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23.2: Gene regulation: Eukaryotic - Biology

23.2: Gene regulation: Eukaryotic - Biology


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Eukaryotic Gene Regulation

Regulation overview

As was previously noted, regulation is all about decision making. Gene regulation, as a general topic, is related to making decisions about the functional expression of genetic material. Whether the final product is an RNA species or a protein, the production of the final expressed product requires processes that take multiple steps. We have spent some time discussing some of these steps (i.e. transcription and translation) and some of the mechanisms that nature uses for sensing cellular and environmental information to regulate the initiation of transcription.

When we discussed the concept of strong and weak promoters we introduced the idea that regulating the amount (number of molecules) of transcript that was produced from a promoter in some unit of time might also be important for function. This should not be entirely surprising. For a protein coding gene, the more transcript that is produced, the greater potential there is to make more protein. This might be important in cases where making a lot of a particular enzyme is key for survival. By contrast, in other cases only a little protein is required and making too much would be a waste of cellular resources. In this case low levels of transcription might be preferred. Promoters of differing strengths can accommodate these varying needs. With regards to transcript number, we also briefly mentioned that synthesis is not the only way to regulate abundance. Degradation processes are also important to consider.

In this section, we add to these themes by focusing on eukaryotic regulatory processes. Specifically, we examine - and sometimes re-examine - some of the multiple steps that are required to express genetic material in eukaryotic organisms in the context of regulation. We want you not only to think about the processes but also to recognize that each step in the process of expression is also an opportunity to fine tune not only the abundance of a transcript or protein but also its functional state, form (or variant), and/or stability. Each of these additional factors may also be vitally important to consider for influencing the abundance of conditionally-specific functional variants.

Structural differences between bacterial and eukaryotic cells influencing gene regulation

The defining hallmark of the eukaryotic cell is the nucleus, a double membrane that encloses the cell's hereditary material. In order to efficiently fits the organism's DNA into the confined space of the nucleus, the DNA is first packaged and organized by protein into a structure called chromatin. This packaging of the nuclear material reduces access to specific parts of the chromatin. Indeed, some elements of the DNA are so tightly packed that the transcriptional machinery cannot access regulatory sites like promoters. This means that one of the first sites of transcriptional regulation in eukaryotes must be the control access to the DNA itself. Chromatin proteins can be subject to enzymatic modification that can influence whether they bind tightly (limited transcriptional access) or more loosely (greater transcriptional access) to a segment of DNA . This process of modification - whichever direction is considered first - is reversible. Therefore DNA can be dynamically sequestered and made available when the "time is right".

The regulation of gene expression in eukaryotes also involves some of the same additional fundamental mechanisms discussed in the module on bacterial regulation (i.e. the use of strong or weak promoters, transcription factors, terminators etc.) but the actual number of proteins involved is typically much greater in eukaryotes than bacteria or archaea.

The post-transcriptional enzymatic processing of RNA that occurs in the nucleus and the export of the mature mRNA to the cytosol are two additional difference between bacterial and eukaryotic gene regulation. We will consider this level of regulation in more detail below.

Depiction of some key differences between the processes of bacterial and eukaryotic gene expression. Note in this case the presence of histone and histone modifiers, the splicing of pre-mRNA, and the export of the mature RNA from the nucleus as key differentiators between the bacterial and eukaryotic systems.
Attribution: Marc T. Facciotti (own work)

DNA Packing and Epigenetic Markers

The DNA in eukaryotic cells is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus. It is also organized so that specific segments of the chromosomes can be easily accessed as needed by the cell. Areas of the chromosomes that are more tightly compacted will be harder for proteins to bind and therefore lead to reduced gene expression of genes encoded in those areas. Regions of the genome that are loosely compacted will be easier for proteins to access, thus increasing the likelihood that the gene will be transcribed. Discussed here are the ways in which cells regulate the density of DNA compaction.

DNA packing

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 nucleosomes, which can control the access of proteins to specific DNA regions. Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string. These beads (nucleosome complexes) can move along the string (DNA) to alter which areas of the DNA are accessible to transcriptional machinery. While nucleosomes can move to open the chromosome structure to expose a segment of DNA, they do so in a very controlled manner.

DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

Histone Modification

How the histone proteins move is dependent on chemical signals found on both the histone proteins and on the DNA. These chemical signals are chemical tags added to histone proteins and the DNA that tell the histones if a chromosomal region should be "open" or "closed". The figure below 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 histone proteins 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.

Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

Suggested discussion

Why do histone proteins normally have a large amount of positive charges (histones contain a high number of lysine amino acids). Would removal of the positive charges cause a tightening of loosening of the histone-DNA interaction?

Suggested discussion

Predict the state of the histones in areas of the genome that are transcribed regularly. How do these differ from areas that do not experience high levels of transcription?

DNA Modification

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) often 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.

Epigenetic changes do not result in permanent changes in the DNA sequence. Epigenetic changes alter the chromatin structure (protein-DNA complex) to allow or deny access to transcribe genes. DNA modification such as methylation on cytosine nucleotides can either recruit repressor proteins that block RNA polymerase's access to transcribe a gene or they can aid in compacting the DNA to block all protein access to that area of the genome. These changes are reversible whereas mutations are not, however, epigenetic changes to the chromosome can also be inherited.
Source: modified from https://researcherblogski.wordpress....r/dudiwarsito/

Regulation of gene expression through chromatin remodeling 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 can be inherited) and alter the chromosomal structure (open or closed) as needed.

External link

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

Eukaryotic gene structure and RNA processing

Eukaryotic gene structure

Many eukaryotic genes, particularly those encoding protein products, are encoded on the genome discontinuously. That is, the coding region is broken into pieces by intervening non-coding gene elements. The coding regions are termed exons while the intervening non-coding elements are termed introns. The figure below depicts a generic eukaryotic gene.

The parts of a typical discontinuous eukaryotic gene. Attribution: Marc T. Facciotti (own work)

Parts of a generic eukaryotic gene include familiar elements like a promoter and terminator. Between those two elements, the region encoding all of the elements of the gene that have the potential to be translated (they have no stop codons), like in bacterial systems, is called the open reading frame (ORF). Enhancer and/or silencer elements are regions of the DNA that serve to recruit regulatory proteins. These can be relatively close to the promoter, like in bacterial systems, or thousands of nucleotides away. Also present in many bacterial transcripts, 5' and 3' untranslated regions (UTRs) also exist. These regions of the gene encode segments of the transcript, which, as their names imply, are not translated and sit 5' and 3', respectively, to the ORF. The UTRs typically encode some regulatory elements critical for regulating transcription or steps of gene expression that occur post-transcriptionally.

The RNA species resulting from the transcription of these genes are also discontinuous and must therefore be processed before exiting the nucleus to be translated or used in the cytosol as mature RNAs. In eukaryotic systems this includes RNA splicing, 5' capping, 3' end cleavage and polyadenylation. This series of steps is a complex molecular process that must occur within the closed confines of the nucleus. Each one of these steps provides an opportunity for regulating the abundance of exported transcripts and the functional forms that these transcripts will take. While these would be topics for more advanced courses, think about how to frame some of the following topics as subproblems of the Design Challenge of genetic regulation. If nothing else, begin to appreciate the highly orchestrated molecular dance that must occur to express a gene and how this is a stunning bit of evolutionary engineering.

5' capping

Like in bacterial systems, eukaryotic systems must assemble a pre-initiation complex at and around the promoter sequence to initiate transcription. The complexes that assemble in eukaryotes serve many of the same function as those in bacterial systems but they are significantly more complex, involving many more regulatory proteins. This added complexity allows for a greater degree of regulation and for the assembly of proteins with functions that occur predominantly in eukaryotic systems. One of these additional functions is the "capping" of nascent transcripts.

In eukaryotic protein coding genes, the RNA that is first produced is called the pre-mRNA. The "pre" prefix signifies that this is not the full mature mRNA that will be translated and that it first requires some processing. The modification known as 5'-capping occurs after the pre-mRNA is about 20-30 nucleotides in length. At this point the pre-RNA typically receives its first post-transcriptional modification, a 5'-cap. The "cap" is a chemical modification - a 7-methylguanosine - whose addition to the 5' end of the transcript is enzymatically catalyzed by multiple enzymes called the capping enzyme complex (CEC) a group of multiple enzymes that carry out sequential steps involved in adding the 5'-cap. The CEC binds to the RNA polymerase very early in transcription and carries out a modification of the 5' triphosphate, the subsequent transfer of at GTP to this end (connecting the two nucleotides using a unique 5'-to-5' linkage), the methylation of the newly transferred guanine, and in some transcripts the additional modifications to the first few nucleotides. This 5'-cap appears to function by protecting the emerging transcript from degradation and is quickly bound by RNA binding proteins known as the cap-binding complex (CBC). There is some evidence that this modification and the proteins bound to it play a role in targeting the transcript for export from the nucleus. Protecting the nascent RNA from degradation is not only important for conserving the energy invested in creating the transcript but is clearly involved in regulating the abundance of fully-functional transcript that is produced. Moreover, the role of the 5'-cap in guiding the transcript for export will directly help to regulate not only the amount of transcript that is made but, perhaps more importantly, the amount of transcript that is exported to the cytoplasm that has the potential to be translated.

The structure of a typical 7-methylguanylate cap. Facciotti (own work)

Transcript splicing

Nascent transcripts must be processed into mature RNAs by joining exons and removing the intervening introns. This is accomplished by a multicomponent complex of RNA and proteins called the spliceosome. The spliceosome complex assembles on the nascent transcript and in many cases the decisions about which introns to combine into a mature transcript are made at this point. How these decisions are made is still not completely understood but involves the recognition of specific DNA sequences at the splice sites by RNA and protein species and several catalytic events. It is interesting to note that the catalytic portion of the spliceosome is made of RNA rather than protein. Recall that the ribosome is another example of a RNA-protein complex where the RNA serves as the primary catalytic component. The selection of which splice variant to make is a form of regulating gene expression. In this case rather than simply influencing abundance of a transcript, alternative splicing allows the cell to make decisions about which form of transcript is made.

The alternative splice forms of genes that result in protein products of related structure but of varying function are known as isoforms. The creation of isoforms is common in eukaryotic systems and is known to be important in different stages of development in multicellular organisms and in defining the functions of different cell types. By encoding multiple possible gene products from a single gene whose transcription initiation is encoded from a single transcriptional regulatory site (by making the decision of which end-product to produce post-transcriptionally) obviates the need to create and maintain independent copies of each gene in different parts of the genome and evolving independent regulatory sites. Therefore, the ability to form multiple isoforms from a single coding region is though to be evolutionarily advantageous because it enables some efficiency in DNA coding, minimizes transcriptional regulatory complexity, and may lower the energy burden of maintaining more DNA and protecting it from mutation. Some examples of possible outcomes of alternative splicing can include: the generation of enzyme variants with differential substrate affinity or catalytic rates; signal sequences that target proteins to various sub-cellular compartments can be changed; entirely new functions, via the swapping of protein domains can be created. These are just a few examples.

One additional interesting possible outcome of alternative splicing is the introduction of stop codons that can, through a mechanism that seems to require translation, lead to the targeted decay of the transcript. This means that, in addition to the control of transcription initiation and 5'-capping, alternative splicing can also be considered one of the regulatory mechanisms that may influence transcript abundance. The effects of alternative splicing are therefore potentially broad - from complete loss of function to novel and diversified function to regulatory effects.

A figure depicting some of the different modes of alternative splicing illustrating how different splice variants can lead to different protein forms.
Attribution: Marc T. Facciotti (own work)

3' end cleavage and polyadenylation

One final modification is made to nascent pre-mRNAs before they leave the nucleus - the cleavage of the 3' end and its polyadenylation. This two step process is catalyzed by two different enzymes (as depicted below) and may decorate the 3' end of transcripts with up to nearly 200 nucleotides. This modification enhances the stability of the transcript. Generally, the more As in the polyA tag the longer lifetime that transcript has. The polyA tag also seems to play a role in the export of the transcript from the nucleus. Therefore, the 3' polyA tag plays a role in gene expression by regulating functional transcript abundance and how much is exported from the nucleus for translation.

A two step process is involved in modifying the 3' ends of transcripts prior to nuclear exports. These include cutting transcripts just downstream of a conserved sequence (AAUAAA) and transferring adenylate groups. Both processes are enzymatically catalyzed.
Attribution: Marc T. Facciotti (own work)

MicroRNAs

RNA Stability and microRNAs

In addition to the modifications of the pre-RNA described above and the associated proteins that bind to the nascent and transcripts, there are other factors that can influence the stability of the RNA in the cell. One example are elements called microRNAs. The microRNAs, or miRNAs, are short RNA molecules that are only 21–24 nucleotides in length. The miRNAs are transcribed in the nucleus as longer pre-miRNAs. These pre-miRNAs are subsequently chopped into mature miRNAs by a protein called dicer. These mature miRNAs recognize a specific sequence of a target RNA through complementary base pairing. miRNAs, however, also associate with a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). RISC binds a target mRNA, along with the miRNA, to degrade the target mRNA. Together, miRNAs and the RISC complex rapidly destroy the RNA molecule. As one might expect, the transcription of pre-miRNAs and their subsequent processing is also tightly regulated.

Nuclear export

Nuclear export

Fully processed, mature transcripts, must be exported through the nucleus. Not surprisingly this process involves the coordination of a mature RNA species to which are bound many accessory proteins - some of which have been intimately involved in the modifications discussed above - and a protein complex called the nuclear pore complex (NPC). Transport through the NPC allows flow of proteins and RNA species to move in both directions and is mediated by a number of proteins. This process can be used to selectively regulate the transport of various transcripts depending on which proteins associate with the transcript in question. This means that not all transcripts are treated equally by the NPC - depending on modification state and the proteins that have associated with a specific species of RNA it can be moved either more or less efficiently across the nuclear membrane. Since the rate of movement across the pore will influence the abundance of mature transcript that is exported into the cytosol for translation export control is another example of a step in the process of gene regulation that can be modulated. In addition, recent research has implicated interactions between the NPC and transcription factors in the regulation of transcription initiation, likely through some mechanism whereby the transcription factors tether themselves to the nuclear pores. This last example demonstrates how interconnected the regulation of gene expression is across the multiple steps of this complex process.

Many additional details of the processes described above are known to some level of detail, but many more questions remain to be answered. For the sake of Bis2a it is sufficient to begin forming a model of the steps that occur in the production of a mature transcript in eukaryotic organisms. We have painted a picture with very broad strokes, trying to present a scene that reflect what happens generally in all eukaryotes. In addition to learning the key differentiating features of eukaryotic gene regulation, we would also like for Bis2a students to begin thinking of each of these steps as an opportunity for Nature to regulate gene expression in some way and to be able to rationalize how deficiencies or changes in these pathways - potentially introduced through mutation - might influence gene expression.

While we did not explicitly bring up the Design Challenge or Energy Story here these formalisms are equally adept at helping you to make some sense of what is being described. We encourage you to try making an Energy Story for various processes. We also encourage you to use the Design Challenge rubric to reexamine the stories above: identify problems that need solving; hypothesize potential solutions and criteria for success. Use there formalisms to dig deeper and ask new questions/identify new problems or things that you don't know about the processes is what experts do. Chances are that doing this suggested exercise will lead you to identify a direction of research that someone has already pursued (you'll feel pretty smart about that!). Alternatively, you may raise some brand new question that no one has thought of yet.

Control of Protein Abundance

After an mRNA has been transported to the cytoplasm, it is translated into protein. Control of this process is largely dependent on the RNA molecule. As previously discussed, the stability of the RNA will have a large impact on its translation into a protein. As the stability changes, the amount of time that it is available for translation also changes.

The initiation complex and translation rate

Like transcription, translation is controlled by proteins complexes of proteins and nucleic acids that must associate to initiate the process. In translation, one of the first complexes that must assembles to start the process is referred to as the initiation complex. The first protein to bind to the mRNA that helps initiate translation is called eukaryotic initiation factor-2 (eIF-2). Activity of the eIF-2 protein is controlled by multiple factors. The first is whether or not it is bound to a molecule of GTP. When the eIF-2 is bound to GTP it is considered to be in an active form. The eIF-2 protein bound to GTP can bind to the small 40S ribosomal subunit. When bound, the eIF-2/40S ribosome complex, bringing with it the mRNA to be translated, also recruits the methionine initiator tRNA associates. At this point, when the initiator complex is assembled, the GTP is hydrolyzed into GDP creating an "inactive form of eIF-2 that is released, along with the inorganic phosphate, from the complex. This step, in turn, allows the large 60S ribosomal subunit to bind and to begin translating the RNA. The binding of eIF-2 to the RNA further controlled by protein phosphorylation. When eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP thus inhibiting the initiation complex from forming - translation is therefore inhibited (see the figure below). In the dephosphorylated state eIF-2 can bind GTP and allow the assembly of the translation initiation complex as described above. The ability of the cell therefore to tune the assembly of the translation invitation complex via a reversible chemical modification (phosphorylation) to a regulatory protein is another example of how Nature has taken advantage of even this seemingly simple step to tuned gene expression.

An increase in phosphorylation levels of eIF-2 has been observed in patients with neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s. What impact do you think this might have on protein synthesis?

Chemical Modifications, Protein Activity, and Longevity

Not to be outdone by nucleic acids, proteins can also be chemically modified with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins can regulate their activity or the length of time they exist in the cell. Sometimes these modifications can regulate where a protein is found in the cell—for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.

Chemical modifications can occur in response to external stimuli such as stress, the lack of nutrients, heat, or ultraviolet light exposure. In addition to regulating the function of the proteins themselves, if these changes occur on specific proteins they can alter epigenetic accessibility (in the case of histone modification), transcription (transcription factors), mRNA stability (RNA binding proteins), or translation (eIF-2) thus feeding back and regulating various parts of the process of gene expression. In the case of modification to regulatory proteins, this can be an efficient way for the cell to rapidly change the levels of specific proteins in response to the environment by regulating various steps in the process.

The addition of an ubiquitin group has another function - it marks that protein for degradation. Ubiquitin is a small molecule that acts like a flag indicating that the tagged proteins should be targeted to an organelle called the proteasome. This organelle is a large multi-protein complex that functions to cleave proteins into smaller pieces that can then be recycled. Ubiquitination (the addition of a ubiquitin tag), therefore helps to control gene expression by altering the functional lifetime of the protein product.

Proteins with ubiquitin tags are marked for degradation within the proteasome.

In conclusion, we see that gene regulation is complex and that it can be modulated at each step in the process of expressing a functional gene product. Moreover, the regulatory elements that happen at each step can act to influence other regulatory steps both earlier and later in the process of gene expression (i.e. the process of chemically altering a transcription factor can influence the regulation of its own transcription many steps earlier in the process). These complex sets of interactions form what are known as gene regulatory networks. Understanding the structure and dynamics of these networks is critical for understanding how different cells function, the basis for numerous diseases, developmental processes, and how cells make decisions about how to react to the many factors that are in constant flux both inside and outside.


82 Eukaryotic Transcription Gene Regulation

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

  • Discuss the role of transcription factors in gene regulation
  • Explain how enhancers and repressors regulate gene expression

Like prokaryotic cells, the transcription of genes in eukaryotes requires the action of an RNA polymerase to bind to a DNA sequence upstream of a gene in order to initiate transcription. However, unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation. RNA polymerase by itself cannot initiate transcription in eukaryotic cells. There are two types of transcription factors that regulate eukaryotic transcription: General (or basal) transcription factors bind to the core promoter region to assist with the binding of RNA polymerase. Specific transcription factors bind to various regions outside of the core promoter region and interact with the proteins at the core promoter to enhance or repress the activity of the polymerase.

View the process of transcription—the making of RNA from a DNA template.

The Promoter and the Transcription Machinery

Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long). The longer the promoter, the more available space for proteins to bind. This also adds more control to the transcription process. The length of the promoter is gene-specific and can differ dramatically between genes. Consequently, the level of control of gene expression can also differ quite dramatically between genes. The purpose of the promoter is to bind transcription factors that control the initiation of transcription.

Within the core promoter region, 25 to 35 bases upstream of the transcriptional start site, resides the TATA box. The TATA box has the consensus sequence of 5’-TATAAA-3’. The TATA box is the binding site for a protein complex called TFIID, which contains a TATA-binding protein. Binding of TFIID recruits other transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH. Some of these transcription factors help to bind the RNA polymerase to the promoter, and others help to activate the transcription initiation complex.

In addition to the TATA box, other binding sites are found in some promoters. Some biologists prefer to restrict the range of the eukaryotic promoter to the core promoter, or polymerase binding site, and refer to these additional sites as promoter-proximal elements, because they are usually found within a few hundred base pairs upstream of the transcriptional start site. Examples of these elements are the CAAT box, with the consensus sequence 5’-CCAAT-3’ and the GC box, with the consensus sequence 5’-GGGCGG-3’. Specific transcription factors can bind to these promoter-proximal elements to regulate gene transcription. A given gene may have its own combination of these specific transcription-factor binding sites. There are hundreds of transcription factors in a cell, each of which binds specifically to a particular DNA sequence motif. When transcription factors bind to the promoter just upstream of the encoded gene, it is referred to as a cis-acting element , because it is on the same chromosome just next to the gene. Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed.

Enhancers and Transcription

In some eukaryotic genes, there are additional regions that help increase or enhance transcription. These regions, called enhancers , are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away.

Enhancer regions are binding sequences, or sites, for specific transcription factors. When a protein transcription factor binds to its enhancer sequence, the shape of the protein changes, allowing it to interact with proteins at the promotor site. However, since the enhancer region may be distant from the promoter, the DNA must bend to allow the proteins at the two sites to come into contact. DNA bending proteins help to bend the DNA and bring the enhancer and promoter regions together ((Figure)). This shape change allows for the interaction of the specific activator proteins bound to the enhancers with the general transcription factors bound to the promoter region and the RNA polymerase.


Turning Genes Off: Transcriptional Repressors

Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Like the transcriptional activators, repressors respond to external stimuli to prevent the binding of activating transcription factors.

Section Summary

To start transcription, general transcription factors, such as TFIID, TFIIB, and others, must first bind to the TATA box and recruit RNA polymerase to that location. Additional transcription factors may also bind to other regulatory elements at the promoter to increase or prevent transcription. In addition to promoter sequences, enhancer regions help augment transcription. Enhancers can be upstream, downstream, within a gene itself, or on other chromosomes. Specific transcription factors bound to enhancer regions may either increase or prevent transcription.

Review Questions

The binding of ________ is required for transcription to start.

What will result from the binding of a transcription factor to an enhancer region?

  1. decreased transcription of an adjacent gene
  2. increased transcription of a distant gene
  3. alteration of the translation of an adjacent gene
  4. initiation of the recruitment of RNA polymerase

A scientist compares the promoter regions of two genes. Gene A’s core promoter plus proximal promoter elements encompasses 70bp. Gene B’s core promoter plus proximal promoter elements encompasses 250bp. Which of the scientist’s hypotheses is most likely to be correct?

  1. More transcripts will be made from Gene B.
  2. Transcription of Gene A involves fewer transcription factors.
  3. Enhancers control Gene B’s transcription.
  4. Transcription of Gene A is more controlled than transcription of Gene B.

Critical Thinking Questions

A mutation within the promoter region can alter transcription of a gene. Describe how this can happen.

A mutation in the promoter region can change the binding site for a transcription factor that normally binds to increase transcription. The mutation could either decrease the ability of the transcription factor to bind, thereby decreasing transcription, or it can increase the ability of the transcription factor to bind, thus increasing transcription.

What could happen if a cell had too much of an activating transcription factor present?

If too much of an activating transcription factor were present, then transcription would be increased in the cell. This could lead to dramatic alterations in cell function.

A scientist identifies a potential transcription regulation site 300bp downstream of a gene and hypothesizes that it is a repressor. What experiment (with results) could he perform to support this hypothesis?

The easiest way to test his hypothesis would be to mutate the site in a cell, and monitor levels of the mRNA transcript made from the gene. If the levels of transcript increase in the mutated cell, then the site was repressing transcription.

Glossary


23.2: Gene regulation: Eukaryotic - Biology

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 1a). Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string (Figure 1b). These beads (histone proteins) can move along the string (DNA) and change the structure of the molecule.

Figure 1. DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

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 2). Nucleosomes can move to open the chromosome structure to expose a segment of DNA, but do so in a very controlled manner.

Practice Question

Figure 2. Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

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 3 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.

Figure 3. Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (credit: modification of work by NIH)

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 3).

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

In Summary: Eukaryotic Epigenetic Gene Regulation

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. These mechanisms control how DNA is packed into the nucleus by regulating how tightly the DNA is wound around histone proteins. The addition or removal of chemical modifications (or flags) to histone proteins or DNA signals to the cell to open or close a chromosomal region. Therefore, eukaryotic cells can control whether a gene is expressed by controlling accessibility to transcription factors and the binding of RNA polymerase to initiate transcription.


Art Connection

Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

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.

Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (credit: modification of work by NIH)

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).


23.2: Gene regulation: Eukaryotic - Biology

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).

Figure 1. DNA is folded around histone proteins to create (a) nucleosome complexes. These nucleosomes control the access of proteins to the underlying DNA. When viewed through an electron microscope (b), the nucleosomes look like beads on a string. (credit “micrograph”: modification of work by Chris Woodcock)

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)).

Art Connection

Figure 2. Nucleosomes can slide along DNA. When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off. When the nucleosomes are spaced far apart (bottom), the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and DNA affect nucleosome spacing.

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?

The nucleosomes would pack more tightly together.

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.

Figure 3. Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (credit: modification of work by NIH)

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)).

Link to Learning

View this video that 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?


Evolution Connection

Prokaryotic cells can only regulate gene expression by controlling the amount of transcription. As eukaryotic cells evolved, the complexity of the control of gene expression increased. For example, with the evolution of eukaryotic cells came compartmentalization of important cellular components and cellular processes. A nuclear region that contains the DNA was formed. Transcription and translation were physically separated into two different cellular compartments. It therefore became possible to control gene expression by regulating transcription in the nucleus, and also by controlling the RNA levels and protein translation present outside the nucleus.

Most gene regulation is done to conserve cell resources. However, other regulatory processes may be defensive. Cellular processes such as developed to protect the cell from viral or parasitic infections. If the cell could quickly shut off gene expression for a short period of time, it would be able to survive an infection when other organisms could not. Therefore, the organism evolved a new process that helped it survive, and it was able to pass this new development to offspring.


Genetic Regulation in Prokaryotes and Eukaryotes

In this article we will discuss about:- 1. The Inducible and Repressible Systems 2. Transcriptional and Translational Control 3. Regulation of E.coli Tryptophan Operon 4. Regulation of Transcription in Eukaryotes 5. Promoters and Enhancers 6. Transcriptional Activators 7. Regulation by Alternative Splicing of RNA Transcript 8. Regulation at the Level of Translation 9. Epigenetic Control of Genetic Regulation.

The Inducible and Repressible Systems:

In the inducible system when inducer is absent, the repressor binds to the operator and blocks it RNA polymerase cannot move along DNA so that very small amounts of mRNA if at all, are synthesised by the structural genes.

But when lactose is present in the medium as an inducer, the operon is induced to synthesise large quantities of the enzymes required in the transport and catabolism of lactose. In this case the operon functions because the repressor gets bound to the inducer molecule, the operator becomes free, and structural genes synthesise mRNA.

The lac operon provides one example of an inducible system in which the existence of the nutrient in the medium induces synthesis of large amounts of enzymes needed for the catabolism of that nutrient. Such systems therefore, operate in the degradation of exogenous substrates in catabolic processes.

In repressible systems the operon controls synthesis of proteins or enzymes needed for anabolic reactions. Such an operon continues to function normally until there is an excess of the products.

When the end product is in excess it functions as a repressing metabolite called co-repressor. In this system the repressor is inactive by itself. But when the co-repressor binds to the repressor to form a repressor-co-repressor complex which attaches to the operator, the structural genes cannot transcribe mRNA (Fig. 16.3).

In a repressible system the operon is negatively controlled. In addition to the histidine operon already described, the tryptophan operon in E. coli also functions as a repressible operon. When there are normal concentrations of tryptophan in a cell, the operon is functional or de-repressed. But when tryptophan is in excess, it acts as a co-repressor and binds to the inactive repressor.

The complex attaches to the operator and prevents mRNA synthesis by structural genes. As the concentration of tryptophan decreases, it causes the repressor molecules to remain free, the operator becomes unbound, and the structural genes transcribe mRNA. Thus the operon again becomes de-repressed.

Transcriptional and Translational Control:

The lactose operon demonstrates that the control of transcription involves interaction of regulatory proteins with specific DNA sequences, and this is broadly applicable to eukaryotes as well. Regulatory sequences such as the operator are called cis-acting control elements because they bring about expression of only linked genes on the same DNA molecule.

In contrast, repressor proteins are called trans-acting factors because they can influence the expression of genes located on other chromosomes within the cell. Furthermore, the lac operon is considered as an example of negative control of gene expression because binding of the repressor inhibits transcription. There are however, other examples where trans-acting factors are activators (positive control) of transcription.

Positive control of transcription has been demonstrated in E. coli through studies on the effect of glucose on the expression of genes encoding enzymes that lead to breakdown (catabolism) of other sugars, such as lactose. Lactose provides an alternative source of carbon and energy.

As long as glucose is available, it is preferentially utilised, with the result that enzymes involved in catabolism of alternative energy sources are not expressed. That means, if E. coli are grown on a medium that provides both glucose and lactose, the lac operon is not induced, and only glucose is used by cells of E.coli. Thus, glucose represses the lac operon even in the presence of the normal inducer, lactose.

Repression by glucose, also called catabolite repression is actually mediated by a positive control system which is determined by levels of cyclic AMP. (cAMP) (Fig. 16.4). In bacteria cAMP is produced from ATP by enzyme adenylyl cyclase. The conversion of ATP to cAMP is regulated in such a way that levels of cAMP increase when glucose levels drop. Cyclic AMP then binds to a transcriptional regulatory protein called catabolite activator protein (CAP).

The binding of cAMP to CAP stimulates the binding of CAP to its specific DNA sequences. In the lac operon this specific DNA sequence is located approximately 60 bases upstream of the transcription start site. CAP then interacts with the alpha subunit of RNA polymerase, and that facilitates the binding of polymerase to the promoter and activating transcription.

Regulation of E.coli Tryptophan Operon:

Genes of the amino acid tryptophan (trp genes) are considered as repressible genes in which the presence of the metabolite (trp) in the environment turns off the expression of its structural genes. Tryptophan acts as a co-repressor. Regulation of the trp operon occurs in two ways.

In the first, the expression of the five structural genes E, D, C, B, and A that code for enzymes involved in the synthesis of tryptophan, is controlled by a specific regulatory gene. The regulatory gene codes for a specific protein called repressor. The repressor by itself is inactive, but when it becomes complexed with tryptophan (co-repressor) it becomes activated.

The activated repressor-co-repressor complex hinds to a specific region of DNA, the operator situated adjacent to the structural genes that are being regulated. This blocks the movement of RNA polymerase towards structural genes.

The structural genes, operator and promoter regions together constitute the operon. Thus, when tryptophan is present in the environment, the repressor forms a complex with tryptophan, then binds with operator and prevents transcription of the structural genes.

On the contrary, when tryptophan is lacking in the environment, the repressor remains free and inactive, does not bind with operator, resulting in transcription of structural genes and synthesis of tryptophan. This is referred to as a negative control system because the repressor which is product of the regulatory gene acts to turn off transcription of structural genes (Fig. 16.5).

The second mechanism called attenuation, regulates the expression of tryptophan structural genes by controlling the ability of RNA polymerase to continue elongation over a specific nucleotide sequence. This mechanism operates when high levels of tryptophan are available. There is attenuation of regulation by a sequence that terminates transcription prematurely.

This sequence or region of attenuation is located 162 nucleotides downstream of the transcription start site, that is the first structural gene. Transcription terminates in this region if tryptophan is available, before RNA polymerase reaches the first structural gene. In other words, attenuation occurs if the specific amino-acylated tRNA is available. If not, transcription continues, producing a functional trp mRNA.

Transcription is initiated at the promoter region producing what is referred to as the leader transcript. The leader RNA contains a start and a stop signal for protein synthesis. Since prokaryotes lack a nuclear membrane, transcription and translation can occur simultaneously, unlike eukaryotes where there is spatial separation of transcription (in nucleus) and translation (in cytoplasm).

Therefore, while the leader RNA is being synthesised, ribosomes begin translation at the 5′ end. This results in a short peptide chain while the RNA polymerase is transcribing the leader region. If tryptophan-tRNA is available, synthesis of the peptide chain will continue, until the ribosome reaches the stop signal present in the leader RNA.

However, if there is not sufficient tryptoph an -tRNA, the leader RNA will not be translated into peptide, and the ribosome will be arrested at the tryptophan codons in the leader RNA, without reaching the stop signal.

Besides the stop and start sequences, the leader RNA contains 4 regions which have complementary sequences which enable formation of stem and loop structures by base pairing.

Region 1 can form base pairs with region 2 region 2 can form base pairs on both sides either with region 1 or with region 3 region 3 can likewise form base pairs with region 2 or with region 4 region 4 can base pair only with region 3. Therefore, 3 possible stem/loop structures can form in the RNA transcript (Fig. 16.6).

When region 3 base pairs with region 4, it generates a signal for attenuation, that is, premature termination of transcription. However, it must be noted that if stem/loop has already been formed in the region preceding region 3, then region 3 will not be available to base pair with region 4.

Another important point to note is that, if the ribosome is translating in region 2, then region 2 would not be available for base pairing with region 1 or with region 3. In that situation region 3 will be free to base pair with region 4.

Base pairing only between regions 3 and 4 to form stem/loop signals RNA polymerase to terminate transcription. This implies that when sufficient amount of tryptophan-tRNA is available to translate the leader RNA, it will stop transcription (attenuation) prematurely, and structural genes will not be transcribed, In contrast, if tryptophan-tRNA is lacking or insufficient to translate the leader RNA, there will be no attenuation.

In that case the ribosome will stop at the two trp codons in region 1, thus leaving region 2 available to base pair with region 3. That means region 3 would not be available to base pair with region 4, which is an essential requirement for signaling RNA polymerase to terminate transcription. In absence of attenuation then structural genes will be transcribed (Fig. 16.7).

Regulation of Transcription in Eukaryotes:

The control of expression of eukaryotic genes is more complex than in prokaryotes, and is primarily at the level of initiation of transcription. In general, transcription in eukaryotic cells is controlled by proteins that bind to specific regulatory sequences and modulate the activity of RNA polymerase.

In the many different cell types of multicellular eukaryotic organisms, regulation of gene expression is accomplished by the combined actions of multiple different transcriptional regulatory proteins, by methylation of DNA, and packaging of DNA into chromatin.

Promoters and Enhancers:

In bacteria, transcription is regulated by the binding of proteins to cis-acting sequences, as in the lac operon, that control transcription of adjacent genes (z, y, a). Similar cis-acting sequences regulate the expression of eukaryotic genes. The method of identifying these sequences is based on the use of gene transfer assays by which the activity of supposed regulatory regions of cloned genes are studied (Fig. 16.8).

The regulatory sequence is ligated to a reporter gene that encodes an easily detectable enzyme. The reporter gene is transferred into cultured cells (transfection). The expression of the reporter gene indicates biological activity of the regulatory sequence and provides a sensitive assay for the ability of the cloned regulatory sequences to direct transcription.

The two core promoter elements, the TATA box and the Inr sequence in genes transcribed by RNA polymerase II serve as specific binding sites for transcription factors. Inr is the initiator sequence spanning the transcription start site in promoters of many genes transcribed by RNA polymerase II.

Other cis-acting sequences function as binding sites for a variety of regulatory factors that control expression of individual genes. These cis-acting regulatory sequences are usually located upstream of the TATA box. Interestingly, two regulatory sequences commonly found in eukaryotic gene were found to be present in the promoter of the herpes simplex virus gene that encodes thymidine kinase.

These two sequences are located about 100 base pairs upstream of the TATA box, and their consensus sequences are CCAAT and GGGCGG (called the GC box). The binding of specific proteins to these sequences has been shown to initiate transcription.

In contrast to the CCAAT and GC boxes in the thymidine kinase promoter of herpes simplex virus, the regulatory sequences of several mammalian genes are located further away, upto 10 kilo-bases, from the transcription start site. These sequences are called enhancers and were first described in the virus SV40. Like promoters, enhancers function by binding transcription factors that act by regulating RNA polymerase.

Transcription factors bound to distant enhancers function by the same mechanisms as those bound adjacent to promoters, that is, the cis-acting regulatory sequences. The binding of specific transcriptional regulatory proteins to enhancers is responsible for the control of gene expression during development, differentiation and in response of cells to hormones and growth factors.

Transcriptional Activators:

Among the most thoroughly studied transcription factors are the transcription activators, which bind to regulatory DNA sequences and stimulate transcription. Transcriptional activators consist of two domains, one region binding to DNA to anchor the factor to the proper site on DNA the other activates transcription by interacting with other components of the transcriptional system.

Detailed studies have revealed that the DNA-binding domains of many of these proteins are related to one another. The zinc finger domains contain repeats of cysteine and histidine residues that bind zinc ions and fold into finger-like loops that bind DNA. Transcription factors of the steroid hormone receptors contain zinc finger domains.

The steroid hormone receptors regulate gene transcription in response to hormones estrogen and testosterone. The activation domains of transcription factors are not as well characterised as their DNA binding domains.

Regulation by Alternative Splicing of RNA Transcript:

The primary transcript of some genes could be spliced in alternative ways to yield different products. Even when the same promoter is used to transcribe a gene, different cell types can produce different quantities of a protein, or even a different protein. This could result from differences in the mRNA produced or from processing of mRNA. This can be achieved when the same transcript from one cell type is spliced differently from the transcript in another type of cell.

The protein- coding exons may be the same in the different cell types, but the splicing pattern of the transcript may be different. In that case the protein is identical, but the rate of synthesis is different, because the mRNA molecules are not translated with the same efficiency.

In other cases, the protein-coding part of the transcript has a different splicing pattern in each cell type, with the result that the mRNA molecules produced code for proteins that are not identical even though they share certain exons.

Transcripts of the human genome are frequently spliced in alternative ways. Owing to this, the approximately 30,000 human genes may encode 64,000 to 96,000 different proteins. Alternative RNA processing is considered to be one of the principle sources of human genetic complexity. For example, the human insulin receptor gene undergoes alternative splicing that results in the inclusion or exclusion of exon number11 in the mRNA (Fig. 16.9).

The resulting forms of the polypeptide chain differ in length by 12 amino acids. In liver cells, all the 20 exons are present in mRNA for the long form of the receptor protein (Fig. 16.9, Part A), whereas in skeletal muscle exon 11 is eliminated along with the flanking introns and excluded from the mRNA for the short form (Fig. 16.9, Part B).

The long form of the receptor shows low affinity for insulin and is expressed in tissues such as the liver that are exposed to relatively higher concentrations of insulin. The short form of the protein has a high affinity for insulin and is expressed preferentially in tissues such as the skeletal muscle that are normally exposed to lower levels of insulin.

Thus, alternative splicing provides a mechanism for generating proteins with different properties from the same gene. The Dscam gene of Drosophila could give rise to approximately 38016 different proteins by alternative splicing. The actual number of proteins synthesised is not known. The human genome that contains 30,000 to 40,000 genes produces different proteins whose number is several times greater, by alternative splicing.

Unlike genes of Drosophila and lower worms, human genes are distributed over a large region of the genome, and the primary mRNA transcripts are very long. Alternative splicing of most human genes leads to multiple protein products. About one third of all human genes are believed to undergo alternative splicing.

Among genes that are alternatively spliced, the average number of distinct mRNAs produced from the primary transcript ranges between 2 and 7. The average number of different mRNAs per gene across the genome is in the range of 2 to 3, which includes genes that produce a single mRNA as well as those that produce multiple different mRNAs. Thus alternative splicing greatly increases the number of protein products that can be encoded from a relatively small number of genes.

Regulation at the Level of Translation:

In eukaryotic cells, transcription and translation are uncoupled in the process of gene expression. This permits regulation at the level of translation independently from transcription.

The major types of translational control are: inability of a mRNA molecule to be translated under certain conditions regulation of the overall rate of protein synthesis inhibition or activation of translation by small regulatory RNAs that undergo base pairing with the mRNA activation of previously un-translated cytoplasmic mRNAs.

In the case of translational control by small regulatory RNAs, usually the regulatory RNAs are complementary in sequence to part of the mRNA whose translation they control. An RNA sequence that is complementary to a mRNA is called an antisense RNA. The antisense regulating RNAs act by pairing with the mRNA. It can either activate or inhibit translation. Small regulatory RNAs can also regulate translation.

Epigenetic Control of Genetic Regulation:

Epigenetic phenomena are alternative states of gene activity that are heritable, but do not follow Mendelian rules of inheritance, are not explained by mutation, changes in gene sequence or normal developmental regulation. They are changes brought about in gene expression by heritable chemical modifications in DNA.

The prefix epi means “besides” or “in addition to”. Therefore, epigenetic refers to heritable changes in gene expression that are not associated with changes in the DNA sequence, but with something “in addition to” the DNA sequence, usually either chemical modification of the DNA bases or proteins bound with DNA.

It is now clear that many epigenetic phenomena occur largely via changes in chromatin structure. In general, methylation of DNA is associated with turning off gene expression. However, some organisms that clearly exhibit epigenetic effects, for example Drosophila, do not have DNA methylation. Modifications of histones and non­-histone chromosomal proteins have also been implicated in epigenetics.


Enhancers and Transcription

In some eukaryotic genes, there are regions that help increase or enhance transcription. These regions, called enhancers , are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away.

Enhancer regions are binding sequences, or sites, for transcription factors. When a DNA-bending protein binds, the shape of the DNA changes ([link]). This shape change allows for the interaction of the activators bound to the enhancers with the transcription factors bound to the promoter region and the RNA polymerase. Whereas DNA is generally depicted as a straight line in two dimensions, it is actually a three-dimensional object. Therefore, a nucleotide sequence thousands of nucleotides away can fold over and interact with a specific promoter.


Control of Eukaryotic Gene Regulation

Eukaryotic Gene Regulation: Genomic Control

Genes coding for the human antibody heavy chains are created by DNA rearrangements involving multiple types of V, D and J segments.
Initially, the DNA of the immune cells is arranged as tandem arrays of V, D and J regions
DNA excision randomly removes several D and J segments to place individual D and J sequences side by side.
A second random excision removes several V and D segments to join a V section to the others to form a VDJ segment.
After transcription, the sequences separating the VDJ segment from the C segment are removed by RNA splicing.

Eukaryotic Gene Regulation: Transcriptional Control

In a typical protein-coding eukaryotic gene, the mRNA is transcribed by RNA polymerase II.
The core promoter is characterized by an initiator sequence surrounding the transcriptional startpoint and a sequence called a TATA box located about 25 bp upstream (to the 5 prime side) of the startpoint.
The core promoter is where the general transcription factors and RNA polymerase assemble for the initiation of transcription.
Within about 100 nucleotides upstream from the core promoter lie several proximal control elements, which stimulate transcription of the gene by interacting with regulatory transcription factors.
The number, identity and location of the proximal elements vary from gene to gene.
The transcription unit includes a 5 prime untranslated region (leader) and a 3 prime untranslated region (trailer) which are transcribed and included in the mRNA but do not contribute sequence information for the protein product.
These untranslated regions may contain expression control sequences.
In the primary transcript, at the end of the last exon is a site directing the cleavage of the RNA and poly(A) addition.

Properties of Enhancers

A Model for Enhancer Action
In this model, an enhancer located at a great distance along the DNA from the protein-coding gene it regulates is brought close to the core promoter by a looping of the DNA.
The influence of an enhancer on the promoter is mediated by regulatory transcription factors called activators.
1) The activator proteins bind to the enhancer elements, forming an enhanceosome.
2) Bending of the DNA brings the enhanceosome closer to the core promoter.
The general transcription factor TFIID is in the promoter's vicinity. For the purpose of this figure, two of the protein subunits of TFIID, which will function as coactivators in step 3, are distinguished from the rest of the factor.
3) The DNA-bound activators interact with specific coactivators that are part of TFIID. This interaction facilitates the correct positioning of TFIID on the promoter.
4) The other general transcription factors and RNA polymerase join the complex, and transcription is initiated.

The gene for the protein albumin, like other genes, is associated with an array of regulatory DNA elements here we show only two control elements, as well as the core promoter.
Cells of all tissues contain RNA polymerase and the general transcription factors, but the set of regulatory transcription factors available varies with the cell type.
Liver cells contain a set of regulatory transcription factors that includes the factors for recognizing all the albumin gene control elements.
When these factors bind to the DNA, they facilitate transcription of the albumin gene at a high level.
Brain cells, however, have a different set of regulatory transcription factors, which does not include all the ones for the albumin gene. Consequently, in brain cells, the transcription complex can assemble at the promoter, but not very efficiently.
The result is that brain cells transcribe the albumin gene only at a low level.

Several structural motifs are commonly found in the DNA-binding domains of regulatory transcription factors.
The parts of these domains that directly interact with specific DNA sequences are usually alpha helices (or recognition helices) which fit into DNA's major groove.
The helix-turn-helix motif contains two alpha helices are joined by a short flexible turn.
The zinc finger motif consists of an alpha helix and a two segment, antiparallel beta sheet, all held together by the interaction of four cysteine residues (or two cysteine & two histidine residues) with a zinc atom.
Zinc finger proteins normally contain a number of zinc fingers.
The leucine zipper motif contains an alpha helix that has a regular arrangement of leucine residues that interacts with a similar region in a second polypeptide to coil around each other.
The helix-loop-helix motif contains a short a helix connected to a longer a helix by a polypeptide loop interacts with a similar region on another polypeptide to create a dimer.
The homeodomain is a helix-turn-helix DNA-binding domain containing three alpha helices encoded by a 180 basepair homeobox.
The homeodomain was originally found in homeotic genes which are very important in development.
The homeodomain Hox genes control the head-to-tail development in animals from flies to mammals.

The DNA response sequences that bind transcription factors are often comprised of inverted repeat elements.
Reading the sequence of the glucocorticoid response element in the 5 prime to 3 prime direction from either end yields the same DNA sequence (5 prime-AGAACA -3 prime).
The thyroid hormone element contains the same inverted repeat sequences as the estrogen element but the three bases that separate the two copies of the sequence in the estrogen element are absent.

The glucocorticoid receptors activate gene transcription.
Cortisol, a hydrophobic steroid hormone, can diffuse through a plasma membrane then bind to the intracellular glucocorticoid receptor.
Binding the steroid causes the release of an inhibitory protein and activates the glucocorticoid receptor molecule's DNA binding site.
The glucocorticoid receptor molecule then enters the nucleus and binds to a glucocorticoid response element in DNA which causes a second glucocorticoid receptor molecule to bind to the same response element.
The resulting glucocorticoid receptor dimer activates transcription of the target gene.

The cAMP response element binding protein (CREB) controls gene expression when cAMP levels increase.
Genes activated by cyclic AMP possess an upstream cyclic AMP response element (CRE) that binds a transcription factor called the CREB protein.
In the presence of cyclic AMP, cytoplasmic protein kinase A is activated and its activated catalytic subunit then moves into the nucleus, where it catalyzes phosphorylation of the CREB protein, thereby stimulating its activation domain.

Eukaryotic Gene Regulation: Translational Control

Translation of ferritin is activated in the presence of iron.
Translation is inhibited by binding of the IRE-binding protein to the hairpin structure of an iron response element (IRE) in the 5 prime untranslated leader sequence of ferritin mRNA.
When iron binds to IRE-binding protein, it contorts into a conformation that does not recognize the IRE.
When iron is available, ribosomes can assemble on the mRNA and proceed to translate ferritin.
The hairpin does not interfere with the ribosome activities.

Degradation of the transferrin receptor mRNA (required for iron uptake) is also regulated by the allosteric IRE-binding protein.
Transferrin receptor mRNA has an IRE in its 3 prime untranslated region.
When intracellular [iron] is low, the IRE-binding protein remains bound to the IRE which 1) protects the mRNA from degradation and 2) allowing more transferrin receptor protein to be synthesized.
When intracellular [iron] is high, iron binds to the IRE-binding protein, it releases the IRE and the mRNA can be degraded.


81 Eukaryotic Epigenetic Gene Regulation

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?

<!– <link window=”new” target-id=”fig-ch16_03_02″ document=””/> The nucleosomes would pack more tightly together. –>

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 this video that 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.

Visual Connection Questions

(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.

Review Questions

What are epigenetic modifications?

  1. the addition of reversible changes to histone proteins and DNA
  2. the removal of nucleosomes from the DNA
  3. the addition of more nucleosomes to the DNA
  4. mutation of the DNA sequence

Which of the following are true of epigenetic changes?

  1. allow DNA to be transcribed
  2. move histones to open or close a chromosomal region
  3. are temporary
  4. all of the above

Critical Thinking Questions

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.


Watch the video: Gene regulation in eukaryotes (September 2022).


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