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In order for a specific gene to be expressed in the mammal's cells, all of the gene's binding sites must be bound by transcriptional activators. The mammal's skin cells contain activators that bind to sites B, D, and E, while the mammal's liver cells contain activators that bind to sites A, C, and E. (From Khan academy)
Why can both Gene 2 and Gene 4 be expressed in skin cells? I think Gene 4 can't since it doesn't have site E.
You understand this wrong: All present enhancers of a gene must be bound by a enhancing factor, not all factors of a certain cell types have to be bound. So when skin cells contain activators for B, D and E they can activate gene 2 and 4 of your example, but not gene 3 since the activators for A and C are missing.
So when you have a gene containing any combination of B, D and E, its activation in skin cells is possible according to this model. The same is true for any combination of A, C and E for liver cells. The special case would be a gene having E as the only enhancer as this could be activated in both, liver and skin cells.
Erythrogenic toxins, also referred to as streptococcal pyrogenic exotoxins, are secreted by strains of the bacterium Streptococcus pyogenes.   SpeA and speC are superantigens, which induce inflammation by nonspecifically activating T cells and stimulating the production of inflammatory cytokines.  SpeB, the most abundant streptococcal extracellular protein, is a cysteine protease.   Pyrogenic exotoxins are implicated as the causative agent of scarlet fever and streptococcal toxic shock syndrome.  There is no consensus on the exact number of pyrogenic exotoxins. Serotypes A-C [ clarification needed ] are the most extensively studied and recognized by all sources, but others note up to thirteen distinct types, categorizing speF through speM as additional superantigens.    
Erythrogenic toxins are known to damage the plasma membranes of blood capillaries under the skin and produce a red skin rash (characteristic of scarlet fever).  Past studies have shown that multiple variants of erythrogenic toxins may be produced, depending on the strain of S. pyogenes in question. Some strains may not produce a detectable toxin at all.  Bacteriophage T12 infection of S. pyogenes enables the production of speA, and increases virulence. 
Figure 2.6.1:lac Operon
- lacZ codes for b -galactosidase, which is an enzyme that cleaves b -galactosides (e.g. lactose).
- lacY codes for permease, which is involved in the transport of b -galactosides into the cell.
- lacA codes for b -galactoside transacetylase, which acetylates b -galactosides.
- A mutation in either lac Z or lac Y can lead to a lac - genotype, i.e. cells which cannot utilize b -galactosides as a nutrient.
- A lac A- mutant, lacking transacetylase activity, can still utilize b -galactosides (it is still lac+ genotype). Its role in the metabolism of b ­galactosides is not clear.
a region of DNA involved in binding of RNA polymerase to initiate transcription.
a sequence of DNA that causes RNA polymerase to terminate transcription.
- The cluster of three genes, lacZYA, is transcribed into a single mRNA (polycistronic message) from a promoter just upstream from the lac Z gene.
- In the absence of an inducer the gene cluster is not transcribed.
- When an inducer is added (e.g. lactose, or the non-hydrolyzable analog isopropyl thiogalactoside - IPTG) transcription starts at a single promoter (lacP) and proceeds through the lac ZYA genes to a terminator sequence located downstream of the lac A gene.
The lac ZYA mRNA has a half life of
3 minutes, which allows induction to be reversed relatively rapidly (i.e. cells stop producing enzymes rapidly after induction stops).
What molecule does the inducer (lactose) interact with to affect transcriptional regulation (i.e. induction of the lac operon)?
It is not b-galactosidase, permase or transacetylase, rather it is a separate protein called a repressor protein.
- The lac genes are controlled by a mechanism called negative regulation.
- This means that they are transcribed unless they are turned off by the regulator protein .
- A mutation that inactivates the regulator protein causes the lacZYA genes to be continually expressed.
There are two types of genes in the lac operon:
- Structural genes - they code for enzymes required for some biochemical pathway (e.g. lac Z, Y and A).
- Regulator genes - they code for proteins involved in regulation of structural genes.
lac I is the regulator gene of the lac operon.
- This gene is located just upstream of the promoter region for the lac structural genes.
- The lac I gene has its own promoter (constitutive) and terminator.
- It makes a monocistronic message, and codes for one protein - the lac repressor protein .
- It can prevent transcription
- It can recognize and bind the small molecule inducer (lactose or IPTG)
Prevention of transcription by the lac repressor
- lac repressor (active as a tetrameric protein) binds to a sequence of DNA called the operator (lac O region).
- The operator region lies between the lac promoter region (site of RNA polymerase binding and transcription initiation) and the lac Z gene.
- The first 26 base pairs of the lac Z gene comprise the operator region.
- It is not that the repressor protein "blocks" the movement of RNA polymerase through the lac Z gene.
- Repressor binding and RNA polymerase binding (to the promoter) are mutually exclusive at the lac promoter/operator (lac PO) region.
How does the repressor/operator interaction change in the presence of the inducer molecule?
- The inducer can bind to the repressor to form a repressor/inducer complex that no longer associates with the operator .
- The key feature of this interaction is that the repressor protein has two binding sites , one for the inducer and one for the operator.
- When the inducer binds at its site, it changes the conformation of the repressor protein such that the operator binding site has a much reduced affinity for the DNA operator region.
- This type of control is called allosteric control .
- The result is that when the inducer is added the repressor is converted to a form which releases from the operator .
Positive control of the lac operon is exerted by cAMP-CAP complex
- E. coli prefers glucose over other carbon sources.
- When E. coli is grown on glucose, if another sugar (e.g. lactose) is added the induction of enzymes to utilize the other sugar does not occur until the glucose is used up.
- When E. coli is starved for glucose it synthesizes an unusual nucleotide: cyclic 3'5' adenosine monophosphate (cyclic AMP, or cAMP ):
- In bacteria an increase in the cAMP level seems to be an "alert" signal indicating a low glucose level:
Figure 2.6.4:Interaction of cAMP level and lac operon
- an analogue of cAMP which can pass through the E. coli membrane and into the cell.
- If this is added to media containing glucose and lactose it will result in the induction of the lac operon.
- Thus, it mimics the chemical message which tricks the E. coli to respond as though glucose levels were low.
- Mutants of E. coli have been isolated which cannot be induced to metabolize any sugar other than glucose. There were two general categories of mutants:
- Class I. Defective in the enzyme adenylate cyclase. These mutants are unable to make cAMP even when the glucose conentration is low.
- Class II. Lacks a particular protein known as cAMP receptor protein (CRP) or, also known as catabolite receptor protein (CRP).
- Maximum transcription from the lac operon requires the presence of a cAMP/CRP complex.
- cAMP/CRP complex binds to a specific sequence in the lac control region called the "CAP" site.
- The CAP site is just upstream from the RNA polymerase binding site.
- Mutations in the CAP site that prevent cAMP-CRP binding also prevent high levels of expression of the lac operon.
- cAMP/CRP complex has affinity for DNA, and RNA pol.
- Enhances complex formation of RNA pol with the DNA promoter region.
Induction of the lac operon with lactose analogues
- The lac operon can be induced with lactose
- b -galactosidase (lacZ gene product) metabolizes the lactose
- When levels of lactose are reduced, the lac operon is again repressed by the lac repressor (lacI gene product)
- isopropyl b -thiogalactoside, or IPTG, is a non-metabolized lactose analogue
DNA "footprinting" experiments
- If a protein binds to a region of DNA, it can protect that region of DNA from digestion by dnase (DNAse I: an endonuclease at sites adjacent to pyrimidine nucleotides).
- A fragment of DNA can be labeled at the 5' ends with 32 P and then the label can be preferentially removed from one end (i.e. the 3' end of a gene) by an appropriate restriction endonuclease.
- If this DNA fragment, with a label at one specific end, forms a complex with a DNA binding protein the protein will protect the region of DNA that it binds to from DNAse I digestion.
- The digestion is done so as to be incomplete, for the purposes of this discussion, imagine that each DNA molecule is cleaved only once. Furthermore, the site of cleavage is randomly chosen from the available sites.
- Fragments of the DNA, separated and analyzed by size (using gel electrophoresis) after digestion will indicate the protected region:
Figure 2.6.5:DNA footprinting
Results of footprinting experiments
lac DNA incubated with either cAMP/CAP protein, or RNA polymerase, or lac I repressor protein:
Figure 2.6.6:lac repressor with cAMP
RNA polymerase interacts with specific promoter sequences and produces a "footprint" over a region of
- This protection was observed to be more obvious on one strand than the other (i.e. if the other strand was labeled the results did not show as much protection).
- This region of DNAse protection included sites in the DNA from which mutagenesis experiments produced either "up" regulation or "down" regulation of promoter strength.
- These mutagenic "hot" spots affecting promoter strength were located at positions either -10 or -30 upstream from the transcription start site (position +1 in the above diagram):
Figure 2.6.7:Promoter Strength Mutations
- Promoters can be classified according to their "strength".
- This refers to the relative frequency of transcription initiation (transcriptional initiation events per minute), and is related to the affinity of RNA polymerase for the promoter region.
- Many promoters in E. coli have been characterized and a "consensus" promoter sequence has been identified:
Figure 2.6.8:Consensus Promoter Strength
Eukaryotic Transcription Gene Regulation
Like prokaryotic cells, the transcription of genes in eukaryotes requires the actions of an RNA polymerase to bind to a sequence upstream of a gene to initiate transcription. However, unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation. Transcription factors are proteins that bind to the promoter sequence and other regulatory sequences to control the transcription of the target gene. RNA polymerase by itself cannot initiate transcription in eukaryotic cells. Transcription factors must bind to the promoter region first and recruit RNA polymerase to the site for transcription to be established.
View the process of transcription&mdashthe making of RNA from a DNA template:
A YouTube element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/biowm/?p=196
The Promoter and the Transcription Machinery
Figure 3. An enhancer is a DNA sequence that promotes transcription. Each enhancer is made up of short DNA sequences called distal control elements. Activators bound to the distal control elements interact with mediator proteins and transcription factors. Two different genes may have the same promoter but different distal control elements, enabling differential gene expression.
Genes are organized to make the control of gene expression easier. The promoter region is immediately upstream of the coding sequence. The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
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 (Figure 3). 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.
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.
To start transcription, transcription factors, must first bind to the promoter and recruit RNA polymerase to that location. In addition to promoter sequences, enhancer regions help augment transcription. Enhancers can be upstream, downstream, within a gene itself, or on other chromosomes. Transcription factors bind to enhancer regions to increase or prevent transcription.
The binding of ________ is required for transcription to start.
[reveal-answer q=&rdquo670222&Prime]Show Answer[/reveal-answer]
[hidden-answer a=&rdquo670222&Prime]Answer c. The binding of RNA polymerase is required for transcription to start.
What will result from the binding of a transcription factor to an enhancer region?
- decreased transcription of an adjacent gene
- increased transcription of a distant gene
- alteration of the translation of an adjacent gene
- initiation of the recruitment of RNA polymerase
[reveal-answer q=&rdquo829037&Prime]Show Answer[/reveal-answer]
[hidden-answer a=&rdquo829037&Prime]Answer b. Increased transcription of a distant gene will result from the binding of a transcription factor to an enhancer region.
A mutation within the promoter region can alter transcription of a gene. Describe how this can happen.
[reveal-answer q=&rdquo332179&Prime]Show Answer[/reveal-answer]
[hidden-answer a=&rdquo332179&Prime]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?
[reveal-answer q=&rdquo162780&Prime]Show Answer[/reveal-answer]
[hidden-answer a=&rdquo162780&Prime]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. [/hidden-answer]
DNA Repair Enzymes: Cell, Molecular, and Chemical Biology
Jacqueline K. Barton , . Elizabeth Oɻrien , in Methods in Enzymology , 2017
3.2 Detection of TBP Binding Activity
The transcriptional activator TBP has been easily detected on DNA-modified electrodes, given the large perturbation in DNA stacking associated with the binding of TBP. TBP binds to a TATA sequence in DNA and kinks the helix 80 degree at that location, leading to a significant DNA-mediated signal attenuation. In the presence of TBP, which binds to the specific TBP binding site (5′-TATAAAG-3′) and kinks the DNA, the charge accumulation is significantly attenuated ( Furst et al., 2013 ). Protein binding, in kinking the DNA, acts essentially as a switch, turning off DNA CT. BSA, which does not bind to DNA, shows no signal change.
MB-modified DNA with the TBP binding sites (5′-TATAAAG-3′)
Modified MB dye for coupling was synthesized as described previously ( Pheeney & Barton, 2012 )
TBP (ProteinOne), stored at − 80°C until use
BSA (New England Biolabs), stored at − 20°C until use
Sixteen-electrode multiplex chip
CH760B Electrochemical Analyzer and a 16-channel multiplexer module (CH Instruments)
Ag/AgCl reference electrode
Pt wire counter electrode
Tris buffer (10 mM Tris, 100 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2, pH 7.6)
DNA phosphate buffer (5 mM sodium phosphate, 50 mM NaCl, pH 7.0)
TBP binding buffer (5 mM sodium phosphate, 50 mM NaCl, 4 mM MgCl2, 4 mM spermidine, 50 μM EDTA, 10% glycerol, pH 7.0)
For the 16-electrode multiplex chip cleaning and preparation for the TBP binding test, see Section 2.2.1 .
For all electrochemistry, CV scans were performed at a 100 mV/s scan rate over the potential window of 0 mV to − 500 mV. SWV was performed at 15 Hz over the same potential range. Signal size was measured as the CV cathodic peak area or the SWV peak area.
For all protein binding experiments, after backfilling with mercaptohexanol, electrodes were backfilled with 3 μM BSA in phosphate buffer for 45 min at room temperature. After thorough rinsing by buffer exchange, background scans were performed in the TBP buffer TBP. After removing blank TBP buffer from the common well over the electrodes, a solution of the target protein in binding buffer was then added (200 μL total volume).
In this electrochemical protein detection scheme, the protein binding buffer is also the electrochemical running buffer.
The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are three ways to control the transcription of an operon: repressive control, activator control, and inducible control. Repressive control, typified by the trp operon, uses proteins bound to the operator sequence to physically prevent the binding of RNA polymerase and the activation of transcription. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains off. Activator control, typified by the action of CAP, increases the binding ability of RNA polymerase to the promoter when CAP is bound. In this case, low levels of glucose result in the binding of cAMP to CAP. CAP then binds the promoter, which allows RNA polymerase to bind to the promoter better. In the last example—the lac operon—two conditions must be met to initiate transcription. Glucose must not be present, and lactose must be available for the lac operon to be transcribed. If glucose is absent, CAP binds to the operator. If lactose is present, the repressor protein does not bind to its operator. Only when both conditions are met will RNA polymerase bind to the promoter to induce transcription.
Figure In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think that this is the case?
Figure Tryptophan is an amino acid essential for making proteins, so the cell always needs to have some on hand. However, if plenty of tryptophan is present, it is wasteful to make more, and the expression of the trp receptor is repressed. Lactose, a sugar found in milk, is not always available. It makes no sense to make the enzymes necessary to digest an energy source that is not available, so the lac operon is only turned on when lactose is present.
Cells express (transcribe and translate) only a subset of their genes. Cells respond and adapt to environmental signals by turning on or off expression of appropriate genes. In multicellular organisms, cells in different tissues and organs differentiate, or become specialized by making different sets of proteins, even though all cells in the body (with a couple of exceptions) have the same genome. Such changes in gene expression, or differential gene expression among cells, are most often regulated at the level of transcription.
There are three broad levels of regulating gene expression:
- transcriptional control (whether and how much a gene is transcribed into mRNA)
- translational control (whether and how much an mRNA is translated into protein)
- post-translational control (whether the protein is in an active or inactive form, and whether the protein is stable or degraded)
Based on our shared evolutionary origin, there are many similarities in the ways that prokaryotes and eukaryotes regulate gene expression however, there are also many differences. All three domains of life use positive regulation (turning on gene expression), negative regulation (turning off gene expression), and co-regulation (turning multiple genes on or off together) to control gene expression, but there are some differences in the specifics of how these jobs are carried out between prokaryotes and eukaryotes.
Similarities between prokaryotes and eukaryotes: promoters and regulatory elements
Promoters are sites in the DNA where RNA polymerase binds to initiate transcription. Promoters also contain, or have near them, binding sites for transcription factors, which are DNA-binding proteins that can either help recruit, or repel, RNA polymerase. A regulatory element is a DNA sequence that certain transcription factors recognize and bind to in order to recruit or repel RNA polymerase. The promoter along with nearby transcription factor binding elements regulate gene transcription.
Regulatory elements can be used for either positive and negative transcriptional control. When a gene is subject to positive transcriptional control, the binding of a specific transcription factor to the regulatory element promotes transcription. When a gene is subject to negative transcriptional control, the binding of a specific transcription factor to a regulator elements represses transcription. A single gene can be subject to both positive and negative transcriptional control by different transcription factors, creating multiple layers of regulation.
Some genes are not subject to regulation: they are constitutively expressed, meaning they are always transcribed. What sorts of genes would you imagine a cell would always need to have on, regardless of the environment or situation?
Differences between prokaryotes and eukaryotes: mechanisms of co-regulation
Often a set of proteins are needed together to respond to a certain stimulus or carry out a certain function (for example, many metabolic pathways). There are often mechanisms to co-regulate such genes such that they are all transcribed in response to the same stimulus. Both prokaryotic and eukaryotic cells have ways of co-regulating genes, but they use very different mechanisms to accomplish this goal.
In prokaryotes, co-regulated genes are often organized into an operon, where two or more functionally related genes are transcribed together from a single promoter into one long mRNA. This mRNA is translated to make all of the proteins encoded by the genes in the operon. Ribosomes start at the 5′ end, begin translating at the first AUG codon, terminate when they run into a stop codon, and then re-initiate at the next AUG codon.
A generic operon in prokaryotes. R = a regulatory protein (transcription factor) P = promoter Pol = RNA polymerase
With a few exceptions (C. elegans and related nematodes), eukaryotic genomes do not have genes arranged in operons. Instead, eukaryotic genes that are co-regulated tend to have the same DNA regulatory element sequence associated with each gene, even if those genes are located on completely different chromosomes. This means that the same transcriptional activator or repressor can regulate transcription of every single gene that has that particular DNA regulatory element associated with it. For example, eukaryotic HSP (heat shock protein) genes are located on different chromosomes. HSPs help cells survive and recover from heat shock (a type of cellular stress). All HSP genes are transcribed simultaneously in response to heat stress, because they all have a DNA sequence element that binds a heat shock response transcription factor.
Additional complexities specific to eukaryotic gene regulation: chromatin and alternative splicing
Another major difference between prokaryotic gene regulation and eukaryotic gene regulation is that the eukaryotic (but not prokaryotic) DNA double helix is organized around proteins called histones which organize the DNA into nucleosomes. This combination of DNA + histones is called chromatin.
Chromatin can be condensed in a 30-nm fiber formation (tightly compacted nucleosomes) or loosely arranged as “beads-on-a-string,” where the DNA between and around nucleosomes is more accessible. This compaction is controlled by post-translational modifications which are added to the histones in the nucleosomes. When histones have acetyl groups added to them by enzymes called histone acetyl transferases (HATs), the acetyl groups physically obstruct the nucleosomes from packing too densely and help to recruit other enzymes that further open the chromatin structure. Conversely, when the acetyl groups are removed by histone deacetylases (HDACs), the chromatin assumes a condensed formation that prevents transcription factors from being able to access the DNA. In the image below, you can clearly see how much more compact and inaccessible the 30-nm fiber is (top) compared to the beads-on-a-string formation (bottom).
Chromatin plays a fundamental role in positive and negative gene regulation, because transcriptional activators and RNA polymerase cannot physically access the DNA regulatory elements when chromatin is in a compact form.
Prokaryotic DNA does have some associated proteins that help to organize the genomes, but it is fundamentally different from chromatin prokaryotic DNA can essentially be thought of as ‘naked’ compared to eukaryotic chromatin, so prokaryotic cells lack this layer of gene regulation.
Another difference between prokaryotic and eukaryotic gene regulation is that eukaryotic mRNAs must be properly processed with addition of the 5′ cap, splicing out of introns, and addition of the 3′ poly(A) tail (discussed in more detail here). Each of these processing steps is also subject to regulation, and the mRNA will be degraded if any of them are not properly completed. The export of mRNAs from the nucleus to the cytoplasm is also regulated, as is stability of the properly processed mRNA in the cytoplasm.
Finally, eukaryotic genes often have different splice variants, where different exons can be included in different mRNAs that are transcribed from the same gene. Here you can see a cartoon of a gene with color-coded exons, and two different mRNA molecules transcribed from this gene. The different mRNAs encode for different proteins because they contain different exons. This process is called alternative splicing and we will discuss it more here.
Often different types of cells in different tissues express different splice variants of the same gene, such that there is a heart-specific transcript and a kidney-specific transcript of a particular gene.
In general, eukaryotic gene regulation is more complex than prokaryotic gene regulation. The upstream regulatory regions of eukaryotic genes have binding sites for multiple transcription factors, both positive regulators and negative regulators, that work in combination to determine the level of transcription. Some transcription factor binding sites, called enhancers and silencers, work at quite a distance, thousands of base pairs away from the promoter. Activators are examples of positive regulation and repressors are examples of negative regulation.
Eukaryotic transcription initiation, from biology.kenyon.edu (after Tjian)
Overall differences and similarities
If you understand the similarities and differences in eukaryotic and prokaryotic gene regulation, then you know which of the following process are exclusive to eukaryotes, which are exclusive to prokaryotes, which occur in both, and how each is accomplished:
- coupled transcription and translation
- 5′ cap and 3′ poly(A) tail
- AUG as the translation initiation codon
- regulation of gene expression by proteins binding to DNA regulatory elements
- alternative mRNA splicing
- regulation of gene expression through chromatin accessibility
Putting it all together: the lac operon in E. coli
The lac operon is a good model gene for understanding gene regulation. You should use the information below to make sure you can apply all of the details of gene regulation described above to a specific gene model.
E. coli lac operon: dual positive and negative regulation
lacI is the gene that encodes the lac Repressor protein CAP = catabolite activator protein O = Operator P = promoter lacZ = gene that encodes beta-galactosidase lacY encodes permease lacA encodes transacetylase. Source: Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Lac_operon-2010-21-01.png)
The lac operon of E. coli has 3 structural genes required for metabolism of lactose, a disaccharide found at high levels in milk:
- lacZ encodes the enzyme beta-galactosidase, which cleaves lactose into glucose and galactose
- lacY encodes permease, a membrane protein for facilitated diffusion of lactose into the cell
- lacA encodes transacetylase, an enzyme that modifies lactose
An mRNA encoding all 3 proteins is transcribed at high levels only when lactose is present, and glucose is absent.
Negative regulation by the Repressor – In the absence of lactose, the lac Repressor protein, encoded by the lacI gene with a separate promoter that is always active, binds to the Operator sequence in the DNA. The Operator sequence is a type of DNA regulatory element as described above. Repressor protein bound to the Operator prevents RNA polymerase from initiating transcription.
When lactose is present, an inducer molecule derived from lactose binds allosterically to the Repressor, and causes the Repressor to leave the Operator site. RNA polymerase is then free to initiate transcription, if it successfully binds to the lac promoter.
Positive regulation by CAP – Glucose is the preferred substrate for energy metabolism. When glucose is present, cells transcribe the lac operon only at very low levels, so the cells obtain most of their energy from glucose metabolism. RNA polymerase by itself binds rather poorly to the lac promoter.
Glucose starvation causes a rise in the level of cyclic adenosine monophosphate (cAMP), an intracellular alarm signal. Cyclic AMP binds to the catabolite activator protein (CAP). The CAP+cAMP complex binds to the CAP binding site near the lac promoter and recruits RNA polymerase to the promoter.
High level transcription of the lac operon requires both that CAP+cAMP be bound to the CAP binding site, and that Repressor is absent from the Operator. These conditions normally occur only in the absence of glucose and presence of lactose.
The lac operon in E. coli is a classic example of a prokaryotic operon which is subject to both positive and negative regulation. Positive regulation and negative regulation are universal themes for gene regulation in both prokaryotes and eukaryotes.
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Human Leukocyte Antigen (HLA) System
The human leukocyte antigen (HLA) system (the major histocompatibility complex [MHC] in humans) is an important part of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized to present antigenic peptides to the T-cell receptor (TCR) on T cells. (See also Overview of the Immune System.)
MHC molecules that present antigen (Ag) are divided into 2 main classes:
Class I MHC molecules are present as transmembrane glycoproteins on the surface of all nucleated cells. Intact class I molecules consist of an alpha heavy chain bound to a beta-2 microglobulin molecule. The heavy chain consists of 2 peptide-binding domains, an immunoglobulin (Ig)-like domain, and a transmembrane region with a cytoplasmic tail. The heavy chain of the class I molecule is encoded by genes at HLA-A, HLA-B, and HLA-C loci. T cells that express CD8 molecules react with class I MHC molecules. These lymphocytes often have a cytotoxic function, requiring them to be capable of recognizing any infected cell. Because every nucleated cell expresses class I MHC molecules, all infected cells can act as antigen-presenting cells for CD8 T cells (CD8 binds to the nonpolymorphic part of the class I heavy chain). Some class I MHC genes encode nonclassical MHC molecules, such as HLA-G (which may play a role in protecting the fetus from the maternal immune response) and HLA-E (which presents peptides to certain receptors on natural killer [NK] cells).
Class II MHC molecules are usually present only on professional antigen-presenting cells (B cells, macrophages, dendritic cells, Langerhans cells), thymic epithelium, and activated (but not resting) T cells most nucleated cells can be induced to express class II MHC molecules by interferon (IFN)-gamma. Class II MHC molecules consist of 2 polypeptide (alpha [ α ] and beta [ β ]) chains each chain has a peptide-binding domain, an Ig-like domain, and a transmembrane region with a cytoplasmic tail. Both polypeptide chains are encoded by genes in the HLA-DP, -DQ, or -DR region of chromosome 6. T cells reactive to class II molecules express CD4 and are often helper cells.
The MHC class III region of the genome encodes several molecules important in inflammation they include complement components C2, C4, and factor B tumor necrosis factor (TNF)-alpha lymphotoxin and three heat shock proteins.
Individual serologically defined antigens encoded by the class I and II gene loci in the HLA system are given standard designations (eg, HLA-A1, -B5, -C1, -DR1). Alleles defined by DNA sequencing are named to identify the gene, followed by an asterisk, numbers representing the allele group (often corresponding to the serologic antigen encoded by that allele), a colon, and numbers representing the specific allele (eg, A*02:01, DRB1*01:03, DQA1*01:02). Sometimes additional numbers are added after a colon to identify allelic variants that encode identical proteins, and after another colon, other numbers are added to denote polymorphisms in introns or in 5' or 3' untranslated regions (eg, A*02:101:01:02, DRB1*03:01:01:02).
The MHC class I and II molecules are the most immunogenic antigens that are recognized during rejection of an allogeneic transplant. The strongest determinant is HLA-DR, followed by HLA-B and -A. These 3 loci are therefore the most important for matching donor and recipient.
Some autoimmune disorders are linked to specific HLA alleles—for example,
We report a high-quality complement of ORs in I. typographus, which allowed us to functionally characterize the two first bark beetle ORs, specifically responding to (S)-(−)-ipsenol and (R)-(−)-ipsdienol. Responses from the ORs correspond well with those of previously characterized OSN classes, and the OR expression levels and antennal distribution are consistent with the antennal frequency and distribution of these OSNs, especially for ItypOR46. Our investigation of the ligand-OR interaction predicted two discrete binding sites and suggested that hydrogen bonding is important for the binding of ipsenol in ItypOR46. It remains to be investigated whether these putative binding sites are conserved across the insect OR family or if the predicted binding cleft may present a continuum of binding sites in different ORs. Also, further work is needed to investigate whether broadly tuned ORs contain single discrete binding sites, or if they interact with their multiple ligands at different sites.
Because ipsenol elicits strong antagonistic effects on pheromone attraction in I. typographus, ItypOR46 could be a target to employ in screenings aimed to identify more potent agonists than the natural ligand. Such a screening can now be directed towards the predicted binding site, and such agonists may be used in the development of more efficient repellents for forest protection. Indeed, agonists that elicit ultra-prolonged activation of the carbon dioxide-sensitive neurons of mosquitos have been identified, with extended effects on host-seeking behavior . Whether or not similar compounds can be identified for ItypOR46, and how effectively they will divert attacks, remains to be investigated. Additionally, the widespread production of ipsenol and ipsdienol across many species of bark beetles makes both ItypOR46 and ItypOR49 suitable candidates to be used in sensitive biosensors  for detection of infestations of different bark beetles. Although there are technical challenges to overcome before such sensors can be used for airborne volatiles in a field situation, detection of infestations and removal of attacked trees before the next generation emerges to infest new trees are crucial to limit bark beetle populations, and hence outbreaks and economic loss.
Why can a gene lack of a binding site be expressed in skin cells? - Biology
Regulation of Gene Expression
Cellular function is influenced by cellular environment. Adaptation to specific environments is achieved by regulating the expression of genes that encode the enzymes and proteins needed for survival in a particular environment. Factors that influence gene expression include nutrients, temperature, light, toxins, metals, chemicals, and signals from other cells. Malfunctions in the regulation of gene expression can cause various human disorders and diseases.
Regulation in Prokaryotes
Bacteria have a simple general mechanism for coordinating the regulation of genes that encode products involved in a set of related processes. The gene cluster and promoter, plus additional sequences that function together in regulation are called an operon.
The Lactose Operon (lac operon)
The lactose operon of E. coli encodes the enzyme b -galactosidase which hydrolyzes lactose into galactose and glucose.
The lac operon contains three cistrons or DNA fragments that encode a functional protein. The proteins encoded by cistrons may function alone or as sub-units of larger enzymes or structural proteins.
The Z gene encodes for b -galactosidase. The Y gene encodes a permease that facilitates the transport of lactose into the bacterium. The A gene encodes a thiogalactoside transacetylase whose function is not known. All three of these genes are transcribed as a single, polycistronic mRNA. Polycistronic RNA contains multiple genetic messages each with its own translational initiation and termination signals.
Regulation of the lac Operon
The activity of the promoter that controls the expression of the lac operon is regulated by two different proteins. One of the proteins prevents the RNA polymerase from transcribing (negative control), the other enhances the binding of RNA polymerase to the promoter (positive control).
Negative Control of the lac Operon
The protein that inhibits transcription of the lac operon is a tetramer with four identical subunits called lac repressor. The lac repressor is encoded by the lacI gene, located upstream of the lac operon and has its own promoter. Expression of the lacI gene is not regulated and very low levels of the lac repressor are continuously synthesized. Genes whose expression is not regulated are called constitutive genes.
In the absence of lactose the lac repressor blocks the expression of the lac operon by binding to the DNA at a site, called the operator that is downstream of the promoter and upstream of the transcriptional initiation site. The operator consists of a specific nucleotide sequence that is recognized by the repressor which binds very tightly, physically blocking (strangling) the initiation of transcription.
The lac repressor has a high affinity for lactose. When a small amount of lactose is present the lac repressor will bind it causing dissociation from the DNA operator thus freeing the operon for gene expression. Substrates that cause repressors to dissociate from their operators are called inducers and the genes that are regulated by such repressors are called inducible genes.
Positive Control of the lac Operon
Although lactose can induce the expression of lac operon, the level of expression is very low. The reason for this is that the lac operon is subject to catabolite repression or the reduced expression of genes brought on by growth in the presence of glucose. Glucose is very easily metabolized so is the preferred fuel source over lactose, hence it makes sense to prevent expression of lac operon when glucose is present.
The strength of a promoter is determined by its ability to bind RNA polymerase and to form an open complex. The promoter for the lac operon is weak and consequently the lac operon is poorly transcribed upon induction. There is a binding site, upstream from the promoter, for a protein called the catabolite activator protein (CAP). When the CAP protein binds it distorts the DNA so that the RNA polymerase can bind more effectively, thus transcription of the lac operon is greatly enhanced. In order to bind the CAP must first bind cyclic AMP (cAMP), a second messenger synthesized from ATP by the enzyme Adenylate Cyclase.
In the presence of glucose circulating cAMP levels are very low and consequently the initiation of transcription from the lac operon is very low. As glucose levels decrease the concentration of cAMP increases activating CAP which in turn binds to the CAP site stimulating transcription. The cAMP-CAP complex is called a positive regulator.
Arabinose is a five-carbon sugar that can serve as an energy and carbon source for E. coli. Arabinose must first be converted into ribulose-5-phosphate before it can be metabolized. The arabinose operon has three genes,araB, araA and araD that encode for three enzymes to carry out this conversion. A fourth gene, araC, which has its own promoter, encodes a regulatory factor called the C protein.
The regulatory sites of the ara operon include four sites that bind the C protein and one CAP binding site. The araO1 and araO2 sites are upstream of the promoter and CAP binding sites. The other two C protein binding sites called araI1 and araI2 are located between the CAP binding site and the promoter.
Negative Control of the araC Operon
In the absence of arabinose, dimers of the C protein bind to araO2, araO1 and araI1. The C proteins bound to araO2 and araI1 associate with one another causing the DNA between them to form a loop effectively blocking transcription of the operon.
Positive Control of the araC Operon
The C protein binds arabinose and undergoes a conformational change that enables it to also bind the araO2 and araI2 sites. This results in the generation of a different DNA loop that is formed by the interaction of C proteins bound to the araO1 and araO2 sites.
The formation of this loop stimulates transcription of the araC gene resulting in additional C protein synthesis, thus the C protein autoregulates its own synthesis. In the absence of glucose, cAMP-CAP is formed which binds to the CAP site. C protein bound at the araI1 and araI2 sites interacts with the bound CAP enabling RNA polymerase to initiate transcription from the ara operon promoter.
The Tryptophan Operon
E. coli can synthesize all 20 of the natural amino acids. Amino acid synthesis consumes a lot of energy, so to avoid wasting energy the operons that encode for amino acid synthesis are tightly regulated. The trp operon consists of five genes, trpE, trpD, trpC, trpB and trpA, that encode for the enzymes required for the synthesis of tryptophan.
The trp operon is regulated by two mechanisms, negative corepression and attenuation. Most of the operons involved in amino acid synthesis are regulated by these two mechanisms.
The trp operon is negatively controlled by the trp repressor, a product of the trpR gene. The trp repressor binds to the operator and blocks transcription of the operon. However, in order to bind to the operator the repressor must first bind to Trp hence tryptophan is a corepressor. In the absence of Trp the trp repressor dissociates and transcription of the trp operon is initiated.
Attenuation regulates the termination of transcription as a function of tryptophan concentration. At low levels of trp full length mRNA is made, at high levels transcription of the trp operon is prematurely halted. Attenuation works by coupling transcription to translation. Prokaryotic mRNA does not require processing and since prokaryotes have no nucleus translation of mRNA can start before transcription is complete. Consequently regulation of gene expression via attenuation is unique to prokaryotes.
a. Attenuation is mediated by the formation of one of two possible stem-loop structures in a 5' segment of the trp operon in the mRNA.
b. If tryptophan concentrations are low then translation of the leader peptide is slow and transcription of the trp operon outpaces translation. This results in the formation of a nonterminating stem-loop structure between regions 2 and 3 in the 5' segment of the mRNA. Transcription of the trp operon is then completed.
c. If tryptophan concentrations are high the ribosome quickly translates the mRNA leader peptide. Because translation is occurring rapidly the ribosome covers region 2 so that it can not attach to region 3. Consequently the formation of a stem-loop structure between regions 3 and 4 occurs and transcription is terminated.
Regulation of Gene Expression in Eukaryotes
The genetic information of a human cell is a thousand fold greater than that of a prokaryotic cell. Things are further complicated by the number of cell types and the fact that each cell type must express a particular subset of genes at different points in an organisms development. Regulating gene expression so that a particular subset of genes is expressed in a specific tissue at specific points of development is very complicated. This increased complexity in regulation lends itself to malfunctions that cause disease. Three ways that eukaryotes regulate gene expression will be discussed: alteration of gene content or position, transcriptional regulation and alternative RNA processing.
1. Alteration of Gene Content or Position
The copy number of a gene or its location on the chromosome can greatly effect its level of expression. Gene content or location can be altered by gene amplification, diminution or rearrangement.
The expression of a particular gene can be augmented by amplifying its copy number. Histone proteins and rRNA are needed in large quantities by almost all eukaryotic cells therefore the genes encoding histones and rRNA exist in a permanently amplified state. Gene amplification can present problems with the use of chemotherapeutic drugs. Methotrexate inhibits dihydrofolate reductase, the enzyme responsible for regenerating the folates used in nucleotide synthesis. Tumor cells often become resistant to the drug because the gene encoding dihydrofolate reductase is amplified by several hundred fold resulting in more enzyme production then the drug can handle.
A gene whose expression is only needed at a particular developmental point or in a particular tissue may be shut off by gene diminution. As reticulocytes mature into red blood cells all of their genes are lost as the nucleus is degraded.
Gene rearrangement is used to generate each of the genes encoding the millions of different antibodies that are produced by B cells. Sometimes bad gene rearrangements occur that lead to improper gene regulation. This frequently occurs in cancer cells. Translocation of a segment from chromosome 8 to chromosomes that encode immunoglobulins leads to activation of a gene that transforms healthy B cells into Burkitt's lymphoma cells (unregulated proliferating B cells).
2. Transcriptional Regulation
Through Chromosomal Packaging
Regions of each of the different chromosomes are either packaged as heterochromatin or euchromatin. In heterochromatin the DNA is very tightly condensed and rendered inaccessible to the transcriptional machinery, consequently heterochromatin is transcriptionally inactive. In human females one of each of the two X chromosomes is completely inactivated by being packaged into a heterochromatin to form a Barr body. The Cys residues in DNA in the heterochromatin are heavily methylated suggesting that methylation may play a role in the maintenance of heterochromatin. Drugs that interfere with methylation cause activation of previously inactive genes found in heterochromatin.
In euchromatin the DNA is not as condensed and is accessible to the transcription machinery. The regions of a chromosome that are maintained as hetero- and eu- chromatin may vary in a cell specific manner. This may enable the cells of specific tissues to express a particular subset of genes required for tissue function.
Through Individual Genes
Proteins that participate in regulating gene expression are often called trans acting elements. At least 100 different proteins, many specific for the regulation of a particular gene, are known. Others play a more general role in regulating gene expression in a manner analogous to the activation of numerous prokaryotic genes by the CAP-cAMP complex. Trans-acting factors have multiple domains required for activity and may include DNA-binding, transcription-activating and ligand-binding domains.
DNA binding domains recognize specific DNA sequences in the regulatory regions of a gene. The DNA-binding domains of a regulatory protein generally consist of one of three motifs: helix-turn-helix, zinc finger or leucine zipper. DNA-binding proteins possessing these motifs bind with high affinity to their recognition sites and with low affinity to other DNA. A very small portion of the protein makes contact with the DNA through H-bonds and van der Waals interactions between amino acid side chains and the functional groups in the major groove and the phosphate backbone of the DNA. The remainder of the protein is involved in proper positioning of the DNA-binding domain and in making protein-protein contacts with other transcriptional proteins.
Proteins with this motif form symmetric dimers that recognize a symmetric palindromic DNA sequence. Each monomer of the dimer contains a region in which two a helices are held at 90 degrees to each other by a turn of four amino acids. One set of helices makes contact with about five base pairs in the major groove. The other set sits atop the phosphate backbone and helps to properly position the set of helices that fits into the major groove.
Proteins possessing this motif contain between 2 to 9 repeated domains that are each centered on a tetrahedrally coordinated zinc ion. Each zinc coordinated domain forms a loop containing an a -helix, this loop is called a zinc-finger. There are two types of zinc fingers: the C2H2 finger and the Cx finger.
Three fingers interact with the major groove and wrap around the DNA. Many transcription factors have this type of domain.
Proteins with this motif bind as dimers to the major groove of the DNA. Many steroid receptors have this type of domain.
Proteins with this type of motif have an amphipathic a -helix at their carboxyl terminus. One side of the helix consists of hydrophobic groups, usually leucine, that are repeated every seventh position for several turns of the helix. The other face consists of charged and polar groups.
Proteins with this motif bind as dimers to the major groove of the DNA. The two a -helices of each arm enter the major groove and wrap around the double helix. Several oncogenes use this type of motif.
These domains generally act separately and independently of the DNA-binding domains. Transcription-activating domains enhance transcription by physically ineracting with other regulatory proteins and/or with RNA polymerase. The actual mechanisms by which these domains activate or enhance transcription are not known.
Steroid hormones, thyroid hormones and retinoic acid are examples of ligands that activate transcription by binding to a specific domain on a receptor protein. Upon binding the receptor undergoes a conformational change that enables it to bind DNA. Once bound to the DNA a receptor protein can activate or repress transcription of the target gene.
Cis-acting elements are DNA sequences that are recognized and bound by the trans-acting elements that regulate transcription. There are two major types of cis-acting elements: promoters and regulatory elements.
Promoters are the sites where RNA polymerase must bind to the DNA in order to initiate transcription (see "RNA Synthesis and Processing" lecture). The rate or efficiency of promoter use by RNA polymerase is affected by the regulatory elements.
Regulatory elements are specific DNA sequences that are recognized and bound by the trans-acting elements that stimulate or inhibit the expression of a particular gene. There are two types: enhancers and response elements.
Enhancers are regulatory elements that increase or repress the rate of gene transcription.
Response Elements are regulatory sequences that facilitate the coordinated regulation of a group of genes. Certain ligands such as steroid hormones and cAMP bind to their receptors which in turn bind to their response element to activate or inhibit transcription.
3. Alternative Processing
Initiating transcription at an alternative start site places a different exon at the 5' end of the transcript. Examples of genes that use alternative start sites as a form of regulation include amylase, myosin and alcohol dehydrogenase.
Alternative Polyadenylation Sites
Immunoglobin (antibody) heavy chains use an alternative polyadenylation site to affect the length of transcripts. The longer transcript encodes the m m form which is localized to the cell membranes of lymphocytes, the shorter transcript encodes the secreted form, m s.
Alternative splice sites are used to generate similar proteins with tissue specific functions called isoforms. Many peptide hormones exist as isoforms such as the calcitonin gene which is differentially spliced to produce calcitonin in the thyroid and calcitonin gene-related peptide in the neurons.
Regulation of mRNA Stability
The stability of mRNA is quite variable form gene to gene. These variations in stability govern the length of time that mRNA is available for translation and hence the amount of protein that is synthesized. The half-lives of mRNA vary from 10 hours to minutes. Sequences in the 3' untranslated region of mRNA which serve as signals for rapid degradation have been identified in some mRNA's with very short half-lives. The length of the poly A tail also affects mRNA stability, with longer tails tending to have longer half-lives.
Watch the video: Κακόηθες μεσοθηλιώμα υπεζωκότα - Δ. Σπυράτος (August 2022).