Term
| Gene expression of eukaryotes differs from that in prokaryotes in 4 significant ways: |
|
Definition
o Highly ordered chromatin DNA in eukaryotes are less accessible for transcription due to histones where as prokaryotes’ DNA are not bound by proteins and are more accessible
o Prokaryotes have a single pol for transcription where as eukaryotes have multiple pol’s
o Initial eukaryotic mRNA must undergo modification before translation
o Transcription and translation are coupled in prokaryotes; in eukaryotes, they are separated as RNA must leave the nucleus for translation |
|
|
Term
o Why are prokaryotes and eukaryotes different?
Note: These differences suggest there are strong reasons why prokaryotes and eukaryotes must regulate their gene repertoire differently |
|
Definition
o The primary function of gene regulation in prokaryotes is to adjust their enzymatic machinery to the immediate nutritional and physical environment to allow for growth and division. Therefore, it is constantly adjusting its genetic repertoire in reaction to environment
o Multicellular eukaryotes are protected from immediate outside influences and experience a constant environment. Therefore, genes devoted to response of environmental change aren’t very many. Genes involved in the execution of precise developmental decisions characterize the majority of gene control |
|
|
Term
| Three points of info flow make up the underlying strategy of bacterial gene control: |
|
Definition
o Initiation of transcription (lac, trp, sigma factor)
o Termination of transcription (trp, rho-dependant)
o Stability of mRNA (lac) |
|
|
Term
| A segment of DNA containing a set of contiguous genes coding for enzymes or other proteins that are closely related in metabolic function |
|
Definition
|
|
Term
| Segments of DNA involved in regulating transcription of operon |
|
Definition
|
|
Term
Most control elements are linked directly to genes (promoters)
• However, codes for a protein which participates in regulation of transcription of other genes of the operon. Regulatory Gene may be separated from the other components of the operon by a region of unrelated DNA (still operon) |
|
Definition
|
|
Term
| Structural genes of operons are transcribed sequentially to produce a ??? molecule containing coding sequences for all of the proteins of the operon. Called coordinate expression. |
|
Definition
|
|
Term
| True or False: Coordinate regulation = Regulation of transcription of this operon controls expression of ALL of the genes |
|
Definition
|
|
Term
| True or False: All genes on the operon genome are not expressed simultaneously. Primarily, environmental stimuli determine what promoters (and thus which operons) are available at any given time (e.g., nutrients or lack of, procreation, survival) |
|
Definition
|
|
Term
The promoter for an operon encoding enzymes to metabolize an environmental nutrient becomes available only when that nutrient is present.
This response is called enzyme induction.
Default state of ??? is off.
Enzymes synthesized in response to their substrates are said to be induced.
What type of operon has these characteristics? |
|
Definition
|
|
Term
A common example of catabolic operon involves enzymes that convert complex sugars into glucose.
• The gal operon codes for enzymes that convert galactose into glucose, the lac operon codes for enzymes converting lactose into glucose, and the ara operon codes for enzymes converting arabinose into a glycolytic intermediate. When any of these molecules are added to the environment, the corresponding operon is made available, and synthesis of these enzymes increases.
Molecules such as lactose, galactose, or arabinose are inducers. |
|
Definition
|
|
Term
The lack of a nutrient in the environment can also determine what promoters (and operons) are available.
The promoter for an operon encoding enzymes to synthesize an essential nutrient becomes unavailable when that nutrient is present in the environment.
In this case, the operon is said to be repressed.
Default state of operon is on.
Essential elements like amino acids, nucleotides, and cofactors fall into this category, and are called corepressors.
What type of operon is this? |
|
Definition
|
|
Term
| Region to which RNA Pol binds to initiate transcription on both catabolic & anabolic operons |
|
Definition
|
|
Term
Binding site for repressor protein, which when bound blocks transcription by RNA Pol.
o Located between promoter & 1st transcribed base |
|
Definition
|
|
Term
- Codes for repressor protein mRNA
o This genes has its own promoter |
|
Definition
|
|
Term
The ??? binding site is the site at which cAMP-bound CRP binds & stimulates initiation of transcription by RNA Pol
o Found on catabolic operons only |
|
Definition
|
|
Term
o Found in some anabolic operons coding for enzymes to synthesize amino acids
o It is normally transcribed & translated
o Under some conditions, ribosome binding to initiate translation at the newly transcribed attenuator mRNA leads to termination of further transcription before the polymerase gets to the first gene |
|
Definition
|
|
Term
| Codes for a permease, which is a protein that occurs in the cell membrane and participates in the transport of sugars, including lactose, across the membrane |
|
Definition
|
|
Term
True or False:
o Induction of the three genes occurs during transcription initiation. Transcription occurs at a lower level w/o the inducer, lactose
o Lactose transcript is very unstable. Transcription ceases as soon as the inducer is no longer present, mRNA is degraded, protein disappears |
|
Definition
|
|
Term
The regulatory gene for the lac operon, codes for the repressor protein
Located just upstream of the other controlling elements for the LacZYA gene cluster. It is not necessary for regulator to be physically close |
|
Definition
|
|
Term
| True or False: Transcription of lacI is not regulated. It is always transcribed from its own promoter at a low rate that is relatively independent of the cell’s status |
|
Definition
|
|
Term
- is a symmetrical tetrameric protein consisting of 4 identical monomeric subunits
- has a strong affinity for the operator, LacO
- has a strong affinity for the inducer molecule of this operon (lactose) |
|
Definition
|
|
Term
o Repressor also has a strong affinity for the inducer molecule of this operon (lactose)
Each repressor monomer has a binding site for an inducer molecule
The binding of inducer to the monomeric subunits of the repressor causes an allosteric change in the repressor that greatly lowers its affinity for the operator
The inducer is a negative allosteric modifier of the repressor, resulting in the repressor no longer binding to the operator, allowing RNA pol to bind the promoter and initiate transcription
The inducer can bind to a repressor molecule already bound to the operator. This causes a conformational change in the operator site and the repressor falls off
o Operator overlaps the promoter partially, so that a bound repressor physically blocks RNA pol from binding to the promoter
o Operator sequence has an axis of dyad symmetry (palindrome)
The symmetry in DNA recognition sequence reflects symmetry in tetrameric repressor |
|
Definition
|
|
Term
o Immediately upstream of the lac operator
o Contains the recognition site for RNA pol and CAP-binding protein
o The sequence of the RNA pol binding site is not composed of symmetrical elements (ie. not palindromic) as it must be able to initiate RNA synthesis in a specified direction from the binding site
o The CAP-binding site does contain some symmetry |
|
Definition
|
|
Term
o Catabolic Repression
oGlucose and cAMP
E.coli prefers to use glucose instead of other sugars as a carbon source.
If the concentrations of glucose and lactose in the environment are the same, bacteria will selectively metabolize glucose and not use lactose until the glucose is gone.
Thus, glucose interferes with the induction of the lac operon.
This effect is called catabolite repression (as it occurs during the catabolism of glucose, and may be due to a catabolite of glucose rather than glucose itself).
This phenomenon is common to a number of inducible operons that metabolize various substances as energy sources. It is probably a general coordinating switch for turning off unwanted genes when the preferred substrate is around. |
|
Definition
|
|
Term
Catabolic Repression
o cAMP binds to the regulatory protein, CAP (CRP)
CAP is an allosteric protein and, when combined with cAMP, is capable of binding to the CAP regulatory site (CBS) in the promoter of the lac (and other) operon.
cAMP is a positive allosteric modifier of CAP. The CAP cAMP complex exerts positive control on the transcription of these operons. Its binding to the CAP site facilitates the binding of RNA polymerase to the promoter. Transcription is enhanced.
If the CAP site is not occupied, RNA polymerase has more difficulty binding to the promoter, and transcription of the operon occurs much less efficiently.
When glucose is present, the cAMP levels drop, the CAP cAMP complex does not form, and the positive influence on RNA polymerase does not occur.
The CRP protein has a helix-turn-helix motif
• Binds to the palindromic sequence
In presence of glucose ---> inhibition
In presence of glu & lactose ---> inhibition
In presence of lactose --> activation
Absence of glu ---> presence of cAMP
Presence of cAMP ---> allosteric activation |
|
Definition
|
|
Term
o Problems and the Lac operon
o Problem: if the lac operon is totally shut off in the absence of lactose, how does the cell get lactose across its membrane with the permease?
o Solution: Even in what would be considered “fully repressed” state there is still a basal level of transcription of the lac operon. This provides 5-6 molecules of permease per cell, which is enough to get a few molecules of lactose inside the cell. |
|
Definition
|
|
Term
o Gene regulation – TRP
o Tryptophan is essential for synthesis of many E.coli proteins and must be made b/c not enough in environment. It is synthesized from chorismic acid by three different enzymes
o The trp operon contains five genes each of which code for subunits making up the three enzymes
o Upstream is a promoter where transcription begins, and an operator which binds to a repressor
o The trp operon is always turned on unless tryptophan is in the environment
o When the trp operon is being actively transcribed, it is said to be derepressed, that is the trp repressor is not preventing RNA polymerase from binding
o The repressor, by itself, does not bind to the trp operator. It must be complexed with tryptophan in order to bind to the operator.
o Repressor of trp operon is a dimer
o Tryptophan is a positive allosteric modifier of the trp corepressor |
|
Definition
|
|
Term
o Attenuation (region of multiple, identical aa, ie. trp-trp)
o The trp operon is regulated by attenuation
o The leader peptide coded by the attenuator sequence contains 14 amino acid residues with two adjacent tryptophan residues at positions 10 and 11. This is unusual as tryptophan is a relatively rare amino acid in E.coli.
o This is a critical feature of the attenuation mechanism. If the tryptophan in the cell is low, the amount of charged trp tRNAtrp will also be low. Ribosomes translating the leader mRNA sequence will be forced to stop and wait, or stall, when they reach these two trp codons.
o The attenuator region of the mRNA can form some secondary structure. Further, this region can adopt several different stem loop structures.
o The position of the ribosome along the nascent mRNA chain determines which stem loop will form.
o This secondary structure, in turn, is recognized, or sensed by the RNA polymerase that has now transcribed through the attenuator region and is a small distance downstream.
o High Tryptophan
The stem loop formed (3-4) when the ribosomes is not stalled at the trp codons is a termination signal for the RNA polymerase.
Transcription stops after the 140 nucleotide leader, before reaching the operon genes.
o Low Tryptophan
If the ribosomes are stalled at the trp codons, the stem loop formed (2-3) is not recognized as a termination signal, and the RNA polymerase continues to transcribe the operon genes |
|
Definition
|
|
Term
| o Eukaryotes regulate gene expression through 5 major mechanisms: |
|
Definition
o Transcription initiation
o Transcription termination
o Processing/transport of mRNA, tRNA
o Message stabilization
o Translation |
|
|
Term
o While prokaryotes used signals from within the cell (the presence or absence of certain molecules), eukaryotes are not as directly in touch with the changes to the organism’s environment, so the signals for gene regulation come from outside the cell in several forms:
o Fat-Soluble (Steroid) hormones – directed to nucleus (by receptors) and act directly on transcription
o Peptide hormones – intracellular signal transduction cascade
o Non-hormonal molecules (NO gas -> signal cascade, others that have autocrine or paracrine effect)
o Direct contact signals from surface receptors on adjoining cells, as happens in embryonic induction
o Environmental/nutritional factors (heat, light, nutrients) |
|
Definition
|
|
Term
Transcription and Gene Control through Promoters
Genetic Switch
Short DNA sequences form one component that
regulate protein transcription
UAS - contains palindromic binding site
-TATA box
50% of genes
About 30 bp upstream of gene
Where RNA pol binds to (if present)
TFIID loads RNA pol II Holoenzyme complex onto TATA box
-Promoter Region (proximal region) – about 300 bp upstream
Regulate on expressor
Examples: CCAAT, GGGCG
-Enhancer (distal region) – can lie 100 kb upstream
Can be upstream, downstream, in introns, palindromic
-Activators and cofactors act on these regions
With out these, there is a low level of transcription (=base level)
Adding these results in highly activated transcription
Possibility of different amounts and types leads to a complexity of ways to regulate |
|
Definition
|
|
Term
Regulatory Protein Structure and DNA Interaction
3-D Structure
o Regulatory proteins are able to recognize target DNA sites b/c protein is complimentary
o Binds weakly to surface features (major or minor grooves) of the wound helix.
o Binding involves hydrogen bonds, ionic bonds, and hydrophobic interactions
o Each contact is weak, but the combined total makes interaction tight and specific
o Proteins contain a small set DNA binding structural motifs (ie. helix-turn-helix) as well as protein binding motifs (protein-protein binding)
o Types of structures
o Helix-turn-Helix
o Zinc Finger
o Leucine Zipper – Amphipathic, dimeric
o Helix-Loop-Helix – Amphipathic, dimeric |
|
Definition
|
|
Term
| Tryptophan is essential for synthesis of many E.coli proteins. Therefore, if it is not supplied in sufficient quantity in the environment, the cell must make it. In E.coli, tryptophan is synthesized from chorismic acid in a five step pathway catalyzed by three different enzymes (Figure 12) The trp operon contains five genes each of which code for subunits making up the three enzymes. Upstream from this gene cluster is a promoter where transcription begins, and an operator to which binds a repressor protein that is coded for by the trpR gene. The trpR gene is located some distance away from the rest of the operon. |
|
Definition
|
|
Term
The trp operon is ALWAYS turned on unless tryptophan is in the environment Figure 13, in which case it acts as the corepressor (use this word instead of repressor to distinguish between the molecule, tryptophan, and the product of the trpR gene). When the trp operon is being actively transcribed, it is said to be derepressed, that is the trp repressor is not preventing RNA polymerase from binding. The trp repressor is a tetramer of four identical subunits. Under normal conditions, about 20 molecules of the repressor tetramer are present per cell. The repressor, by itself, does NOT bind to the trp operator. It must be complexed with tryptophan in order to bind to the operator. Tryptophan is a positive allosteric modifier of the trp corepressor
Attenuation.
The attenuator lies within the 162 nucleotides between the start of transcription at trpP, and the initiator codon of the trpE gene. |
|
Definition
|
|
Term
The eucaryotic RNA polymerases are very inefficient at binding to their promoter. Initiation of transcription is dependent on the action of multiple activator proteins.
Nonspecific DNA binding becomes a problem in the much larger genomes of higher eucaryotes. The chance of a single specific binding domain occuring randomly at an inappropriate site increases with genome size. Specificity for transcriptional activation is improved if each of several positive–regulatory proteins must bind specific DNA domains and form a complex in order to make that gene active. The average number of regulatory sites for a gene in a multicellular organism appears to be at least 5. This vastly reduces the probability of the random occurrence of a functional juxtaposition of all the necessary sites to activate a DNA region.
This could be true for negative regulation except that positive regulation is just plain more efficient. To maintain negative regulation of all the genes in a eucaryote, that organism would have to continuously make a lot of negative regulating proteins that keep the genes off. Under positive regulation, the default state for a gene is off, and you only need make a positive regulator for the subset of genes you want on. This is energetically more efficient. |
|
Definition
|
|
Term
| CTCF: CCCTC-binding factor: Is a member of the BORIS + CTCF gene family and encodes a transcriptional regulator protein with 11 highly conserved zinc finger (ZF) domains. This nuclear protein is able to use different combinations of the ZF domains to bind different DNA target sequences and proteins. Depending upon the context of the site, the protein can bind a histone acetyltransferase (HAT)-containing complex and function as a transcriptional activator or bind a histone deacetylase (HDAC)-containing complex and function as a transcriptional repressor. If the protein is bound to a transcriptional insulator element, it can block communication between enhancers and upstream promoters, thereby regulating imprinted expression. Mutations in this gene have been associated with invasive breast cancers, prostate cancers, and Wilms' tumors. |
|
Definition
|
|
Term
| A genetic boundary element that plays two distinct roles in gene expression, either as an enhancer-blocking element, or more rarely as a barrier against condensed chromatin proteins spreading onto active chromatin. The need for them arises where two adjacent genes on a chromosome have very different transcription patterns, and it is critical that the inducing or repressing mechanisms of one do not interfere with the neighboring gene |
|
Definition
|
|
Term
| A DNA sequence capable of binding transcription regulation factors termed repressors. Upon binding, RNA polymerase is prevented from initiating transcription thus decreasing or fully suppressing RNA synthesis. |
|
Definition
|
|
Term
| A short region of DNA that can bind proteins called an activator (genetics), binding of activators to this region can initiate the transcription of a gene that may be some distance away, or can even be on a different chromosome. The increase in transcription is due to the activators recruiting transcription factors, which enhances the binding of RNA polymerase. |
|
Definition
|
|
Term
| A DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription. |
|
Definition
|
|
Term
| Repressor proteins are coded for by regulator genes. Repressor proteins then attach to a DNA segment known as the operator. By binding to the operator, the repressor protein prevents the RNA polymerase from creating messenger RNA. |
|
Definition
|
|
Term
If an inducer, a molecule that initiates the gene expression, is present, then it can interact with the repressor protein and detach it from the operator. RNA polymerase then can transcribe the message (expressing the gene). A corepressor is a molecule that can bind to repressor and make it binds to operator tightly, which decreases transcription.
The above mechanism of repression is a type of a feedback mechanism because it only allows transcription to occur if a certain condition is present: the presence of specific inducer(s). |
|
Definition
|
|
Term
| A response element is a short sequence of DNA within the promoter of a gene that is able to bind a specific hormone receptor complex and therefore regulate transcription |
|
Definition
|
|
Term
Three a-helicies where helix 2 and 3 are in a “helix-turn-helix” motif.
Two a-helices connected by a short extended chain of aa constituting the turn. |
|
Definition
|
|
Term
Insulin-like growth factor 2 receptor (Igf2r) that is inherited from the father synthesizes an antisense RNA that appears to block synthesis of the mRNA for Igf2r. An inherited difference in the expression of a gene depending on whether it is inherited from the mother or the father is called genomic or parental imprinting.
In the mother's (maternal) copy of the gene, there is an upstream (left) promoter that is unmethylated and active binding of transcription factors to this upstream promoter enables transcription of the sense strand of the gene to produce Igf2r messenger RNA. There is also a downstream set of CpG islands that are methylated
In the father's (paternal) copy of the IGF2r gene (the imprinted version) the promoter for IGF2r transcription is methylated (and inactive), but the downstream promoter is unmethylated and active. Transcription of the antisense strand from the downstream promoter produces an antisense RNA that may participate in shutting his gene down. |
|
Definition
|
|
Term
Accidental (in humans) inheritance of two copies of a particular chromosome from one parent and none from the other parent is usually fatal (even though a complete genome is present).
Inheritance of two copies of one of mother's genes and no copy of the father's (or vice versa) can produce serious developmental defects.
Failure of imprinting in somatic cells may lead to cancer. |
|
Definition
|
|
Term
siRNA genes are transcribed into ~70bp RNA precursors that contain inverted repeats that permit ds hairpin RNA formation. The cell has a specific enzyme (in Drosophila it is called Dicer) that recognizes the double stranded RNA and chops it up into small fragments between 21-25 base pairs in length. These short RNA fragments (called small interfering RNA, or siRNA) bind to the RNA-induced silencing complex (RISC). The RISC is activated
when the siRNA unwinds and the activated complex binds to the corresponding mRNA using the antisense RNA. The RISC contains an enzyme to cleave the bound mRNA (called Slicer in Drosophila) and therefore cause gene suppression. Once the mRNA has been cleaved, it can no longer be translated into functional protein.
Human cells can physiologically silence HIV-1 RNA —
a natural example of RNA silencing in mammalian
host–virus interactions. |
|
Definition
|
|
Term
miRNAs are implicated in a wide range of functions such as cell growth and apoptosis, development, neuronal plasticity and remodeling, and even insulin secretion. miRNAs have also been implicated in disease: e.g. an overabundance of miRNA has been reported in cases of Fragile X Mental Retardation,
while some cancers are associated with up- and downregulation of certain miRNA genes. |
|
Definition
|
|
Term
Riboswitch is a form of RNA that act as a precision genetic switch. Produced in many cases from noncoding DNA between known genes, a riboswitch folds into a complex shape. One part of the folded RNA can bind to a specific target protein or chemical. Another part contains the RNA code for a protein product. The riboswitch turns "on" and produces the protein it encodes only when in the presence of its target.
Conceptually divided into two parts: an aptamer (oligonucleic acid or peptide molecules that bind
a specific target molecule ) and an expression platform. The aptamer directly binds the small molecule, and usually undergoes structural changes in response. These structural changes also affect the expression platform, which is the mechanism by which gene expression is regulated.
Most known riboswitches occur in eubacteria, but functional riboswitches of one type (the TPP riboswitch) have been discovered in eukaryotes. The TPP riboswitch in Neurospora crassa (a fungus) controls alternative splicing to conditional produce a uORF, thereby affecting expressing of downstream genes. |
|
Definition
|
|
Term
The dystrophin gene (Duchenne Muscalar Dystrophy) is the longest gene in the human genome. It codes for
slightly different proteins—isoforms— that are tissue specific, a different isoform expressed in a variety of differentiated cell types. The dystrophin gene is thought to have eight promoters, each with its own initial exon and as many as seventy-nine downstream exons.
The isoforms are encoded by a range of different mRNA's which are generated by three processes:
the use of different, unique and often tissue-specific promoters alternative splicing
the use of different polyA-addition signals |
|
Definition
|
|
Term
Stable, heritable changes in the DNA of cells.
Stepwise accumulation of these changes in regulatory genes leads to the capacity for malignant growth.
Frequency:
1016 cell divisions/human body/lifetime.
Spontaneous mutations = 10-6/gene/cell division.
THUS: Every gene will have about 1010 spontaneous mutations in a lifetime.
Points out fidelity of the cellular correction machinery!!!!!!!!!
Requires several, independent events that are not corrected.
From 3 (in leukemias) to 7 (in carcinomas) events are required to turn a normal cell into a malignant cell. |
|
Definition
|
|
Term
For any particular cell there will be a finite number of ways in which dysfunction can occur (depends on the regulatory mechanisms active in that cell type).
Implies that in any cell there are a finite number of genes that can be responsible for this dysfunction (this means regulatory proteins). |
|
Definition
|
|
Term
Regulatory proteins can be classified into those that:
Stimulate cell growth (inducers).
Repress cell growth (repressors).
Protooncogene: normal gene whose product involved in stimulation of cell cycle.
Oncogene: Caused by a mutation that makes an inducer regulatory protein become hyper stimulatory.
Requires mutation in only one allele.
Antioncogene (Tumor Suppressor): a gene whose product involved in suppression of the cell cycle.
Abnormal tumor suppressor can no longer repress/suppress a stimulatory process leading to unscheduled stimulation of the cell cycle.
Requires mutation in both alleles. |
|
Definition
|
|
Term
| LOH: loss of heterogeneity: Loss of one of two alleles on a chromosome due to increasing aberrant behaviour of replication and recombination machinery. Thus, one allele will have a mutation introduced into it (like APC). Through nondisjunct recombination, gene conversion, etc the second allele will be loss leading to only the mutated allele expressing a product or fixing homogeneity of that locus. |
|
Definition
|
|
Term
Normal p53.
Functions as a tetramer (dimer of dimers).
Normal product causes a pause in the cell cycle at G1/S when the cell senses DNA damage. Does this by inducing transcription of p21 (and other molecules like MDM-2 (HDM), and Bax) which binds and blocks phosphorylation of G1 cyclin/Cdk2 complexes.
This allows DNA repair to restore genome integrity, followed by release from the arrest.
Alternatively, it induces a sensor molecule which, if cell cannot repair damage, promotes permanent exit from the cell cycle and apoptosis. |
|
Definition
|
|
Term
Mutated p53.
Mutations disable this “emergency brake” allowing the cell to replicate its defective genome, and stably introduce those mutations into the genome.
Li Fraumeni syndrome (Clinical Correlate):
Inheritance of mutated allele (germline mutation).
Predisposed to increased risk of breast, brain, adrenocortical tumors, sarcomas, and leukemia. |
|
Definition
|
|
Term
P53 mutant protein, even though inactive, interacts with wt p53 (heterooligomer) leading to an inactive complex, causing transdominant phenotype and inactivating all p53.
Having joined to a free end of DNA, ATM then functions as a kinase to transmit the damage signals. Among other proteins in the p53 damage network, active ATM phosphorylates MDM2 and p53 disrupting the negative feedback loop. ATM phosphorylates human MDM2 on Ser 395 and this probably compromises MDM2's ability to ubiquitinate p53 [29]. ATM also phosphorylates p53 at Ser 15 and dephosphorylates p53 at Ser 376 [7]. The former prevents MDM2 from binding to p53 but allows cofactors to bind and the latter also encourages the activation of p53.
ATM is very important in the ability of humans to prevent tumours. Ataxia-telangiectasia (a progressive neurodegenerative disease) patients have a predisposition to cancer and have extreme cellular radio-sensitivity [30].
Also, although the normal function of p53 is altered, in some cases the mutant p53 acquires an oncogenic activity that is directly involved in transformation. |
|
Definition
|
|
Term
Thus transcription activation by p53 can be disrupted by mutations which:
prevent sequence specific DNA binding.
Prevent formation of active oligomers.
Prevent interaction with the transcription machinery.
Also, overexpression of other gene products (e.g. Mdm2) can prevent p53 interaction with the initiation complex. |
|
Definition
|
|
Term
Mouse double minute two (MDM2) is an E3 ring-finger ubiquitin ligase that is the key negative regulator of p53.
MDM2 regulates p53 in a variety of ways to control p53 activity. By far the most significant regulation mechanism is the ubiquitin-mediated proteolysis of p53 by MDM2 which causes p53 to be degraded rapidly and hence keep the concentration of p53 low. MDM2 attaches several ubiquitin molecules to the p53 protein and these act as a label to the protein-degrading machinery to phagocytise the p53 protein [3]. For the efficient degradation of the p53 protein it is required that it is localised outside the nucleus (even though some degradation does occur within the nucleus) [15]. MDM2 ubiquitin ligase activity contributes to the efficient nuclear export of p53 [24][25] possibly by changing the form of the p53 protein to allow access to the nuclear export sequence [26]. Furthermore, the removal of p53 protein from the nucleus has an additional suppressing effect on p53; its transcription activity is repressed. When p53 is in the cytoplasm it has no access to the genes. Interestingly, in certain tumours wildtype p53 is found trapped outside the nucleus [15].
MDM2 also suppresses p53 activity by binding to p53 in its transactivation domain which is located on the N terminus [7]. This means that when MDM2 is bound to p53 it directly blocks the ability of p53 to suppress or activate the transcription of genes, in effect deactivating it. The final way that MDM2 suppresses the activities performed by p53 is by inhibiting the acetylation of p53. Cofactors such as p300 bind to the N terminus of p53 and cause the acetylation of the C terminus which negatively affects the ability of MDM2 to ubiquitinate p53 [7]. Hence by preventing this acetylation, MDM2 ensures that p53 is less likely to stabilise and become active.
It is clear that MDM2 suppresses the amount of active p53. What is interesting is that p53 activates the expression of MDM2, its own negative regulator [27]. This means that there is a negative feedback loop between MDM2 and p53. If there is a rise in p53, this will cause a rise in MDM2 which in turn will suppress p53 and reduce its amount. This negative feedback loop works to keep the equilibrium level of p53 low. When there is a stress placed on the cell these interactions need to be suppressed so that p53 levels can increase and set off the necessary pathways. This is done in two main ways: the modification of the p53 protein or the modification of the MDM2 protein. Both of these negatively affect the binding of MDM2 to p53 and hence prevent the ubiquitination and degradation of p53. In p53 there are numerous phosphorylation sites within or near the N-terminal MDM2-binding region of p53 and phosphorylation at many of these sites can help prevent the binding of p53 to MDM2 [15]. |
|
Definition
|
|
Term
Transcription activation by p53 can be disrupted by p53 mutations which:
1. prevent sequence specific DNA binding.
2. formation of active p53 oligomers.
3. interaction with the transcription machinery.
Also, overexpression of other gene preoducts, like Mdm2 protein can prevent p53 interaction with initiation complex.
A. Normal Mdm2 protein (light blue), which binds p53(dark blue) and prevents interaction with the transcription initiation complex.
B. Mutant p53 fails to bind its elements in the promoters and fails to stimulate transcription.
C. When mdm2 gene is amplified, its product is overexpressed and most of p53 is tied up in complexes with Mdm2 and not available for transcription activation. |
|
Definition
|
|
Term
| When can one see the basal rate of expression of the lac operon? |
|
Definition
| High Glucose & No Lactose |
|
|
