Transcriptional Regulation: From Operons to Gene Regulatory Networks Flashcards
1
Q
What is the major mechanism for controlling production of the protein encoded by a given gene?
A
Transcriptional control
2
Q
Fill in the blanks:
Transcription of a gene can be _______ (little or no mRNA is synthesized) or _______ (up to 1000x or more mRNA is synthesized)
A
Repressed; Activated
3
Q
What is the difference between single-celled and multicellular organisms in terms of gene regulation?
A
Single celled organisms: Genes are regulated to adjust to CHANGES in the nutritional and physical ENVIRONMENT. A cell usually produces only the proteins required for survival and proliferation under the particular conditions it experiences.
Multicellular organisms: Genes are regulated to ensure CO-ORDINATION during embryonic development and tissue differentiation (more complex).
4
Q
(T/F) About half the genes in E.coli are organized into operons.
A
True!
5
Q
What does the lac operon encode?
A
The lac operon encodes 3 enzymes required for the catabolism of lactose.
6
Q
What does the trp operon encode?
A
The trp operon encodes 5 enzymes required for the biosynthesis of tryptophan (anabolism).
The 5 enzymes work together.
7
Q
Fill in the blanks:
Transcription of operons and isolated genes in prokaryotes is controlled by interplay between ____ _________ and specific ________ and ________ proteins.
A
RNA polymerase; repressor; activator
8
Q
Which factor does the RNA polymerase must associate with to initiate transcription in prokaryotes?
A
Sigma factor; most commonly sigma 70
9
Q
Match the following parts of the lac operon to what they bind to:
1) Promoter
2) Operator
3) Cap site
A) Lac repressor binds in the absence of lactose, and blocks start site.
B) Cap-cAMP
C) Sigma factor 70 binds and positions the RNA polymerase
A
1) Promoter: Sigma factor 70 binds and positions the RNA polymerase
2) Operator: Lac repressor binds in the absence lactose, and blocks start site
3) Cap site: Cap-cAMP
10
Q
What is LacI? How is it transcribed?
A
LacI encodes the lac repressor in the lac operon.
It is transcribed from its own promoter.
11
Q
(T/F) Transcription of the lac operon is repressed when lactose is absent.
A
True!
12
Q
What does the lac repressor do?
Where does it bind?
A
The lac repressor prevents RNA polymerase from binding to promoter. It reduces transcription by 1000 folds.
It binds to the main operator (O1) and secondary operator (O2 and O3).
13
Q
What happens if there is a mutation in O2 and O3, the secondary operators?
A
Repression by the repressor is not possible and the transcription reduces by 100 fold.
14
Q
What happens to the lac operon when lactose (allolactose) is present?
A
Lactose binds to the lac repressor, changing its conformation and releasing it from the operator sequence.
Transcription is DE-REPRESSED (foot is neither on the breaks or accelerator).
Now, there is LOW TRANSCRIPTION.
15
Q
Answer the questions regarding allolactose:
1) What is allolactose?
2) What enzyme is involved in the production of allolactose?
3) What gene encodes this protein?
A
1) Allolactose is a metabolite of lactose. It serves as a cue that lactose is present and being metabolized as a food source (inducer/ligand).
2) BETA-GALACTOSIDASE metabolizes lactose into allolactose.
3) LacZ encodes the beta-galactosidase.
16
Q
(T/F) Glucose is a better energy source than lactose and other complex sugars. When glucose levels are low, transcription of operons like lac is activated.
A
True!
17
Q
Fill in the blanks:
E.coli synthesizes __________ in response to low glucose levels, which binds and activates a transcriptional activator protein called _____.
A
CYCLIC-AMP
CAP (cAMP receptor protein)
18
Q
What causes high transcription of the lac operon?
A
1) Lactose is present to bind to the lac repressor and causes de-repression where RNA polymerase binds to the promoter.
2) CAP complexed with cAMP binds to the CAP site and interacts with RNA polymerase and stimulates the rate of transcription initiation.
Overall, the presence of lactose and absence of glucose (which leads to high cAMP) causes high transcription of the lac operon.
19
Q
Why is there low transcription of the lac operon at high glucose levels?
A
Catabolites produced by the breakdown of glucose prevent the production of cAMP.
20
Q
What is a regulon? What is its function? Give an example.
A
Network of operons with a common regulator.
It allows coordinated shifts in cellular functions that can require hundreds of genes.
CAP and cAMP regulate many operons, thus being a regulon.
21
Q
What happens to the rate of gene expression (transcription) of the lac operon in each of these scenarios?
1) Glucose high, cAMP low, lactose absent
2) Glucose low, cAMP high, lactose absent
3) Glucose high, cAMP low, lactose present
4) Glucose low, cAMP high, lactose present
A
Glucose high, cAMP low, lactose absent: NO gene expression! Repressor is still bound to the operator and blocks start site.
Glucose low, cAMP high, lactose absent: NO gene expression! Repressor is still bound to the operator and blocks start site.
Glucose high, cAMP low, lactose present: LOW LEVEL of gene expression! Repressor is no longer bound to the operator as it binds to the lactose but cAMP-CAP is not present to activate the RNA polymerase and stimulate the rate of transcription.
Glucose low, cAMP high, lactose present: HIGH LEVEL of gene expression; repressor is not bound to the operator and RNA polymerase is activated and stimulated by cAMP-CAP.
22
Q
What kind of mechanism is the trp operon?
A
REPRESSION mechanism
Trp operon is always on and has to be turned off. Whereas, the lac operon is always off and has to be turned on.
23
Q
What happens when there is low levels of tryptophan vs high levels of tryptophan?
A
Low levels of tryptophan: trp repressor is INACTIVE. This allows RNA polymerase to transcribe the trp genes.
High levels of tryptophan: trp binds to its repressor and ACTIVATES it, and transcription of the genes do not occur.
24
Q
What are the four processes to control cell protein concentration?
A
1) Gene transcription: regulation of the frequency of an mRNA synthesis.
2) mRNA degradation: rate at which mRNA is degraded.
3) Protein translation: rate at which mRNA is translated into protein.
4) Protein degradation: rate at which a protein is degraded.
25
Out of the four processes to control cell protein concentration, which one contributes to it the most?
Gene transcription (accounts for 73% of the protein concentration).
This is followed by mRNA degradation (11%) and the other two (8% each).
26
Match the following methods use to determine the rates of the 4 processes that control cell protein concentration to their usages:
1) Mass spectrometry
2) mRNA sequencing
3) Ribosome footprinting (mRNA protection from ribonuclease digestion by associated ribosomes)
4) Stable isotope labeling
5) Statistical analysis of the data
A) to measure mRNA levels
B) to correct for inherent biases and errors in these methods
C) to measure protein concentrations
D) to estimate translation rates
E) to determine degradation rates
1) Mass spectrometry: to measure protein concentrations
2) mRNA sequencing: to measure mRNA levels
3) Ribosome footprinting (mRNA protection from ribonuclease digestion by associated ribosomes): to estimate translation rates
4) Stable isotope labeling: to determine degradation rates
5) Statistical analysis of the data: to correct for inherent biases and errors in these methods
*all in cultured mouse fibroblasts
27
Match the RNAs to their functions:
1) Pre-rRNA
2) mRNA
3) snRNA
4) siRNA
5) miRNA
6) tRNA
7) 5S rRNA
8) snRNA U6
9) 7S RNA
A) Chromatin-mediated repression, translation control
B) Ribosome components, protein synthesis
C) RNA splicing
D) Encodes protein
E) Signal recognition particle for insertion of polypeptides into the endoplasmic reticulum
F) Protein synthesis
G) Translational control
1) Pre-rRNA: Ribosome components, protein synthesis
2) mRNA: Encodes protein
3) snRNA: RNA splicing
4) siRNA: Chromatin-mediated repression, translation control
5) miRNA: Translational control
6) tRNA: Protein synthesis
7) 5S rRNA: Ribosome component, protein synthesis
8) snRNA U6: RNA splicing
9) 7S RNA: Signal recognition particle for insertion of polypeptides into the endoplasmic reticulum
28
While RNA pol ___ transcribes pre-rRNA, RNA pol ___ transcribes mRNA, snRNA, siRNA, and miRNAs, and RNA pol ____ transcribes tRNA, 5S, 7S RNA.
I, II, III
29
(T/F) Improper regulation of mechanisms controlling transcription causes pathological processes.
True!
A.k.a a mutation in transcription causes pathological processes.
30
What does a dominant mutation in a human HOXD13 gene cause?
Polydactyly - development of extra digits
31
What does a recessive mutation in Drosophila Ubx gene cause?
It prevents Ubx expression in the third thoracic segment. This transforms the segment, which normally has a balancing organ (haltere) into wings. (double wings)
32
Approximately ____ of eukaryotic protein-coding genes (trans-acting element) are transcriptional regulators.
10%
33
Match the following terms to their definitions:
1) Transcriptional activator
2) Transcriptional repressor
A) NEGATIVE REGULATOR - turns genes off, decreasing the frequency of transcription initiation
B) POSITIVE REGULATOR - turns genes on, increasing the frequency of transcription initiation up to 1,000x
1) Transcriptional activator: POSITIVE REGULATOR - turns genes on, increasing the frequency of transcription initiation up to 1,000x
2) Transcriptional repressor: NEGATIVE REGULATOR - turns genes off, decreasing the frequency of transcription initiation
34
Chromatin condensation __________ gene transcription.
Inactivates
*It blocks RNA polymerase and general TFs from interacting with gene promoters.
35
Match the following terms to their definition:
1) Repressor proteins
2) Pioneer transcription factor
3) Activator proteins
4) Transcriptional activation
A) Bind to a specific regulatory sequence within the condensed chromatin. They interact with chromatin-remodelling enzymes and histone acetylases that DECONDENSE the chromatin.
B) Done by RNA polymerase II.
C) May bind to transcriptional control elements to INHIBIT transcription initiation by Pol II. They also can interact with multiprotein co-repressor complexes to CONDENSE CHROMATIN.
D) Bind to specific TRANSCRIPTIONAL-CONTROL ELEMENTS in both promoter-proximal sites and distant enhancers. They interact with one another and with the multisubunit MEDIATOR COMPLEX to assemble general TFs and RNA pol on promoters.
1) Repressor proteins: may bind to transcriptional control elements to INHIBIT transcription initiation by Pol II. They also can interact with multiprotein co-repressor complexes to CONDENSE CHROMATIN.
2) Pioneer transcription factor: bind to a SPECIFIC REGULATORY SEQUENCE within the condensed chromatin. They interact with chromatin-remodelling enzymes and histone acetylases that DECONDENSE the chromatin.
3) Activator proteins: bind to specific TRANSCRIPTIONAL-CONTROL ELEMENTS in both promoter-proximal sites and distant enhancers. They interact with one another and with the multisubunit MEDIATOR COMPLEX to assemble general TFs and RNA pol on promoters.
4) Transcriptional activation: done by RNA polymerase II.
36
Which genes have a TATA box starting 35 bp upstream of the start site? What do these boxes do?
Genes transcribed at HIGH levels (strong promoters).
The TATA box functions similarly to an E.coli promoter, positioning RNA polymerase for transcription initiation.
37
What are promoters?
The term PROMOTERS refers to the TATA box or other sequences that:
1) Direct binding of RNA polymerase II to DNA
2) Determine the site of transcription initiation
3) Influence the frequency of transcription initiation
38
What are promoter-proximal elements?
Sequences near the promoter (TATA box) that can regulate transcription. They can be cell type specific.
*they are close to the gene; can be up or downstream of the promoter.
*they are in equal frequency in mammalian genes.
39
What kind of mutations can be used to find promoter-proximal elements?
Linker-scanning mutations.
*this approach is good for detailed analysis of a short stretch of DNA.
40
(T/F) Most eukaryotic genes are regulated only by one transcriptional-control element.
False!
Most are regulated by multiple transcription-control elements.
41
(T/F) Mammalian genes with a TATA box promoter are regulated by promoter-proximal elements and enhancers.
True.
42
Where are enhancers located?
What do they do?
They may be either upstream or downstream or in introns and as far away as hundreds of kilobases from the transcription start site; and are often cell type-specific.
They include binding sites for several transcription factors. They can also produce loops in the DNA between their binding sites. Their length is ~50-200bp.
43
Most S. cerevisiae genes contain ____ upstream activating sequence (UAS) and a ______ box, ~90 bp upstream from the transcription start site.
one; TATA
*UAS in yeast = enhancer in a mammalian gene
*mammalian gens have multiple enhancers.
44
Monomers (transcription regulator) typically bind _____ nucleotide pairs of DNA.
These nucleotide sequence would occur every ______ nucleotides.
6-8
6-nucleotide sequence would occur once every 4,096 nucleotides (4^6).
*transcription regulators = transcription factors
45
What does dimerization of monomers (transcription regulators) do? What kinds are there?
It increases AFFINITY and SPECIFICITY.
There are HOMODIMERS and HETERODIMERS.
46
Match the following steps of how to map enhancers:
1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5
A) Ligate into vector carrying reporter gene
B) Transfect each type of plasmid (1-5) separately into cultured cells
C) Recombinant DNA techniques
D) Prepare cell extract and assay activity of reporter enzyme
E) Transform E. coli and isolate plasmid DNAs
Step 1: Recombinant DNA techniques
Step 2: Ligate into vector carrying reporter gene
Step 3: Transform E. coli and isolate plasmid DNAs
Step 4: Transfect each type of plasmid (1-5) separately into cultured cells
Step 5: Prepare cell extract and assay activity of reporter enzyme
47
What is a reporter gene?
A reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants.
It is a gene that generally encodes an easily measured enzyme (like beta-Gal).
It allows us to measure the regulatory sequence s/promoters activity.
48
Transcription activators and repressors are modular PROTEINS that contain a single _________ domain and one or a few ______ or ________ domains + intrinsically disordered protein domain.
DNA binding domain; activation; repression
49
Match the following transcription activators to their definitions:
1) GAL4
2) GCN4
3) GR
A) promotes transcription of target genes when glucocorticoid hormones bind to the C-terminal activation domain
B) yeast transcription activator
C) yeast transcription activator
1) GAL4: yeast transcription activator
2) GCN4: yeast transcription activator
3) GR: promotes transcription of target genes when glucocorticoid hormones bind to the C-terminal activation domain
50
(T/F) There are several classes of DNA binding domains.
True!
*Transcription factors (activators/repressors) usually contain ONE DNA binding domain.
51
What are the 4 major classes of activation (transactivation) domains?
1) Acidic (lots of D and E residues)
2) Glutamine-rich
3) Proline-rich
4) Isoleucine-rich
52
What are intrinsically disordered domains and what can they do?
Intrinsically disordered protein domains are another kind of domain that is found in a transcription factor.
They lack tertiary structure (due to minimal hydrophobic acids) and can:
1) Enable scaffolding
2) Target for post-translational modifications
3) Enable flexible conformational changes
53
(T/F) Repressors are the functional converse of activators and therefore with a DNA binding domain, they have a repression domain.
True!
54
Fill in the blanks regarding proteins binding to specific DNA sequences:
It commonly involves interactions between atoms in a DNA-binding domain ________ ______ and atoms on the edges of the bases within the _______ groove in the DNA.
They may involve interactions between ______ charged repressor residues (arginine & lysine) and negatively charged phosphates in the sugar phosphate backbone and with atoms in the DNA ______ groove.
alpha helix; major
positively; minor
55
Bacteriophage 434 repressor is a ________ protein, with a ______-___ ______ motif, which interacts with one side of the DNA over a length of ____ turns.
A recognition/sequence-reading alpha helix from each monomer inserts in the ____ groove.
DIMERIC; helix-turn helix; 1.5
major
56
Match the following eukaryotic DNA binding domains (a domain of TFs) that use an ALPHA helix to interact with the MAJOR groove of DNA:
1) C2H2 zinc finger
2) C4 zinc-finger proteins
3) Leucine-zipper proteins
4) bHLH proteins
5) GL1 DNA-binding domain
A) A monomeric structure with FIVE C2H2 zinc fingers, where finger 2-5 interact with DNA.
B) DNA binding helices at N-termini of the monomers are separated by nonhelical loops from a leucine zipper-like region containing a COILED-COIL DIMERIZATION. can form HETERODIMERS.
C) Most common DNA-binding motif. Contains TWO conserved CYSTEINE and TWO conserved HISTIDINE residues, whose side chains bind one zinc ion. 23-26 residues long.
D) Basic residues in the alpha helical region of the monomers bind to the DNA. COILED-COIL DIMERIZATION domain, stabilized by HYDROPHOBIC interactions between the monomers. They can form HETERODIMERS.
E) Found in 50 human TFs, including nuclear hormone receptors. It contains FOUR conserved CYSTEINES that bind a zinc ion. It binds DNA as a HOMODIMER, where one alpha helix in each monomer interacts wth the DNA.
C2H2 zinc finger: Most common DNA-binding motif. Contains TWO conserved CYSTEINE and TWO conserved HISTIDINE residues, whose side chains bind one zinc ion. 23-26 residues long.
C4 zinc-finger: Found in 50 human TFs, including nuclear hormone receptors. It contains FOUR conserved CYSTEINES that bind a zinc ion. It binds DNA as a HOMODIMER, where one alpha helix in each monomer interacts wth the DNA.
Leucine-zipper proteins: Basic residues in the alpha helical region of the monomers bind to the DNA. COILED-COIL DIMERIZATION domain, stabilized by HYDROPHOBIC interactions between the monomers. They can form HETERODIMERS.
bHLH proteins: DNA binding helices at N-termini of the monomers are separated by nonhelical loops from a leucine zipper-like region containing a COILED-COIL DIMERIZATION. can form HETERODIMERS.
GL1 DNA-binding domain: A monomeric structure with FIVE C2H2 zinc fingers, where finger 2-5 interact with DNA.
57
(T/F) Activation domains (a domain of transcription factors) may be random coils until they interact with co-activator proteins or folded protein domains.
True!
58
CREB (cyclic AMP response element-binding protein) acidic activation domain is activated by __________ at ______ 133 (this then activates the TF).
Interaction with a CBP co-activator domain folds random coil structure into two __________ __ helices.
phosphorylation; serine
amphipathic alpha
*mutation of serine leads to a broken CREB
59
In an estrogen receptor ligand-binding activation domain, what happens when estrogen is present and what happens when estrogen is absent?
Presence of Estrogen: alpha helix interacts with the estrogen and generates a hydrophobic groove in the ligand-binding domain that BINDS CO-ACTIVATOR subunit alpha helix.
Absence of Estrogen: Receptor folds into a conformation that sterically BLOCKS CO-ACTIVATOR BINDING. It is stabilized by the binding of estrogen antagonist tamoxifen.
60
Transcriptional factor interactions ________ gene-control options.
Increase
*Combinatorial possibilities due to formation of heterodimeric transcriptional factors
*1400 TFs encoded in the human genome can bind to DNA through a much larger number of cooperative interactions to provide unique transcriptional control for each of the ~21,000 human genes.
61
What are the three different types of heterodimeric TFs?
1) Heterodimeric TFs in which the DNA binding domain of each monomer recognize the SAME DNA sequence (different activation domains)
2) Heterodimeric TFs in which the DNA binding domain of each monomer recognize DIFFERENT DNA sequences (different activation domains)
3) Heterodimeric TFs in which one DNA binding domain is INHIBITORY (different activation domains)
*if there is 3 different TF monomers, for 1 and 2 there is 6 different possibilities.
*for 2, the 6 combinations bind at 6 different DNA sequences
*for 3, if there is an inhibitory factor that interacts with domain of A, transcription is activated in combinations that do not include the domain
62
(T/F) Single bZIP- or bHLH-binding DNA regulatory element in the transcription-control region of a gene may elicit different transcriptional responses depending on which bZIP or bHLH monomers are expressed in the cell and how their activities are regulated.
True!
63
How is combinatorial transcription regulation achieved of two unrelated TFs?
Give examples.
It is achieved through the interaction of structurally unrelated TFs bound to closely spaced binding sites in DNA.
First example: TFs NFAT and AP1 interacting with one another to regulate IL-2 gene.
Second example: binding of SRF and SAP1
64
______ NFAT and _____ AP1 TFs bind cooperatively to the composite site.
Each TF has a _____ affinity for their respective binding sites in the IL-2 promoter-proximal region.
The interaction between NFAT and AP1 increases overall ______ of the NFAT-AP1-DNA complex, allowing for the regulation of IL-2 gene.
Monomeric; heterodimeric
Low
Stability
65
______ SRF and _____ SAP1 cooperatively bind DNA, when their binding sites are separated by ___ to ___ bp.
It contains an ______ SAP1 binding site.
The SAP1 domain that interacts with SRF is connected to the DNA-binding domain of SAP1 by a _______ ______ region of the SAP1 polypeptide chain.
Dimeric; monomeric; 5; 30
inverted; flexible linker
66
What is beta-interferon enhancer?
What is it composed of?
What complex forms on the enhancer?
It is very important for immune response regulated by lots of TFs.
It has six OVERLAPPING binding sites that bind two HETERODIMERIC factors, jun/ATF-2 and p50/RelA and two copies each of the MONOMERIC factors, IRF-3 and IRF-7. These all bind highly cooperatively.
ENHANCESOME complex forms on the enhancer.
67
In the enhancesome complex that forms on the beta-interferon enhancer, HMGI binds the ______ groove of DNA regardless of the sequence and bends the molecule, allowing _________ _______ to interact.
minor; transcription factors
68
What are the two ways for regulation of transcription factor activity?
1) Transcription factor activities can be indirectly regulated by cell-surface receptor activation of intracellular signal transduction pathways
2) Lipid-soluble hormones can diffuse into the cell and activate receptors that bind to specific response elements in genes to regulate gene expression.
69
Which one of the statements regarding nuclear receptors and hormones that bind to them is false?
1) Lipid soluble hormones interact directly with the TFs including the nuclear-receptor superfamily.
2) Lipid soluble hormones include steroid hormones, retinoids, and thyroid hormones.
3) Lipid soluble hormones diffuse through the plasma and nuclear membranes.
4) Nuclear receptors are regulated by intracellular hormone signals.
5) Ligand-receptor complex functions as a transcription activator.
4! Nuclear receptors are regulated by extracellular hormone signals.
70
Match the following regions of nuclear receptors to their definitions:
1) N-terminal region
2) DNA-binding domain
3) Hormone-binding domain
A) in the centre of the receptor primary sequence; is 68 aa long. It contains a repeat of C4 ZINC-FINGER motif.
B) in the C-terminal end. It contains a hormone-dependent activation domain. In some receptors, this domain can function as a repression domain when ligand is absent.
C) contains variable region, that is 100-150 aas long. It functions as activation domains in most receptors.
1) N-terminal region: contains variable region, that is 100-150 aas long. It functions as activation domains in most receptors.
2) DNA-binding domain: in the centre of the receptor primary sequence; is 68 aa long. It contains a repeat of C4 ZINC-FINGER motif. (receptor + ligand bind to DNA here)
3) Hormone-binding region: in the C-terminal end. It contains a hormone-dependent activation domain. In some receptors, this domain can function as a repression domain when ligand is absent. (hormone binds to receptor here)
71
What happens when there is no hormone (cortisol) to bind to the nuclear receptor (glucocorticoid receptor)?
Receptor complex is retained in the cytoplasm by interaction between its ligand-binding domain (LBD) and chaperone proteins.
72
What happens when there is a hormone (cortisol) to bind to the nuclear receptor (glucocorticoid receptor)?
1) Hormone complex diffuses through the plasma membrane and binds to the receptor ligand-binding domain.
2) That causes a conformational change that releases chaperone proteins.
3) Receptor-hormone complex translocates into the nucleus.
4) DNA Binding Domain binds to response elements.
5) Ligand-binding domain and an additional activation domain (AD) at the N-terminus stimulate transcription of target genes
73
Nuclear-receptor RESPONSE ELEMENTS (DNA binding sites) contain _________ or ______ repeats.
Glucocorticoid and estrogen receptors, both are twofold _________ _______ that bind to the glucocorticoid/estrogen receptor response element. These response elements are inverted repeats separated by _____ base pairs.
Inverted; direct
Symmetric dimers; three
74
Heterodimeric nuclear receptor contains one ______ subunit associated with another nuclear-receptor subunit that defines the hormone response.
RXR
75
Match the following heterodimeric nuclear receptors:
1) RXR-VDR
2) RXR-TR
3) RXR-RAR
A) mediates retinoic acid responses by binding to a direct response element separated by FIVE base pairs (RARE)
B) mediates vitamin D3 responses by binding to a direct repeat response element separated by THREE base pairs (VDRE)
C) mediates thyroid hormone responses by binding to a direct repeat response element separated by FOUR base pairs (TRE)
1) RXR-VDR: mediates vitamin D3 responses by binding to a direct repeat response element separated by THREE base pairs (VDRE)
2) RXR-TR: mediates thyroid hormone responses by binding to a direct repeat response element separated by FOUR base pairs (TRE)
3) RXR-RAR: mediates retinoic acid responses by binding to a direct response element separated by FIVE base pairs (RARE)
76
(T/F) Tissue specificities come from transcription factor specific combinations in cell types.
True!
77
What's the difference between class I and class II nuclear receptors?
Class 1 - Hormone interacts with receptor in the CYTOPLASM (estrogen + glucocorticoid)
Class 2 - Hormone interacts with receptor in NUCLEUS; the receptor is already in the nucleus and bound to response elements and hetero-dimerizes with RXR. The default state is repression!
78
What are the four different families of receptors involved in the four different signal transduction pathways?
Pathway A: GPCR (G-protein coupled receptors)
Pathway B: TGF-β
Pathway C: Notch/Delta
Pathway D: Wnt, Hedgehog, IL-1, TNF α
79
Fill in the blanks regarding the four signal transduction pathways:
1) Many GPCRs activate the heterotrimeric ____-BINDING PROTEIN, this activates a ________ which activates TFs such as CREB.
2) The ________ domains of receptors of the TGF-β family of signaling proteins contain a ______-_______ kinase, which activates ____ class of TFs, unmasking a nuclear localization signal.
3) Binding of a delta ligand to the ______________ domain of a notch receptor triggers _________ _________ of the receptor, releasing its _______ domain which moves to the nucleus and regulates gene expression.
4) The pathways activated by binding of Wnt, Hedgehog, IL-1 families of ligands to their receptors lead to ____________ and _______ of the multi-proteins in the cytosol, releasing a TF that translocates into the nucleus.
GTP; kinase
Cytosolic; serine-threonine; Smad
Extracellular; proteolytic cleavage; cytosolic
Ubiquitination; degradation
80
What is the key signaling molecule in the canonical Wnt pathway?
How is its stability controlled?
The cytoplasmic protein, β-catenin.
Its stability is controlled by a large multiprotein, β-catenin destruction complex.
81
Briefly describe the Wnt signaling pathway in THE ABSENCE OF WNT.
In the absence of WNT, the transcription factor TCF is bound to the promoters/enhancers of the Wnt genes.
TCF's activator, β-catenin is absent from the nucleus, and TCF is associated with repressor proteins like GROUCHO (Gro).
In the cytoplasm, CK1 and GSK3 phosphorylate β-catenin at multiple serine and threonine residues.
Then, the E3 (βTrCP) ubiquitin ligase binds to phosphorylated residues on the β-catenin, leading ubiquitinylation of β-catenin and its degradation in proteasomes.
This prevents the β-catenin to enter the nucleus.
82
Briefly describe the canonical Wnt signaling pathway.
Wnt proteins bind to a complex of two receptor proteins on the cell surface, FRIZZLED (Fz) and LRP.
Binding of Wnt to Fz induces PHOSPHORYLATION of the LRP cytosolic domain by several kinases.
Then, AXIN binds to the LRP.
This causes part of the AXIN-CK1-GSK3-b-catenin complex to fall apart, preventing phosphorylation of b-catenin and inhibiting ubiquintination, allowing b-catenin to accumulate in the cytosol.
After translocation to the nucleus, b-catenin binds to TCF, displaces Gro repressor and recruits co-activator proteins to activate expression of Wnt target genes.
83
What are the seven forms of regulation of TF activity?
1) Synthesis of TFs (most important)
2) Ligand/hormone binding of TF (activates TF)
3) Post-transcriptional modification of TF (e.g., phosphorylation)
4) Addition of a critical subunit (activates TF)
5) Removal/unmasking of a repressor (need to remove inhibitor to activate TF)
6) Stimulation of nuclear entry
7) Release from membrane
*extracellular information leads to the activation of TFs!
84
Differential binding of TFs to enhancer and suppressor regulatory elements can influence the expression of __________ ____.
Tfs can provide identity to cells and tissues in a highly _______ manner and this is possible by their expression being regulated in a ______-specific manner.
This upstream/downstream interactions can form into _____ _______ ________ that are responsible for the proper patterning, growth and development of tissues + organs.
Downstream TFs
Specific; tissue
Gene Regulatory Networks (GRN)
85
Briefly answer some of the questions regarding the SALL1 gene?
1) What does the SALL1 gene encode?
2) What is located ~500kb of the SALL1 gene in the human DNA?
3) When human DNA with the conserved region is inserted into a plasmid next to E.coli b-galactosidase reporter gene, what happened?
4) Orthologs of developmental genes like SALL1 are important for?
1) A zinc-finger transcription repressor
2) A highly conserved region between humans, mice, chickens, frogs and fish. Here, TFs can bind to and control gene expression regulation of the SALL1 gene.
3) It was found that the conserved region contained an ENHANCER that stimulated strong, specific transcription of the b-galactosidase reporter gene in limb buds. This means that the TFs are being synthesized in the limbs!
4) Appendage development from insects to humans
Also, other transcriptional-control regions control expression of this gene in other types of cells, where it function sin the normal development of the ears, the lower intestine, and kidneys.
86
Gene A is expressed in the limbs and the brain, thus it has a brain-specific enhancer and a limb-specific enhancer.
Does the limb-specific enhancer function in the developing brain cells?
In the developing brain cells, brain specific TFs bind to the brain enhancer, causing it to bind to the MEDIATOR, stabilize RNA polymerase II at the PROMOTER, and modify nucleosomes in the region of the promoter.
The gene is transcribed in the brain cells only! The limb enhancer does not function.
Gene is not transcribed in any cell type whose TFs and enhancers can not bind!
87
What is the Pax6 protien?
Pax6 protein is required for the development of the eye, certain regions of the brain and spinal cord, and the pancreas cells that secrete hormones such as insulin.
There are different enhancers in different tissues.
88
(T/F) The pancreas-specific enhancer element of the Pax6 has binding sites for the Pbx1 and Meis transcription factors. Both must be present to activate Pax6 in the pancreas.
True!
89
(T/F) There are several alternative Pax6 promoters and these regulate expression of the gene at different embryogenesis times in different tissues.
True!
*10.5 days after fertilization, there was an expression of a b-galactosidase reporter gene in the eye lens and pancreas
*13.5 days after fertilization, there was expression in the retina
90
What is eyeless?
The insect ortholog of the vertebrate Pax6.
It is expressed in the lens and retina of the mouse and the nervous system.
It is expressed in the developing eye and nervous system of the fly.
Their (eyeless and pax6) expression pattern is evolutionarily conserved (similar in vertebrates + insects)!
91
How does the UAS/GAL4 system work?
GAL4 is a transcription factor of yeast. It can be placed downstream of an enhancer of interest.
When the enhancer is actively bound by a TF, the GAL4 gene will be activated (transcribed) leading to the production of GAL4 protein.
This protein will bind to yeast UPSTREAM ACTIVATING SEQUENCE (UAS) that is inserted upstream of a gene of interest.
This enables over expression of gene X in a temporally and spatially controlled manner.
92
GAL4 driven ectopic expression of ey induces the formation of _____ structures with _____________.
eye; photoreceptors
*whether it is being expressed in the wing, the distal antennae, leg, etc.
93
(T/F) The pax6 gene is highly conserved across animals.
True!
By studying them in various groups, we can get a high resolution idea about the ways that TFs (like PAX-6) play in critical development processes like the formation of eyes.
94
What are the two logic circuits for gene expression? Describe.
Double-negative gate circuit: a single gene encodes a repressor of an entire battery of genes. When the repressor is repressed, the battery of genes is expressed.
Feedforward circuit: gene product A activates gene product B and C, and gene B also activates gene C. It provides an efficient way to amplify a signal in one direction.
95
(T/F) The gene regulatory networks (lots of circuits) in the sea urchin embryo progresses through time. For example, different genes are expressed during fertilization than when the cell divides to become multicellular.
True!
*not just about where it is expressed, but also about when it is expressed.
