from the octomer whenever DNA binding proteins have to act on it. These DNA binding proteins or enzymes recognize their binding sites on DNA only when it is released from histone octomer or when present on linker DNA. The DNA is unwrapped from nucleosomes.

    This unwrapping of DNA from nucleosomes is performed by nucleosome modifier enzymes or nucleosome remodelling complex. They act in various ways. They may remodel the structure of octomer or slide the octomer along DNA, thus uncover the DNA binding sites for the action of regulatory proteins. Thus the genes are activated.

    Some of these nucleosome modifiers add acetyl groups (acetylation) to the tails of histones, thus loosen the DNA wrapping and in the process exposes the DNA binding sites. All these lead to the expression of genes. Similarly, deacetylation by deacetylases causes inactivation of DNA.

    Nucleosomes are entirely absent in the regions that are active in transcription like rRNA genes.

    Dense form of chromatin is called heterochromatin in eukaryotes. It leads to gene inhibition or gene silencing. Heterochromatin is densely packaged part of chromatin which does not allow gene expression. Densely packaged chromatin cannot be easily transcribed. Some enzymes make the chromatin more dense. Telomeres and contromeres are in the form of heterochromatin.

    In higher animals about 50% of the genome is in the form of heterochromatin. Enzymes are capable of changing the density of chromatin by chemically modifying the tails of histones. This affects transcription.

    In this way, both activation and repression of transcription is performed by modification of chromatin into heterochromatin and euchromatin.

    Methylation of certain sequences of DNA prevents the transcription of genes in mammals. It has been observed that genes, which are heavily methylated are not transcribed, therefore not expressed. DNA methylase enzymes cause methylation of certain DNA sequences thereby silencing of genes.

    Control of Transcription by Hormones:

    Various intercellular and intracellular signals regulate the gene expression.

    Hormones exercise considerable control over transcription. Hormones are extracellular substances synthesized by endocrine glands. They are carried to the distant target cells. Various hormones like insulin, estrogen, progesterone, testosterone etc. often act by “switching on” transcription of DNA.

    The hormone on entering a target cell forms a complex with the receptor present in the cytoplasm. This hormone-receptor complex enters the nucleus and binds to a particular chromosome by means of specific proteins. This initiates the transcription. Hormone-receptor complex can enhance or suppress the expression of genes.

    It has been observed in chickens that when hormone estrogen is injected, the oviduct responds by synthesizing mRNA, which is responsible for synthesis of albumen. The hormone directly binds to DNA and acts as an inducer.

    Regulation of Processing of mRNA:

    Genes of eukaryotes have non-coding regions (introns) in between coding regions (exons). Such genes are called split genes. The entire gene is transcribed to produce mRNA which is called precursor mRNA or primary transcript (pre-mRNA). Before translation takes place, the introns are spliced out by excision and discarded. This is known as processing of mRNA and the processed mRNA is called mature mRNA. This takes part in protein synthesis. Mature mRNA is considerably smaller than precursor mRNA.

    Higher eukaryotes have various mechanisms by which pre-mRNA is processed in alternate or differential ways to produce different mRNAs which encode different proteins. Multiple proteins are produced from one gene by alternate mRNA processing. Many cells take advantage of different splicing pathways to alter the expression of genes and synthesize different polypeptides. Alternate mRNA splicing increases the number of proteins expressed by a single eukaryotic gene.

    Alternate processing of pre-mRNA is accomplished by exon skipping, by retaining certain introns etc.

    These alternate processing pathways are highly regulated.

    In drosophilla mRNA is processed in four different ways, therefore produces four different kinds of muscle protein myosin. Different kind of myosin is produced in larva, pupa and late embryonic stages.

    Control of Life Span of mRNA:

    In prokaryotes the life span of an mRNA molecule is very brief, lasting only for a minute or less. The mRNA immediately degenerates after the protein synthesis.

    But as the mRNA in eukaryotes is transported to cytoplasm through the nucleopores, this mRNA is repeatedly translated. This repeated translation of mRNA is achieved by increasing the life span of mRNA. In a highly differentiated cell, single mRNA molecule having long life span is able to produce large amount of single protein. Life span of a eukaryotic mRNA varies from a few hours to several days.

    Chicken oviduct cells have a single copy of ovalbumen gene but produce large amount of albumen.

    Silk gland of silkworm produces a very long thread made of protein fibroin, which forms cocoon. Silk gland is a single polyploid cell. It produces large number of mRNA molecules, which have long life span of several days.

    Gene Amplification:

    A mechanism exists in various organisms whereby the number of genes is increased many fold without mitosis division. This is called gene amplification.

    During amplification DNA repeatedly undergoes replication without mitotic separation into daughter DNA molecules or chromatids. This enables the cell to produce large amount of protein in a short time.

    Post Translation Regulation:

    In prokaryotes, a single polycistronic mRNA molecule codes for many different proteins. But in eukaryotes having mono-cistronic mRNA, synthesis of different proteins is achieved in a different way. A single mRNA yields a large polypeptide called polyprotein. This polyprotein is then cleaved in alternate ways to produce different proteins. Each protein is regarded as the product of a single gene. In this system, there are many cleaving sites on the polyprotien.

    Post Transcription Gene Silencing:

    Many small RNAs exist in eukaryotes that play their role in silencing of genes. These small RNAs act on mRNA resulting in disruption of translation. These small RNAs are micro RNAs (miRNAs), small interfering RNAs (siRNAs) and many others.


    Regulation of Gene Expression in Eukaryotes | Gene Regulation

    The variation in the rate of transcription often regulates gene expression. Interactions between RNA polymerase II and basal trans­cription factors leading to the formation of the transcription initiation complex influence the rate of transcription. Other transcription factors change the rate of transcription initiation by binding to promoter sequences. The rate of transcription is also influenced by enhancers and silencers.

    This is a site for regulation of transcription. Every structural gene in eukaryotes has the promoter site which consists of several hundred nucleotide sequences that serve as the recognition point for RNA polymerase binding, located at a fixed distance from the site where transcription is initiated.

    Eukaryotic pro­moters require the binding of a number of protein factors to initiate transcription. Promoter regions are recognized by RNA polymerase II, which transcribes primarily mRNA, consists of short DNA sequences usually located within 100 bp upstream (in the 5′ direction) of the gene.

    The promoter regions of most eukaryotic gene contain several specific regions such as:

    Variation in the rate of transcrip­tion often regulates gene expression. Interactions between RNA polymerase II and basal transcrip­tion factors lead to formation of transcription initiation complex (TIC) at the TATA box.

    It is located about 25-30 bases upstream from the initial point of transcription, it consists of an 8 bp consensus sequence composed of A = T base pairs (TATAAA) only, but flanked on either side by G=C rich regions. Mutation in TATA box reduces transcription or may alter the initiation point. TATA box is also known as Hogness box.

    Many promoters contain other components and also bear the consensus sequence like GGCCAATC which is situated at the region 70-80 bp from the start site, it can function in both 5-3′ or a 3-5′ orientation. Mutational analysis showed that CAAT box plays the strongest role in determining the efficiency of the promoter.

    Another element often seen in some promoter regions, called the GC box, has the consensus sequence GGGCGG and is found at about position -110, often occurs in multiple copies, the GC elements bind transcription fac­tors and function more like enhancer.

    Binding of RNA Polymerase II to Promoters:

    The binding of RNA polymerase II to its promo­ter site requires a number of transcriptional factors (TPs).

    Promoters have multiple binding sites for transcription factors each of which can influence transcription. TF IID is the first transcriptional factor to bind close to the promoter at an initiator site about -20 to -10 base pairs before the transcrip­tional start site, i.e., at the TATA boxes, so it is also called TATA box binding protein (TBP).

    TF IID may also interact with other transcriptional factors like TF IIA, TF MB and TF ME. A complex consisting of all transcriptional factors determine which RNA polymerase binds and which gene can be transcribed, and the complex is called pre-initiation complex.

    The transcription factors have a modular structure containing DNA binding, dimerization and transactivation domains.

    DNA binding domains contain three motifs: helix-turn-helix, zinc fingers and basic domains which occur in combination with dimerization domains.

    Dimeri­zation domains contain two motifs: leucine zippers and helix-loop-helix.

    Dimerization allows the formation of homo- and heterodimers creating transcription factors with diverse func­tions. Transactivation domains have no motifs but are often enriched with acidic amino acid, glutamines or pro-lines. They interact with a variety of proteins at different stages during trans­cription. Transcription factors can also repress transcription by direct or indirect mechanisms.

    The transcriptional factors are produced constitutively, but except these there are some transcriptional activators (TAs) which bind to the enhancer site situated many hundreds base pairs from the promoter site.

    These transcriptional activators are induced proteins, i.e., synthesized only in response to specific signals, which on binding with DNA forms the loop back on itself when they interact with the TFs near the promoter. This interaction between enhancer site and initiation site is usually necessary for transcription above a basal level (Fig. 17.10).

    Co-activators are activator proteins that often connect TFs and TAs and may be essential for expression of gene at high level.

    There are many ways by which negative control of transcription takes place in eukaryotes.

    These can be divided into 3 main categories:

    (1) Inhibition of DNA binding

    (ii) Blocking of activation

    (iii) Silencing, i.e., transcriptional activation factor (TAP) cannot bind with transcrip­tion initiation complex (TIC) due to pre­sence of silencer factor.

    Like an enhancer, a silencer also functions irrespective of its position (many thousands base pairs away) and orientation relative to the gene, whose expression it controls. The silencer factor (a protein) either locks the transcription initiation complex or makes it unavailable for activating factors or it disorganizes the transcription initia­tion complex (Fig. 17.11).

    Among the various models, the Britten and Davidson model for regulation of protein synthesis in eukaryotes is most popular. This model is also called genes controlled by one sensor site is termed as battery.

    This model assumes the presence of four classes of sequences (Fig. 17.12a):

    It is comparable with the structural gene of a prokaryotic operon.

    It is comparable to operator gene in bacterial operon and one such receptor site is always assumed to be present adjacent to each producer gene or a set of producer gene.

    It is comparable to regu­lator gene and is responsible for synthesis of an activator RNA that may or may not give rise to proteins before it activates the receptor site.

    A sensor site regulates the activity of integrator gene, which can be transcribed only when the sensor site is activated by agents like hormones and proteins, changes the pattern of gene expression. In this model the genes (producer gene and integrator gene) are involved in RNA synthesis whereas receptor and sensor sites are those sequences which help only in recognition with­out taking part in RNA synthesis.

    It is proposed in this model that receptor sites and integrator genes may be repeated a number of times to control the activity of a large number of genes in the same cell. Repetition of receptor ensures that same activator recognises all of them and several enzymes of one pathway are simultaneously synthesized.

    When the transcription of same gene is needed at different developmental stages, it can be achieved by multiplicity of receptor sites and integrator genes.

    Each producer gene may have several recep­tor sites, each responding to one activator (Fig. 17.12b) so that a single activator thus can recog­nize several genes at a time. One sensor site may regulate the activity of several integrators and different activators may activate the same gene at different times. An inte­grator gene may also fall in cluster with same sensor site (Fig. 17.12c).

    Regulation of Gene Expression by Hor­mones:

    Hormones influence target cells by activating gene transcription. Steroid hormones on entering cells, bind steroid hormone recep­tor protein, releasing it from an inhibitory pro­tein. The receptor dimerizes and is trans-located to the nucleus where it binds to target gene promoters activating transcription.

    Polypeptide hormones bind receptor proteins on the surface of target cells. Signal transduction triggers gene activation in which a sequential activation of several proteins by phosphorylation takes place.

    Post-Transcriptional Regulation of Gene Expression in Eukaryotes:

    Post-transcriptional regulation of gene expression may occur in different ways.

    Regulation of Processing:

    Post-transcriptional modes of regulation also occur in many organisms where the eukaryotic nuclear RNA transcripts are modified prior to translation, non-coding introns are removed, the remaining exons are precisely spliced together and the mRNA is modified by the addition of cap at the 5′ end and a poly-A tail after end.

    The message is then complexed with proteins and exported to the cytoplasm. Each of these pro­cessing steps offers several possibilities for regulation, for example, several alternative splicing pathways of a single pre-mRNA trans­cript to give multiple mRNAs and regulation of the stability of mRNA itself. This leads to the synthesis of different proteins or isoforms in the same time and space.

    Regulation of Translation:

    Regulation at translational level occurs in different ways:

    (i) Activation and repression of translation:

    In eukaryotes the activator protein binds to mRNA and leads to the formation of hairpin structure which helps in ribosome binding with mRNA by the exposure of 5′ end. The translational repressor protein (IRE-BP) controls ferritin synthesis by down-regulation and transferring receptor synthesis by up-regulation.

    (ii) Regulation by phosphorylation machi­nery:

    Translational repressor protein may regulate the translation in eukaryotic system or regulation of translation is brought about by modification of general components of translational machinery.

    Reversible phosphorylation machinery is involved in the regulation of gene expres­sion, as the phosphorylated or dephosphorylated forms of the components of translational machinery should identify a specific mRNA from the bulk mRNA population.


    Free Response

    Describe how controlling gene expression will alter the overall protein levels in the cell.

    The cell controls which protein is expressed, and to what level that protein is expressed, in the cell. Prokaryotic cells alter the transcription rate to turn genes on or off. This method will increase or decrease protein levels in response to what is needed by the cell. Eukaryotic cells change the accessibility (epigenetic), transcription, or translation of a gene. This will alter the amount of RNA, and the lifespan of the RNA, to alter the amount of protein that exists. Eukaryotic cells also change the protein’s translation to increase or decrease its overall levels. Eukaryotic organisms are much more complex and can manipulate protein levels by changing many stages in the process.


    Why is the gene regulation in eukaryotic cells needs multiple level of control than in prokaryotic cells? - Biology

    THE ORGANIZATION AND CONTROL OF EUKARYOTIC GENOMES

    Gene expression in eukaryotes has two main differences from the same process in prokaryotes.

    The typical multicellular eukaryotic genome is much larger than that of a bacterium.
    Cell specialization limits the expression of many genes to specific cells.
    The estimated 35,000 genes in the human genome includes an enormous amount of DNA that does not program the synthesis of RNA or protein.

    Eukaryotic DNA is precisely combined with large amounts of protein.
    During interphase, chromatin fibers are highly extended.
    If extended, each DNA molecule would be about 6 cm long.

    First level - Histone proteins
    Their positively charged amino acids bind tightly to negatively charged DNA.
    The five types of histones are very similar from one eukaryote to another and are even present in bacteria.
    Unfolded chromatin has the appearance of beads on a string, a nucleosome , in which DNA winds around a core of histone proteins.
    The beaded string seems to remain essentially intact throughout the cell cycle.
    Histones leave the DNA only transiently during DNA replication.
    They stay with the DNA during transcription.
    By changing shape and position, nucleosomes allow RNA-synthesizing polymerases to move along the DNA.

    Level two - As chromosomes enter mitosis the beaded string coils to form the 30- nm chromatin fiber .
    Level three - This fiber forms looped domains attached to a scaffold of nonhistone proteins.
    Level four - the looped domains coil and fold to produce the characteristic metaphase chromosome.
    Interphase chromatin is generally much less condensed than the chromatin of mitosis with the 30-nm fibers and looped domains remaining intact.
    The chromatin of each chromosome occupies a restricted area within the interphase nucleus.
    Interphase chromosomes have areas that remain highly condensed, heterochromatin , and less compacted areas, euchromatin .

    Genome Organization at the DNA Level

    In eukaryotes, most of the DNA (about 97% in humans) does not code for protein or RNA.
    1. noncoding regions are regulatory sequences.
    2. introns.
    3. repetitive DNA , present in many copies in the genome.
    In mammals about 10 -15% of the genome is tandemly repetitive DNA , or satellite DNA .
    These differ in density from other regions, so they form a separate band after differential ultracentrifugation.
    There are three types of satellite DNA, differentiated by the total length of DNA at each site. Table 19.1.
    Some genetic disorders are caused by abnormally long stretches of tandemly repeated nucleotide triplets within the affected gene.
    Fragile X syndrome is caused by hundreds to thousands of repeats of CGG in the fragile X gene.
    Huntington's disease occurs due to repeats of CAG that are translated into a proteins with a long string of glutamines.
    The severity of the disease and the age of onset of these diseases are correlated with the number of repeats.
    About 25-40% of most mammalian genomes consists of interspersed repetitive DNA.
    Appear at multiple sites in the genome.
    Are similar but usually not identical to each other.

    While most genes are present as a single copy per haploid set of chromosomes, multigene families exist as a collection of identical or very similar genes.
    These likely evolved from a single ancestral gene.
    The members of multigene families may be clustered or dispersed in the genome.
    Identical genes are multigene families that are clustered tandemly. Fig 19.2.
    Usually consist of the genes for RNA products or those for histone proteins.
    The three largest rRNA molecules are encoded in a single transcription unit that is repeated tandemly hundreds to thousands of times.
    This transcript is cleaved to yield three rRNA molecules that combine with proteins and one other kind of rRNA to form ribosomal subunits.
    Nonidentical genes
    Two related families of globin genes, a (alpha) and ß (beta), of hemoglobin, which are located on different chromosomes. Fig 19.3.
    The different versions of each globin subunit are expressed at different times in development.
    Within both families are sequences that are expressed during the embryonic, fetal, and/or adult stage of development.
    The embryonic and fetal hemoglobins have higher affinity for oxygen than do adult forms, ensuring transfer of oxygen from mother to developing fetus.
    The differences in genes arise from mutations that accumulate in the gene copies over generations.
    These mutations may even lead to enough changes to form pseudogenes , DNA segments that have sequences similar to real genes but that do not yield functional proteins.

    Gene amplification, loss, or rearrangement

    The nucleotide sequence of an organism's genome may be altered in a systematic way during its lifetime.
    Does not affect gametes
    Their effects are confined to particular cells and tissues.
    In gene amplification, certain genes are replicated as a way to increase expression of these genes.
    In amphibians, the genes for rRNA not only have a normal complement of multiple copies but millions of additional copies are synthesized in a developing ovum.
    This assists the cell in producing enormous numbers of ribosomes for protein synthesis after fertilization.
    In some insect cells, whole or parts of chromosomes are lost early in development.
    Rearrangement of the loci of genes in somatic cells may have a powerful effect on gene expression.
    Transposons are genes that can move from one location to another within the genome.
    10% of the human genome are transposons.
    If one "jumps" into a coding sequence of another gene, it can prevent normal gene function.
    If the transposon is inserted in a regulatory area, it may increase or decrease transcription.
    Most transposons are retrotransposons (Fig 19.5), in which the transcribed RNA includes the code for an enzyme that catalyzes the insertion of the retrotransposon and may include a gene for reverse transcriptase .
    Reverse transcriptase uses the RNA molecule originally transcribed from the retrotransposon as a templete to synthesize a double stranded DNA copy.
    This can populate the eukaryotic genome with multiple copies of its sequence.
    Major rearrangements of at least one set of genes occur during immune system differentiation.
    B lymphocytes produce immunoglobins , or antibodies , that specifically recognize and combat viruses, bacteria, and other invaders. Fig 19.6.
    Each differentiated cell produces one specific type of antibody that attacks a specific invader.
    Functional antibody genes are pieced together from physically separated DNA regions.
    Each immunoglobin consists of four polypeptide chains, each with a constant region and a variable region , giving each antibody its unique function.
    As a B lymphocyte differentiates, one of several hundred possible variable segments is connected to the constant section by deleting the intervening DNA.
    The random combinations of different variable and constant regions create an enormous variety of different polypeptides, which combine with others to form complete antibody molecules.
    As a result, the mature immune system can make millions of different kinds of antibodies from millions of subpopulations of B lymphocytes.

    The Control of Gene Expression

    Each cell expresses only a small fraction of its genes
    Are continually turned on and off in response to signals from their internal and external environments.
    Gene expression must be controlled on a long-term basis during cellular differentiation.
    Highly specialized cells express only a tiny fraction of their genes.
    Problems with gene expression and control can lead to imbalance and diseases, including cancers.

    The control of gene expression can occur at any step in the pathway from gene to functional protein. Fig 19.7
    These levels of control include chromatin packing , transcription , RNA processing , translation , and various alterations to the protein product.

    Chromatin packing modifications

    Genes of densely condensed heterochromatin are usually not expressed.

    Chemical modifications of chromatin play a key role in chromatin structure and transcription regulation.
    DNA methylation
    Inactive DNA is generally highly methylated compared to DNA that is actively transcribed.
    For example, the inactivated mammalian X chromosome in females is heavily methylated.
    Methylation enzymes correctly methylate the daughter strands.
    This accounts for genomic imprinting in which methylation turns off either the maternal or paternal alleles.
    Histone acetylation and deacetylation appear to play a direct role in the regulation of gene transcription.
    Acetylated histones grip DNA less tightly, providing easier access for transcription proteins in this region.
    Some of the enzymes responsible for acetylation or deacetylation are associated with or are components of transcription factors that bind to promotors.
    DNA methylation and histone deacetylation may cooperate to repress transcription.

    Initiation of transcription is the most important and universally used control point in gene expression.

    Control elements are noncoding DNA segments that regulate transcription by binding transcription factors. Fig 19.8
    Eukaryotic RNA polymerase is dependent on transcription factors before transcription begins.
    One transcription factor recognizes the TATA box.

    Distal control elements, enhancers , may be thousands of nucleotides away from the promoter or even downstream of the gene or within an intron. Fig 19.9.
    Bending of DNA enables transcription factors, activators , bound to enhancers to contact the protein initiation complex at the promoter.

    Eukaryotic genes also have repressor proteins that bind to DNA control elements called silencers.
    Repression may operate mostly at the level of chromatin modification.

    Each protein generally has a DNA-binding domain that binds to DNA and a protein-binding domain that recognizes other transcription factors.

    Genes coding for the enzymes of a metabolic pathway may be scattered over different chromosomes.
    Coordinate gene expression depends on the association of a specific control element or collection of control elements with every gene of a dispersed group.
    A common group of transcription factors bind to them, promoting simultaneous gene transcription.

    Post-transcriptional mechanisms
    Gene expression may be blocked or stimulated by any post-transcriptional step.

    In alternative RNA splicing , different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Fig 19.11. Movie! Regulation of mRNA degradation .
    Prokaryotic mRNA molecules may be degraded after only a few minutes.
    Eukaryotic mRNAs typically endure for hours and can even last days or weeks.
    For example, in red blood cells the mRNAs for the hemoglobin polypeptides are unusually stable and are translated repeatedly in these cells.
    A common pathway of mRNA breakdown begins with enzymatic shortening of the poly(A) tail.
    This triggers the enzymatic removal of the 5' cap.
    This is followed by rapid degradation of the mRNA by nucleases.

    Control of translation Translation of specific mRNAs can be blocked by regulatory proteins that bind to specific sequences or structures within the 5' leader region of mRNA. Movie!
    This prevents attachment to ribosomes.
    Protein factors required to initiate translation in eukaryotes offer targets for simultaneously controlling translation of all the mRNA in a cell.
    This allows the cell to shut down translation if environmental conditions are poor
    Eukaryotic polypeptides must often be processed to yield functional proteins. Movie!
    Regulation may occur at cleavage, chemical modifications , and transport to the appropriate destination .
    For example, cystic fibrosis results from mutations in the genes for a chloride ion channel protein that prevents it from reaching the plasma membrane.
    The defective protein is rapidly degraded.
    The cell limits the lifetimes of normal proteins by selective degradation.

    Proteins intended for degradation are marked by the attachment of ubiquitin proteins . Fig 19.12.
    Giant proteosomes recognize the ubiquitin and degrade the tagged protein.

    The Molecular Biology of Cancer

    Cancer is a disease in which cells escape from the control methods that normally regulate cell growth and division.
    Changes can be random spontaneous mutations or environmental influences such as chemical carcinogens or physical mutagens.
    Cancer-causing genes, oncogenes , are products of proto-oncogenes , that code for proteins that stimulate normal cell growth and division and have essential functions in normal cells. Fig 19.13.
    An oncogene arises from a genetic change that leads to an increase in the proto-oncogene's protein or the activity of each protein molecule.
    These genetic changes include movements of DNA within the genome , amplification of proto-oncogenes , and point mutations in the gene .
    Malignant cells frequently have chromosomes that have been broken and rejoined incorrectly.
    This may translocate a fragment to a location near an active promotor or other control element.
    Amplification increases the number of gene copies.
    A point mutation may lead to translation of a protein that is more active or longer-lived. Mutations to genes whose normal products inhibit cell division, tumor-suppressor genes , also contribute to cancer.
    Some tumor-suppressor proteins normally repair damaged DNA.
    Others control the adhesion of cells to each other or to an extracellular matrix, crucial for normal tissues.
    Still others are components of cell-signaling pathways that inhibit the cell cycle.

    Oncogene proteins and faulty tumor-suppressor proteins interfere with normal signaling pathways. Fig 19.14.

    Mutations in the products of two key genes, the ras proto-oncogene , and the p53 tumor suppressor gene occur in 30% and 50% of human cancers respectively.
    Both are components of signal-transduction pathways that convey external signals to the DNA.
    Ras , the product of the ras gene, is a G protein that provides the synthesis of a protein that stimulates the cell cycle.
    Many ras oncogenes have a point mutation that leads to a hyperactive version of the Ras protein that can issue signals on its own, resulting in excessive cell division.
    The tumor-suppressor protein encoded by the normal p53 gene is a transcription factor that promotes synthesis of growth-inhibiting proteins.
    A mutation that knocks out the p53 gene can lead to excessive cell growth and cancer.

    The p53 gene is often called the " guardian angel of the genome ".
    Damage to the cell's DNA leads to expression of the p53 gene.
    The p53 protein can:
    activate the p21 gene, which halts the cell cycle.
    turn on genes involved in DNA repair.
    activate "suicide genes" whose protein products cause cell death.

    Multiple mutations underlie the development of cancer

    If cancer results from an accumulation of mutations, and if mutations occur throughout life, then the longer we live, the more likely we are to develop cancer.


    Prokaryotic Gene Regulation

    In bacteria and archaea, structural proteins with related functions are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript (Figure 1). In this way, regulation of the transcription of all of the structural genes encoding the enzymes that catalyze the many steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time, or none will be needed. For example, in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine in 1965. Although eukaryotic genes are not organized into operons, prokaryotic operons are excellent models for learning about gene regulation generally. There are some gene clusters in eukaryotes that function similar to operons. Many of the principles can be applied to eukaryotic systems and contribute to our understanding of changes in gene expression in eukaryotes that can result pathological changes such as cancer.

    Figure 1. In prokaryotes, structural genes of related function are often organized together on the genome and transcribed together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing transcription.

    Each operon includes DNA sequences that influence its own transcription these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.

    In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers.

    Think about It

    • What are the parts in the DNA sequence of an operon?
    • What types of regulatory molecules are there?

    Perspectives

    The development of the eukaryotic type of genome organization—with multiple chromosomes and many scattered origins of replication—was probably important for the expansion of genome size that allowed the development of complex organisms. Taken at face value, control of replication seems to be organized differently in prokaryotic and eukaryotic cells however, the control mechanisms found in the two systems seem to regulate the same steps in the process. First, in both prokaryotes and eukaryotes the crucial step in the establishment of a replication origin is loading of the replicative helicase. This process is mediated when the concentration of the helicase-loading AAA + ATPases builds up to a certain threshold in the cell. In E. coli, this seems to be the rate-limiting step loading of the replicative polymerase and initiation immediately follows. In S. pombe, further progress requires the action of S-phase-activating kinases. Second, once an origin of replication has fired, re-firing is prevented for a period of time. In both systems, this is accomplished by a combination of physical modification of the origin and/or associated protein factors (by sequestration or by phosphorylation), such that the helicase loader cannot access it, and by removing the helicase-loader activity. Eukaryotic cells literally get rid of the protein by switching on ubiquitin-mediated degradation. The prokaryotic cell does not have this option and therefore it is dependent on several other methods of reducing the active concentration of the helicase loader, such as through hydrolysis of its bound ATP, binding of the loader to unproductive sites or downregulation of its expression. The development of ubiquitin-mediated protein degradation made these mechanisms redundant.

    In this review, we have attempted to draw parallels between the basic mechanisms that prevent re-replication in two simple uni-cellular model organisms. Failure to restrict replication to once per cell cycle leads to DNA damage through the generation of double-stranded breaks and can result in development of tumours (reviewed by Arias & Walter, 2007 ). It is therefore not surprising that metazoans have evolved additional mechanisms—such as inactivation of Cdt1 by Geminin binding—to minimize the likelihood of untimely replication initiations.


    Perspective

    Whatever the link between nuclear architecture and regulation of gene expression is, it is an important but still poorly understood aspect of genome structure and function (O'Brien et al., 2003). Understanding its molecular basis will be essential if we want to understand the orchestration of gene expression in eukaryotes.

    What therefore are the major questions that we must answer in order to begin to understand how the genome functions in the confined space of the cell nucleus? The most prominent one concerns the relationship between 3D chromatin structure and function. We lack information about the static and dynamic 3D arrangement of nucleosomes in different types of chromatin. It is attractive to start from the idea that chromatin can adopt a limited set of different spatial configurations reflecting different functional states and that gene regulation in part is due to the controlled switching between these defined states. This process appears to be closely related to histone modification and is ultimately encrypted in the DNA sequence itself.

    Chromatin structure must be studied in the living cell, since it probably changes dramatically if the cell is disrupted. A breakthrough has been the development by Belmont and coworkers of methods for GFP tagging of specific genomic sites (Belmont, 2001). As the spatial resolution of light microscopy is approaching the size of a few nucleosomes, there is hope that detailed information about chromatin structure can be obtained in the next few years (Esa et al., 2000 Kano et al., 2002).

    We also need quantitative models of genome function that link transitions between defined structural and functional chromatin states to protein binding and histone modification. Such models should constitute a critical guide for rational experiments, whose results can be fed back into the model. Finally, we urgently need tools to manipulate nuclear organization. This should allow us to rearrange chromatin and chromosomes and interfere with the assembly or function of nuclear domains and subsequently establish the effects on gene expression. Clearly, we first have to learn more about which proteins and possibly RNA molecules are essential for nuclear organization and chromatin structure. Subsequently, they can become targets for experiments that interfere with nuclear structure and function.


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