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I am doing research on inherited risk of Autism Spectrum Disorders(ASD) due to common Copy Number Variants(CNVs) One of the mutations is the 'CC' variant of Rs1858830 in the promoter region of the MET gene. (http://www.snpedia.com/index.php/Rs1858830)
After digging a bit deeper I found that hepatocyte growth factor is a pretty ubiquitous compound in the body and it's necessary for growth/repair of many tissues, namely the liver and digestive tract. I am trying to separate the direct causal effect of the mutation itself from the effects it has on other tissue which may themselves have an effect on ASD risk.
My question comes from a study which measured reduced serum HGF levels in autistic children with this mutation. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694825/)
I am confused about the implied causal effect of the 'CC' variant on HGF levels even though it is merely the ligand for the MET gene? Please help me understand where in the gene signalling process does HGF come in, what does it do, what proteins are transcribed by the MET gene, and how this mutation effects the transcription of these proteins.
HGF is the ligand for Met, which is a membrane receptor involved in multiple transduction pathways. Gene ID 4233 we're looking at a single gene product for MET.
If we're just reading through the wikipedia page for c-Met, when HGF binds to Met, it activates the tyrosine kinase activity of Met. Met recruits Gab1, and this complex mediates interactions with Ras, P13K, ß-catenin… not just pathways associated w/ Autism, but also associated with invasive growth cancers. An interesting note about the C allele, it's associated with decreased Met transcription. Quoting a recent article,
The present morphological and functional studies have revealed a highly novel mechanism conferred by MET receptor signaling, which we show serves a pleiotrophic role in controlling both neuronal and spine morphology, and the time course of glutamatergic synapse maturation on CA1 hippocampal neurons. Such findings are consistent with the possibility that mistimed maturation of glutamatergic synapses underlie aberrant function of neural circuits that are enriched in MET during development.
Source
So the critical results from the same study showed that when Met got knocked down or deleted, we saw maturation of neural tissue. So now, if i'm thinking about Met and HGF, your article notes that HGF is decreased in autism brains. If we (1) need HGF for Met function, and (2) ASD may be attributed to decreased Met function, and (3) the C allele decreases Met levels, we're basically looking at a system that's modulating for ASD.
80 Prokaryotic Gene Regulation
By the end of this section, you will be able to do the following:
- Describe the steps involved in prokaryotic gene regulation
- Explain the roles of activators, inducers, and repressors in gene regulation
The DNA of prokaryotes is organized into a circular chromosome, supercoiled within the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons . For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon, and transcribed into a single mRNA.
In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors and activators are proteins produced in the cell. Both repressors and activators regulate gene expression by binding to specific DNA sites adjacent to the genes they control. In general, activators bind to the promoter site, while repressors bind to operator regions. Repressors prevent transcription of a gene in response to an external stimulus, whereas activators increase the transcription of a gene in response to an external stimulus. Inducers are small molecules that may be produced by the cell or that are in the cell’s environment. Inducers either activate or repress transcription depending on the needs of the cell and the availability of substrate.
The trp Operon: A Repressible Operon
Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesize using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon ((Figure)). The genes are transcribed into a single mRNA, which is then translated to produce all five enzymes. If tryptophan is present in the environment, then E. coli does not need to synthesize it and the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA is transcribed, the enzyme proteins are translated, and tryptophan is synthesized.
The trp operon includes three important regions: the coding region, the trp operator and the trp promoter. The coding region includes the genes for the five tryptophan biosynthesis enzymes. Just before the coding region is the transcriptional start site . The promoter sequence, to which RNA polymerase binds to initiate transcription, is before or “upstream” of the transcriptional start site. Between the promoter and the transcriptional start site is the operator region.
The trp operator contains the DNA code to which the trp repressor protein can bind. However, the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes the shape of the repressor protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding to the promoter and transcribing the downstream genes.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator, the polymerase can transcribe the enzyme genes, and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is said to be negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators .
Watch this video to learn more about the trp operon.
Catabolite Activator Protein (CAP): A Transcriptional Activator
Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the promoter sequences that act as positive regulators to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate sugars must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. Accumulating cAMP binds to the positive regulator catabolite activator protein (CAP) , a protein that binds to the promoters of operons which control the processing of alternative sugars. When cAMP binds to CAP, the complex then binds to the promoter region of the genes that are needed to use the alternate sugar sources ((Figure)). In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. CAP binding stabilizes the binding of RNA polymerase to the promoter region and increases transcription of the associated protein-coding genes.
The lac Operon: An Inducible Operon
The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. The Z gene of the lac operon encodes beta-galactosidase, which breaks lactose down to glucose and galactose.
However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed ((Figure)). In the absence of glucose, the binding of the CAP protein makes transcription of the lac operon more effective. When lactose is present, it binds to the lac repressor and changes its shape so that it cannot bind to the lac operator to prevent transcription. This combination of conditions makes sense for the cell, because it would be energetically wasteful to synthesize the enzymes to process lactose if glucose was plentiful or lactose was not available.
In E. coli, the trp operon is on by default, while the lac operon is off. Why do you think this is the case?
If glucose is present, then CAP fails to bind to the promoter sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these conditions is met, then transcription remains off. Only when glucose is absent and lactose is present is the lac operon transcribed ((Figure)).
Signals that Induce or Repress Transcription of the lac Operon | ||||
---|---|---|---|---|
Glucose | CAP binds | Lactose | Repressor binds | Transcription |
+ | – | – | + | No |
+ | – | + | – | Some |
– | + | – | + | No |
– | + | + | – | Yes |
Watch an animated tutorial about the workings of lac operon here.
Section Summary
The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. There are two majors kinds of proteins that control prokaryotic transcription: repressors and activators. Repressors bind to an operator region to block the action of RNA polymerase. Activators bind to the promoter to enhance the binding of RNA polymerase. Inducer molecules can increase transcription either by inactivating repressors or by activating activator proteins. In the trp operon, the trp repressor is itself activated by binding to tryptophan. Therefore, if tryptophan is not needed, the repressor is bound to the operator and transcription remains off. The lac operon is activated by the CAP (catabolite activator protein), which binds to the promoter to stabilize RNA polymerase binding. CAP is itself activated by cAMP, whose concentration rises as the concentration of glucose falls. However, the lac operon also requires the presence of lactose for transcription to occur. Lactose inactivates the lac repressor, and prevents the repressor protein from binding to the lac operator. With the repressor inactivated, transcription may proceed. Therefore glucose must be absent and lactose must be present for effective transcription of the lac operon.
Visual Connection Questions
(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.
Review Questions
If glucose is absent, but so is lactose, the lac operon will be ________.
Prokaryotic cells lack a nucleus. Therefore, the genes in prokaryotic cells are:
- all expressed, all of the time
- transcribed and translated almost simultaneously
- transcriptionally controlled because translation begins before transcription ends
- b and c are both true
The ara operon is an inducible operon that controls the production of the sugar arabinose. When arabinose is present in a bacterium it binds to the protein AraC, and the complex binds to the initiator site to promote transcription. In this scenario, AraC is a(n) ________.
Critical Thinking Questions
Describe how transcription in prokaryotic cells can be altered by external stimulation such as excess lactose in the environment.
Environmental stimuli can increase or induce transcription in prokaryotic cells. In this example, lactose in the environment will induce the transcription of the lac operon, but only if glucose is not available in the environment.
What is the difference between a repressible and an inducible operon?
A repressible operon uses a protein bound to the promoter region of a gene to keep the gene repressed or silent. This repressor must be actively removed in order to transcribe the gene. An inducible operon is either activated or repressed depending on the needs of the cell and what is available in the local environment.
Glossary
What Happens If The Promoter Is Mutated
Answer Wiki. A mutation in a promoter sequence could make the promoter unable do perform its usual function, which is recruiting enzymes and other proteins to the site of the gene, to promote transcription. A promoter’s sequence is essential for it to work, so changes in the sequence would very likely make it nonfunctional.
How does a mutation in the promoter affect the gene?
Depending on the location and the nature of the genetic defect, a mutation in the promoter region of a gene may disrupt the normal processes of gene activation by disturbing the ordered recruitment of TFs at the promoter. As a result a promoter mutation can decrease or increase the level of mRNA and thus protein.
Why are promoter mutations so difficult to study?
In addition, promoter mutation analysis is complex, and the assays that are needed to investigate the functional relationship between the mutation and disease are laborious and difficult to perform. Therefore, thorough studies of promoter mutations are scarce and often confined to research laboratories.
How does a mutation in a promoter affect transcription?
The mutation may also affect binding site for transcription factors, and may thus affect it's regulation. A mutation in a promoter sequence could make the promoter unable do perform its usual function, which is recruiting enzymes and other proteins to the site of the gene, to promote transcription.
Regulation of Lac Operon
- Glucose must be absent: The level of cyclic AMP must be high enough so that the CAP protein binds to the CAP binding site. Bound CAP helps to attach RNA polymerase efficiently to the lac operon promoter.
- Lactose must be present: There must be an inducer (such as lactose) so that lactose repressor does not block transcription by binding to the operator.
Each of the regulatory proteins (CAP and lac repressor) responds to one environmental signal and communicates it to the lac genes. The combined effect of these two regulators ensures that the genes are expressed at significant levels only when lactose is present and glucose is absent. Now, let’s observe the transcription of the operon in various environmental conditions:
1. Glucose present, lactose absent
As glucose is present, cAMP level is low so activator CAP remains inactive. Lac repressor remains bound to the operator and prevents binding of RNA polymerase. In this condition, no transcription of the lac operon occurs.
2.Glucose present, lactose present
Activator CAP remains inactive. The lac repressor is not functional because the inducer (lactose) is present. In this condition, the basal level transcription of the lac operon occurs.
3.Glucose absent, lactose absent
Activator CAP is active as a high level of cAMP is present (as glucose is absent) but lac repressor is functional (active). Lac repressor remains bound to the operator and prevents transcription.
4.Glucose absent, lactose present
cAMP levels are high so CAP is active and bound to the DNA. CAP helps the efficient binding of RNA polymerase to the promoter. lac repressor is inactive due to the presence of inducer (lactose/allolactose). In this condition, strong transcription of the lac operon occurs.
Health Conditions Related to Genetic Changes
Achondroplasia
Two mutations in the FGFR3 gene cause more than 99 percent of cases of achondroplasia, which is a form of short-limbed dwarfism. Both mutations lead to the same change in the FGFR3 protein. Specifically, the protein building block (amino acid) glycine is replaced with the amino acid arginine at protein position 380 (written as Gly380Arg or G380R). Researchers believe that this genetic change causes the receptor to be overly active, which leads to the disturbances in bone growth that occur in this disorder.
Crouzon syndrome with acanthosis nigricans
A single FGFR3 gene mutation has been identified in people with Crouzon syndrome with acanthosis nigricans. This rare condition causes premature joining of the bones of the skull (craniosynostosis), leading to a misshapen head and distinctive facial features, and a skin abnormality called acanthosis nigricans that is characterized by thick, dark, velvety skin in body folds and creases. The genetic change that causes Crouzon syndrome with acanthosis nigricans replaces the amino acid alanine with the amino acid glutamic acid at position 391 of the FGFR3 protein (written as Ala391Glu or A391E). The altered receptor is more easily turned on than normal and can trigger signaling pathways even without attachment of growth factors. The resulting overactivity of the FGFR3 protein disrupts the normal growth of the skull bones and skin, leading to the features of Crouzon syndrome with acanthosis nigricans.
Epidermal nevus
Mutations in the FGFR3 gene have been found in approximately 30 percent of people with a certain type of epidermal nevus (plural: nevi). Specifically, FGFR3 gene mutations are associated with some keratinocytic epidermal nevi, which are abnormal skin growths that are composed of skin cells called keratinocytes. FGFR3 gene mutations have not been found in other types of epidermal nevi.
The most common FGFR3 gene mutation in epidermal nevi changes a single amino acid in the FGFR3 protein. The amino acid arginine is replaced with the amino acid cysteine at position 248 (written as Arg248Cys or R248C). This mutation creates a protein that is turned on without attachment of a growth factor, which means that the FGFR3 protein is constantly active. Studies suggest that cells with this FGFR3 gene mutation grow and divide more than normal cells. The resulting overgrowth of skin cells leads to epidermal nevi.
The FGFR3 gene mutations found in epidermal nevi also occur in people with another skin abnormality called seborrheic keratosis and in people with skeletal disorders known as thanatophoric dysplasia, Crouzon syndrome with acanthosis nigricans, and SADDAN (each described in another section on this page). However, in contrast with the skeletal conditions, the mutations associated with epidermal nevi (and seborrheic keratosis) are somatic mutations that arise randomly. In epidermal nevi, the mutations occur during the early stages of development before birth and are present only in the cells of the nevus, not in the normal skin cells surrounding it.
Hypochondroplasia
More than 25 mutations in the FGFR3 gene have been identified in people with hypochondroplasia, another form of short-limbed dwarfism that is milder than achondroplasia. Many cases are caused by one of two specific FGFR3 gene mutations, both of which lead to the same change in the FGFR3 protein. Specifically, the amino acid asparagine is replaced with the amino acid lysine at protein position 540 (written as Asn540Lys or N540K). Other FGFR3 gene mutations probably cause a small number of cases of hypochondroplasia. Although the effects of these hypochondroplasia-associated mutations have not been explained, they probably cause the receptor to be mildly overactive, which leads to the disturbances in bone growth that occur in this disorder.
Lacrimo-auriculo-dento-digital syndrome
At least one mutation in the FGFR3 gene has been found to cause lacrimo-auriculo-dento-digital (LADD) syndrome. The main features of LADD syndrome are abnormal tear production, malformed ears with hearing loss, decreased saliva production, small teeth, and hand deformities. The FGFR3 gene mutation that causes LADD syndrome replaces the amino acid aspartic acid with the amino acid asparagine at position 513 in the FGFR3 receptor protein (written as Asp513Asn or D513N). This mutation most likely reduces the ability of the FGFR3 receptor protein to trigger chemical signaling within cells when it is attached to its growth factor. These defects in cell signaling disrupt cell maturation and development, which results in abnormal formation of the ears, skeleton, and glands in the eyes and mouth in people with LADD syndrome.
Muenke syndrome
A single mutation in the FGFR3 gene has been shown to cause Muenke syndrome, which is a condition that causes craniosynostosis, leading to a misshapen head and distinctive facial features. Additional signs and symptoms can include hearing loss, subtle hand and foot abnormalities, and developmental delay. The mutation that causes Muenke syndrome substitutes the amino acid arginine for the amino acid proline at position 250 in the FGFR3 protein (written as Pro250Arg or P250R). This mutation results in the production of a receptor that is overly active, which allows the bones of the skull to fuse sooner than normal.
The Pro250Arg mutation has also been identified in some people with apparently isolated coronal craniosynostosis. This condition is characterized by a premature fusion of the growth line that runs across the top of the head from ear to ear (the coronal suture). People with isolated coronal craniosynostosis do not have the other features that are sometimes associated with Muenke syndrome (such as hand and foot abnormalities).
SADDAN
One mutation in the FGFR3 gene has been identified in people with SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans). SADDAN is characterized by short-limb dwarfism (achondroplasia) profound developmental delay and thick, dark, velvety skin. The genetic change that causes this condition substitutes the amino acid methionine for the amino acid lysine at position 650 of the FGFR3 protein (written as Lys650Met or K650M). Researchers believe that this mutation strongly overactivates the FGFR3 protein, which leads to severe problems with bone growth. It remains uncertain how the mutation causes developmental delay or acanthosis nigricans.
Thanatophoric dysplasia
Mutations in the FGFR3 gene have been identified in people with thanatophoric dysplasia, which is a severe skeletal disorder characterized by extremely short limbs and a narrow chest. More than 10 FGFR3 gene mutations have been found to cause type I thanatophoric dysplasia. Most of these mutations change a single amino acid in the FGFR3 protein. The most common mutation substitutes the amino acid cysteine for the amino acid arginine at protein position 248 (written as Arg248Cys or R248C). Other mutations cause the protein to be longer than normal.
Only one mutation has been shown to cause type II thanatophoric dysplasia. This mutation replaces the amino acid lysine with the amino acid glutamic acid at position 650 of the FGFR3 protein (written as Lys650Glu or K650E). This change affects a different part of the FGFR3 protein than the mutations that cause type I thanatophoric dysplasia.
The genetic changes responsible for both types of thanatophoric dysplasia cause the FGFR3 receptor to be overactive, which leads to the severe problems with bone growth that occur in this condition.
Bladder cancer
Some gene mutations are acquired during a person's lifetime and are present only in certain cells. These changes, which are called somatic mutations, are not inherited. Somatic mutations in the FGFR3 gene have been found in some cases of bladder cancer. Bladder cancer is a disease in which certain cells in the bladder become abnormal and multiply uncontrollably to form a tumor. Bladder cancer may cause blood in the urine, pain during urination, frequent urination, the feeling of needing to urinate without being able to, or lower back pain.
Bladder cancer is generally divided into two types, non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), based on where in the bladder the tumor is located. Approximately 80 percent of NMIBC tumors have FGFR3 gene mutations. FGFR3 gene mutations change single amino acids in the FGFR3 protein, which appears to overactivate protein signaling. As a result, bladder cells are likely directed to grow and divide abnormally. This uncontrolled cell division leads to the formation of bladder cancer.
Multiple myeloma
MedlinePlus Genetics provides information about Multiple myeloma
Other disorders
At least two FGFR3 gene mutations have been found to cause a rare disorder called camptodactyly, tall stature, hearing loss syndrome (CATSHL syndrome). Individuals with this condition are taller than average and typically have hearing loss. They can also have permanently bent fingers or toes (camptodactyly) and other skeletal abnormalities. Researchers suggest that the FGFR3 gene mutations involved in CATSHL syndrome reduce the function of the FGFR3 protein, although it is unclear how the mutations lead to the signs and symptoms of the condition.
Mutations in the FGFR3 gene have been found in 30 to 70 percent of people with seborrheic keratoses, which are small, dark, noncancerous (benign) tumors of the skin caused by overgrowth of skin cells. Seborrheic keratoses develop in adulthood and are seen in a majority of people older than age 50. The FGFR3 gene mutations associated with seborrheic keratoses are somatic mutations and are not inherited. At least nine FGFR3 gene mutations have been identified in people with seborrheic keratoses. These mutations change single amino acids in the FGFR3 protein. The mutated FGFR3 proteins are abnormally active, which results in the overgrowth of skin cells, leading to seborrheic keratosis. It has been suggested that the mutations involved in seborrheic keratosis may be caused by exposure to ultraviolet (UV) light.
The somatic Arg248Cys FGFR3 gene mutation found in epidermal nevus (described above) can also cause Garcia-Hafner-Happle syndrome (also known as fibroblast growth factor receptor 3 epidermal nevus syndrome). This condition is characterized by a soft, velvety keratinocytic epidermal nevus and neurological problems, such as seizures, intellectual disability, underdevelopment of the tissue that connects the two halves of the brain (corpus callosum), and a loss of brain cells (cortical atrophy). It is thought that the neurological problems occur because the somatic mutation affects brain cells in addition to those in the skin.
Other cancers
In addition to bladder cancer, somatic mutations in the FGFR3 gene have been associated with a cancer of white blood cells (multiple myeloma) and cervical cancer. Some cases of multiple myeloma are related to a rearrangement of genetic material (a translocation) involving chromosome 14 and the region of chromosome 4 containing the FGFR3 gene. Mutations that have been associated with cervical cancer are changes in single nucleotides in the FGFR3 gene.
FGFR3 gene mutations that lead to multiple myeloma and cervical cancer are thought to overactivate the FGFR3 protein in certain cells. The mutated receptor directs the cells to grow and divide in the absence of signals from outside the cell. This uncontrolled division can lead to the overgrowth of cancer cells.
Methylation Status of Arabidopsis DNA Repair Gene Promoters During Agrobacterium Infection Reveals Epigenetic Changes in Three Generations
Agrobacterium tumefaciens is a unique pathogen with the ability to transfer a portion of its DNA, the T-DNA, to other organisms. The role of DNA repair genes in Agrobacterium transformation remains controversial. In order to understand if the host DNA repair response and dynamics was specific to bacterial factors such as Vir proteins, T-DNA, and oncogenes, we profiled the expression and promoter methylation of various DNA repair genes. These genes belonged to nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. We infected Arabidopsis plants with different Agrobacterium strains that lacked one or more of the above components so that the influence of the respective factors could be analysed. Our results revealed that the expression and promoter methylation of most DNA repair genes was affected by Agrobacterium, and it was specific to Vir proteins, T-DNA, oncogenes, or the mere presence of bacteria. In order to determine if Agrobacterium induced any transgenerational epigenetic effect on the DNA repair gene promoters, we studied the promoter methylation in two subsequent generations of the infected plants. Promoters of at least three genes, CEN2, RAD51, and LIG4 exhibited transgenerational memory in response to different bacterial factors. We believe that this is the first report of Agrobacterium-induced transgenerational epigenetic memory of DNA repair genes in plants. In addition, we show that Agrobacterium induces short-lived DNA strand breaks in Arabidopsis cells, irrespective of the presence or absence of virulence genes and T-DNA.
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Contents
RNA polymerase is composed of a core and a holoenzyme structure. The core enzymes contains the catalytic properties of RNA polymerase and is made up of ββ′α2ω subunits. This sequence is conserved across all bacterial species. The holoenzyme is composed of a specific component known as the sigma factor. The sigma factor functions in aiding in promoter recognition, correct placement of RNA polymerase, and beginning unwinding at the start site. After the sigma factor performs its required function, it dissociates, while the catalytic portion remains on the DNA and continues transcription. [4] Additionally, RNA polymerase contains a core Mg+ ion that assists the enzyme with its catalytic properties. RNA polymerase works by catalyzing the nucleophilic attack of 3’ OH of RNA to the alpha phosphate of a complementary NTP molecule to create a growing strand of RNA from the template strand of DNA. Furthermore, RNA polymerase also displays exonuclease activities, meaning that if improper base pairing is detected, it can cut out the incorrect bases and replace them with the proper, correct one. [8]
Initiation of transcription requires promoter regions, which are specific nucleotide consensus sequences that tell the σ-factor on RNA polymerase where to bind to the DNA. [1] The promoters are usually located 15 to 19 bases apart and are most commonly found upstream of the genes they control. [2] [1] RNA polymerase is made up of 4 subunits, which include two alphas, a beta, and a beta prime (α, α, β, and β'). A fifth subunit, sigma (called the σ-factor), is only present during initiation and detaches prior to elongation. Each subunit plays a role in the initiation of transcription, and the σ-factor must be present for initiation to occur. When all σ-factor is present, RNA polymerase is in its active form and is referred to as the holoenzyme. When the σ-factor detaches, it is in core polymerase form. [4] [1] The σ-factor recognizes promoter sequences at -35 and -10 regions and transcription begins at the start site (+1). The sequence of the -10 region is TATAAT and the sequence of the -35 region is TTGACA. [1]
- The σ-factor binds to the -35 promoter region. At this point, the holoenzyme is referred to as the closed complex because the DNA is still double stranded (connected by hydrogen bonds). [4]
- Once the σ-factor binds, the remaining subunits of the polymerase attach to the site. The high concentration of adenine-thymine bonds at the -10 region facilitates the unwinding of the DNA. At this point, the holoenzyme is called the open complex. [9] This open complex is also called the transcription bubble. [7] Only one strand of DNA, called the template strand (also called the noncoding strand or nonsense/antisense strand), gets transcribed. [2]
- Transcription begins and short "abortive" nucleotide sequences approximately 10 base pairs long are produced. These short sequences are nonfunctional pieces of RNA that are produced and then released. [1] Generally, this nucleotide sequence consists of about twelve base pairs and aids in contributing to the stability of RNA polymerase so it is able to continue along the strand of DNA. [8]
- The σ-factor is needed to initiate transcription but is not needed to continue transcribing the DNA. The σ-factor dissociates from the core enzyme and elongation proceeds. This signals the end of the initiation phase and the holoenzyme is now in core polymerase form. [4]
The promoter region is a prime regulator of transcription. Promoter regions regulate transcription of all genes within bacteria. As a result of their involvement, the sequence of base pairs within the promoter region is significant the more similar the promoter region is to the consensus sequence, the tighter RNA polymerase will be able to bind. This binding contributes to the stability of elongation stage of transcription and overall results in more efficient functioning. Additionally, RNA polymerase and σ-factors are in limited supply within any given bacterial cell. Consequently, σ-factor binding to the promoter is affected by these limitations. All promoter regions contain sequences that are considered non-consensus and this helps to distribute σ-factors across the entirety of the genome. [10]
During elongation, RNA polymerase slides down the double stranded DNA, unwinding it and transcribing (copying) its nucleotide sequence into newly synthesized RNA. The movement of the RNA-DNA complex is essential for the catalytic mechanism of RNA polymerase. Additionally, RNA polymerase increases the overall stability of this process by acting as a link between the RNA and DNA strands. [11] New nucleotides that are complementary to the DNA template strand are added to the 3' end of the RNA strand. [4] The newly formed RNA strand is practically identical to the DNA coding strand (sense strand or non-template strand), except it has uracil substituting thymine, and a ribose sugar backbone instead of a deoxyribose sugar backbone. Because nucleoside triphosphates (NTPs) need to attach to the OH- molecule on the 3' end of the RNA, transcription always occurs in the 5' to 3' direction. The four NTPs are adenosine-5'-triphosphate (ATP), guanoside-5'-triphosphate (GTP), uridine-5'-triphosphate (UTP), and cytidine-5'-triphosphate (CTP). [9] The attachment of NTPs onto the 3' end of the RNA transcript provides the energy required for this synthesis. [2] NTPs are also energy producing molecules that provide the fuel that drives chemical reactions in the cell. [4]
Multiple RNA polymerases can be active at once, meaning many strands of mRNA can be produced very quickly. [2] RNA polymerase moves down the DNA rapidly at approximately 40 bases per second. Due to the quick nature of this process, DNA is continually unwound ahead of RNA polymerase and then rewound once RNA polymerase moves along further. [11] [1] The polymerase has a proofreading mechanism that limits mistakes to about 1 in 10,000 nucleotides transcribed. [12] RNA polymerase has lower fidelity (accuracy) and speed than DNA polymerase. [2] DNA polymerase has a very different proofreading mechanism that includes exonuclease activity, which contributes to the higher fidelity. The consequence of an error during RNA synthesis is usually harmless, where as an error in DNA synthesis could be detrimental. [2]
The promoter sequence determines the frequency of transcription of its corresponding gene. [1]
In order for proper gene expression to occur, transcription must stop at specific sites. Two termination mechanisms are well known:
- Intrinsic termination (also called Rho-independent termination): Specific DNA nucleotide sequences signal the RNA polymerase to stop. The sequence is commonly a palindromic sequence that causes the strand to loop which stalls the RNA polymerase. [9] Generally, this type of termination follows the same standard procedure. A pause will occur due to a polyuridine sequence that allows the formation of a hairpin loop. This hairpin loop will aid in forming a trapped complex, which will ultimately cause the dissociation of RNA polymerase from the template DNA strand and halt transcription. [8]
- Rho-dependent termination: ρ factor (rho factor) is a terminator protein that attaches to the RNA strand and follows behind the polymerase during elongation. [5] Once the polymerase nears the end of the gene it is transcribing, it encounters a series of G nucleotides which causes it to stall. [1] This stalling allows the rho factor to catch up to the RNA polymerase. The rho protein then pulls the RNA transcript from the DNA template and the newly synthesized mRNA is released, ending transcription. [5][1] Rho factor is a protein complex that also displays helicase activities (is able to unwind the nucleic acid strands). It will bind to the DNA in cytosine rich regions and when RNA polymerase encounters it, a trapped complex will form causing the dissociation of all molecules involved and end transcription. [8]
The termination of DNA transcription in bacteria may be stopped by certain mechanisms wherein the RNA polymerase will ignore the terminator sequence until the next one is reached. This phenomenon is known as antitermination and is utilized by certain bacteriophages. [13]
Prime editing enables precise gene editing without collateral damage
The latest gene editing technology, prime editing, expands the "genetic toolbox" for more precisely creating disease models and correcting genetic problems, scientists say.
In only the second published study of prime editing's use in a mouse model, Medical College of Georgia scientists report prime editing and traditional CRISPR both successfully shut down a gene involved in the differentiation of smooth muscle cells, which help give strength and movement to organs and blood vessels.
However, prime editing snips only a single strand of the double-stranded DNA. CRISPR makes double-strand cuts, which can be lethal to cells, and produces unintended edits at both the work site as well as randomly across the genome, says Dr. Joseph Miano, genome editor, molecular biologist and J. Harold Harrison, MD, Distinguished University Chair in Vascular Biology at the MCG Vascular Biology Center.
"It's actually less complicated and more precise than traditional CRISPR," Miano says of prime editing, which literally has fewer components than the game-changing gene-editing tool CRISPR.
Miano was among the first wave of scientists to use CRISPR to alter the mouse genome in 2013. Two scientists were awarded the 2020 Nobel Prize in Chemistry for the now 9-year-old CRISPR, which enabled rapid development of animal models, as well as the potential to cure genetic diseases like sickle cell, and potentially reduce the destruction caused by diseases like cancer, in which environmental and genetic factors are both at play.
Prime editing is the latest gene-editing technology, and the MCG scientists report in the journal Genome Biology that they were able to use it to remove expression of a gene in smooth muscle tissue, illustrating prime editing's ability to create cell-specific knockout mice without extensive breeding efforts that may not result in an exact model, says Dr. Xiaochun Long, molecular biologist in the Vascular Biology Center. Miano and Long are corresponding authors of the new study.
Long, Miano and their colleagues did a comparative study using traditional CRISPR and prime editing in the gene Tspan2, or tetraspan-2, a protein found on the surface of cells. Long had earlier found Tspan2 was the most prominent protein in smooth muscle cell differentiation and was likely mutated in cardiovascular disease. She also had identified the regulatory region of this gene in cultured cells. However, it was unclear whether this regulatory region was important in mice.
They used CRISPR to create a subtle change in a snippet of DNA within the promoter region of Tspan2, in this case a three-base change, their standard approach to inactivating control regions of genes. DNA has four base pairs -- adenine, cytosine, guanine and thymine -- which pair up in endless different combinations to make us, and which gene-editing tools alter.
CRISPR created a double-strand break in the DNA and following the three-base change, the Tspan2 gene was no longer turned on in the aorta and bladder of mice.
They then used prime editing to make a single-strand break, or nick, and a single-base change -- like most of the gene mutations that occur in our body -- and found this subtle change also turned the Tspan2 gene off in the aorta and bladder, but without the collateral damage of CRISPR.
"We were trying to model what could happen with a single nucleotide change," says Miano. "We asked the question if we incorporate a single-base substitution, if we just make one base change, what happens to Tspan2 expression? The answer is it did the same thing as the traditional CRISPR editing: It killed the gene's expression."
But there were also important differences. Using CRISPR, they found evidence of significant "indels," short for insertions or deletions of bases in genes, which were unintended, both near the site where the intended edit was made and elsewhere.
The published paper includes a chart with numerous black bars illustrating where multiple nucleotides, the building blocks of DNA and RNA, are gone after using CRISPR. Indels are those unintended changes that genome editors strive to avoid because they can create deficits in gene expression and possible disease. With off-targeting, you could end up substituting one disease for another, Miano says.
But with prime editing, they saw essentially no indels either at the Tspan2 promoter region or elsewhere.
A Manhattan plot illustrated the off-targeting across all chromosomes using both techniques, with the CRISPR skyline stacking up like a real city while the prime editing skyline is comparatively flat.
"Prime editing is a less intrusive cut of the DNA. It's very clean," Miano says. "This is what we want: No detectable indels, no collateral damage. The bottom line is that unintended consequences are much less and it's actually less complicated to use."
Traditional CRISPR has three components, the molecular scissors, Cas9, the guide RNA that takes those scissors to the precise location on DNA and a repair template to fix the problem. Traditional CRISPR cuts both strands of the DNA, which also can happen in nature, can be catastrophic to the cell and must be quickly mended.
Prime editing has two arms, with a modified Cas9, called a Cas9 nickase, that will only make a single-strand cut. The scissors form a complex called the "prime editor" with a reverse transcriptase, an enzyme that can use an RNA template to produce a piece of DNA to replace the problematic piece in the case of a disease-causing mutation. PegRNA, or prime editing guide RNA, provides that RNA template, gets the prime editor where it needs to work and helps stabilize the DNA strands, which are used to being part of a couple.
During the repair of the nicked strand of targeted DNA, the prime editor "copies" a portion of the pegRNA containing the programmed edit, in this case a single-base substitution, so that the repaired strand will now carry the single base edit. In the case of creating a disease model, that enables scientists to "bias" the repair so the desired mutation is created, Miano says.
Dr. David Liu, chemical biologist, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at Harvard University and the Massachusetts Institute of Technology, and his colleagues developed the first major gene editing technology to follow CRISPR. They reported on base editing technology in 2016, which uses "base editors" Liu described as "pencils, capable of directly rewriting one DNA letter into another by actually rearranging the atoms of one DNA base to instead become a different base." Liu and his postdoctoral fellow Dr. Andrew Anzalone, first reported on prime editing in the journal Nature in October 2019. Liu is a coauthor on the newly published study in Genome Biology on prime editing in mice.
Liu's original work on prime editing was done in culture, and others have shown its efficacy in plants. This is more proof of principle, Miano says.
The MCG scientists hope more of their colleagues will start using prime editing in their favorite genes to build experience and hasten movement toward its use in humans.
Their long-term goals including using safe, specific gene editing to correct genetic abnormalities during human development that are known to result in devastating malformations and disease like heart defects that require multiple major surgeries to correct.
Allison Yang, senior research assistant in the Miano lab, is preparing to use prime editing to do an in utero correction of the rare and lethal megacystis-microcolon-intestinal hypoperistalsis syndrome, which affects muscles of the bladder and intestines so you have difficulty moving food through the GI tract and emptying the bladder. In early work with CRISPR on vascular smooth muscle cells, Miano and colleagues inadvertently created a near-perfect mouse model of this human disease that can kill babies.
Collaborators on the new study include scientists from Albany Medical College, St. Jude Children's Research Hospital, Cornell University, Synthego, and Harvard University. The research was supported by the National Institutes of Health.
Changes in just one DNA building block, or nucleotide, called single nucleotide polymorphisms, or SNPs, are the most common type of genetic variation in people, according to MedlinePlus, and each person has millions in their genome. A tiny proportion of SNPs, like the one that causes sickle cell disease, occur in parts of the DNA that produce proteins, which determine cell function. However, the vast majority of SNPs, such as the artificial one generated with prime editing here, occur in the human genome where no protein-coding genes are found. This noncoding portion of the genome, the so called 'dark matter,' comprises 99% of our entire DNA blueprint of life. Noncoding parts of the genome include regulatory elements like the one controlling Tspan2 expression.
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How does the MET gene work and what happens when the promoter region gets mutated? - Biology
This page takes a very brief look at what happens if the code in DNA becomes changed in some way, and the effect that would have on the proteins it codes for. It is designed for 16 - 18 year old chemistry students. In fact, most chemistry students won't need this - check your syllabus and past papers before you go on.
Note: If you have come straight to this page from a search engine, you should be aware that this is the final page in a sequence of pages about DNA, RNA and protein synthesis, starting with the structure of DNA.
Random changes to the genetic code
Copying errors when DNA replicates or is transcribed into RNA can cause changes in the sequence of bases which makes up the genetic code. Radiation and some chemicals can also cause changes. The examples which follow show some of the easier-to-understand effects of this.
Changes to individual bases
Remember that a set of three bases in a gene in DNA codes for a particular amino acid. If you have followed this sequence of pages from the beginning, you will have come across this table showing the codons in DNA:
A gene will be made up of a string of these codes rather like a string of 3-letter words in a sentence. We'll use that as a simple analogy. Take the sentence:
the big fox bit the dog but not the boy
Suppose one letter got changed in this by accident. Suppose, for example, the "d" in dog got replaced by a "p". The sentence would now read:
the big fox bit the pog but not the boy
Clearly this doesn't make complete sense any more. Would that matter if the same thing happened in a gene? It depends!
If you look back at the table, there are several amino acids which are coded for by more than one base combination. For example, glycine (Gly) is coded for by GGT, GGC, GGA and GGG. It doesn't matter what the last base is - you would get glycine whatever base followed the initial GG.
That means that a mutation at the end of a codon like this wouldn't make any difference to the protein chain which would eventually form. These are known as silent mutations.
Alternatively, of course, you could well get a code for a different amino acid or even a stop codon.
If a stop codon was produced in the middle of the gene, then the protein formed would be too short, and almost certainly wouldn't function properly.
If a different amino acid was produced, how much it mattered would depend on whereabouts it was in the protein chain. If it was near the active site of an enzyme, for example, it might stop the enzyme from working entirely.
On the other hand, if it was on the outside of an enzyme, and didn't affect the way the protein chain folded, it might not matter at all.
Inserting or deleting bases
The situation is more dramatic if extra bases are inserted into the code, or some bases are deleted from the code. Using our example sentence from above, and keeping the three letter word structure:
If you insert a single extra base:
the big fro xbi tth edo gbu tno tth ebo y
An extra "r" is inserted in "fox". If the sentence still has to be read three letters at a time (as in DNA), everything from then on becomes completely meaningless.
If you delete a single base:
the big fxb itt hed ogb utn ott heb oy
This time the "o" in "fox" has been deleted. And again, because we have to read the letters in groups of three, the rest of the sentence becomes completely wrecked.
So does this matter? Well, of course it does! Large chunks of the protein will consist of completely wrong amino acid residues.
We've looked so far at inserting or deleting one base. What if you do it for more than one?
The effect is the same unless you add or delete multiples of three bases - without changing any other codons. If you added an extra three bases between two existing codons, then essentially you are just adding an extra word.
the big fox bit the xjy dog but not the boy
That extra word represents an extra codon in the DNA, and so an extra amino acid residue in the protein chain. Does this matter? It depends where it is in the chain (Is it important to the active site of an enzyme, for example?), and whether it affects the folding of the chain.
What if the three bases were inserted so that they broke up an existing codon? Here is the same extra "word", "xjy", dropped in the word "bit". Everything is then reshuffled into groups of three letters.
the big fox bxj yit the dog but not the boy
You can see that the effect is again fairly limited. It will change one codon completely, and introduce an extra codon. That would give you one different amino acid and one extra amino acid in the chain. Again, how much that would affect the final protein depends on where it happens in the chain.
Deleting a whole codon again leaves most of the protein chain unchanged. Again, whether the function of the protein is affected depends on where the missing amino acid should have been and how critical it was to the way the protein folded.
Important: The material on remainder of this page was written for a specific UK syllabus which no longer requires it. I have left it here in case it is useful to anyone else. Read it out of interest if you want to, but check whatever syllabus you are doing before you waste any time learning it.
Some diseases caused by mutation
The following examples illustrate some of the changes we've looked at above and how they can result in disease.
Cystic fibrosis is an inherited disease which affects the lungs and digestive system. It results from mutation in a gene responsible for making a protein which is involved in the transport of ions across cell boundaries.
The effect is to produce a sticky mucus which clogs the lungs and can lead to serious infection. A similar sticky mucus also blocks the pancreas (a part of the digestive system) which provides enzymes for breaking down food. This gets in the way of the processes which convert the food into molecules which can be absorbed by the body.
There are lots of different mutations which can cause this, but we'll just have a quick look at the one which accounts for about 70% of cystic fibrosis cases.
The base sequence in the part of the gene affected ought to look like this:
The phenylalanine (Phe) in red is the amino acid which is missing from the final protein in many sufferers from cystic fibrosis. However, it isn't quite as simple as just losing the TTT codon.
Instead, the three bases lost are:
Notice that the amino acid sequence is identical to before but without the phenylalanine. How did that happen when we didn't actually remove the whole of the phenylalanine codon?
If you look carefully, you will see that the codon for the second isoleucine (Ile) is different from before. It so happens that isoleucine is coded for by both ATC and ATT. Once the second T (the red one) has joined the existing AT, all the rest of the base sequence is exactly what it was before.
Sickle cell anaemia (US: anemia)
This is so called because red blood cells change their shape from the normal flexible doughnut shape to a much more rigid sickle shape - rather like a crescent moon.
It results from the change of a single base in a gene responsible for making one of the protein chains which makes up haemoglobin (US: hemoglobin).
The affected part of the gene should read:
What it actually reads in someone suffering from sickle cell anaemia is:
The effect of this single change is to make the haemoglobin temporarily polymerise to make fibres after it has released the oxygen that it carries around the body. This changes the shape of the red blood cells so that they don't flow so easily - it makes them sticky, especially in small blood vessels. This can cause pain and lead to organ damage.
Haemophilia (US: hemophilia)
Sufferers from haemophilia lack a protein in the blood which allows it to clot. That means that if someone with haemophilia cuts themselves, the wound will just continue to bleed.
There are all sorts of mutations which cause haemophilia. One which is easy to understand is caused by changing a single base at the beginning of a codon for argenine (CGA) somewhere in the gene to give TGA. If you look back to the table higher up the page, you will find that TGA is a stop codon.
All that will be produced is a useless fragment of the intended protein.
Questions to test your understanding
If this is the first set of questions you have done, please read the introductory page before you start. You will need to use the BACK BUTTON on your browser to come back here afterwards.
Health Conditions Related to Genetic Changes
Huntington disease
The inherited mutation that causes Huntington disease is known as a CAG trinucleotide repeat expansion. This mutation increases the size of the CAG segment in the HTT gene. People with Huntington disease have 36 to more than 120 CAG repeats. People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with 40 or more repeats almost always develop the disorder.
The expanded CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. It has also been suggested that loss of the huntingtin protein's DNA repair function may result in the accumulation of DNA damage in neurons, particularly as damaging molecules increase during aging. Regions of the brain that help coordinate movement and control thinking and emotions (the striatum and cerebral cortex) are particularly affected. The dysfunction and eventual death of neurons in these areas of the brain underlie the signs and symptoms of Huntington disease.
As the altered HTT gene is passed from one generation to the next, the size of the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. This phenomenon is called anticipation. People with the adult-onset form of Huntington disease (which appears in mid-adulthood) typically have 40 to 50 CAG repeats in the HTT gene, while people with the less common, juvenile form of the disorder (which appears in childhood or adolescence) tend to have more than 60 CAG repeats.
Individuals who have 27 to 35 CAG repeats in the HTT gene do not develop Huntington disease, but they are at risk of having children who will develop the disorder. As the gene is passed from parent to child, the size of the CAG trinucleotide repeat may lengthen into the range associated with Huntington disease (36 repeats or more).