Transcriptional Regulation: From Operons to Gene Regulatory Networks

Question 1

What is the major mechanism for controlling production of the protein encoded by a given gene?

Answer 1

Transcriptional control

Question 2

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)

Answer 2

Repressed; Activated

Question 3

What is the difference between single-celled and multicellular organisms in terms of gene regulation?

Answer 3

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).

Question 4

(T/F) About half the genes in E.coli are organized into operons.

Answer 4

True!

Question 5

What does the lac operon encode?

Answer 5

The lac operon encodes 3 enzymes required for the catabolism of lactose.

Question 6

What does the trp operon encode?

Answer 6

The trp operon encodes 5 enzymes required for the biosynthesis of tryptophan (anabolism).

The 5 enzymes work together.

Question 7

Fill in the blanks:

Transcription of operons and isolated genes in prokaryotes is controlled by interplay between ____ _________ and specific ________ and ________ proteins.

Answer 7

RNA polymerase; repressor; activator

Question 8

Which factor does the RNA polymerase must associate with to initiate transcription in prokaryotes?

Answer 8

Sigma factor; most commonly sigma 70

Question 9

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

Answer 9

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

Question 10

What is LacI? How is it transcribed?

Answer 10

LacI encodes the lac repressor in the lac operon.

It is transcribed from its own promoter.

Question 11

(T/F) Transcription of the lac operon is repressed when lactose is absent.

Answer 11

True!

Question 12

What does the lac repressor do?

Where does it bind?

Answer 12

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).

Question 13

What happens if there is a mutation in O2 and O3, the secondary operators?

Answer 13

Repression by the repressor is not possible and the transcription reduces by 100 fold.

Question 14

What happens to the lac operon when lactose (allolactose) is present?

Answer 14

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.

Question 15

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?

Answer 15

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.

Question 16

(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.

Answer 16

True!

Question 17

Fill in the blanks:

E.coli synthesizes __________ in response to low glucose levels, which binds and activates a transcriptional activator protein called _____.

Answer 17

CYCLIC-AMP

CAP (cAMP receptor protein)

Question 18

What causes high transcription of the lac operon?

Answer 18

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.

Question 19

Why is there low transcription of the lac operon at high glucose levels?

Answer 19

Catabolites produced by the breakdown of glucose prevent the production of cAMP.

Question 20

What is a regulon? What is its function? Give an example.

Answer 20

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.

Question 21

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

Answer 21

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.

Question 22

What kind of mechanism is the trp operon?

Answer 22

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.

Question 23

What happens when there is low levels of tryptophan vs high levels of tryptophan?

Answer 23

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.

Question 24

What are the four processes to control cell protein concentration?

Answer 24

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.

Question 25

Out of the four processes to control cell protein concentration, which one contributes to it the most?

Answer 25

Gene transcription (accounts for 73% of the protein concentration).

This is followed by mRNA degradation (11%) and the other two (8% each).

Question 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

Answer 26

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

Question 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

Answer 27

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

Question 28

While RNA pol ___ transcribes pre-rRNA, RNA pol ___ transcribes mRNA, snRNA, siRNA, and miRNAs, and RNA pol ____ transcribes tRNA, 5S, 7S RNA.

Answer 28

I, II, III

Question 29

(T/F) Improper regulation of mechanisms controlling transcription causes pathological processes.

Answer 29

True!

A.k.a a mutation in transcription causes pathological processes.

Question 30

What does a dominant mutation in a human HOXD13 gene cause?

Answer 30

Polydactyly - development of extra digits

Question 31

What does a recessive mutation in Drosophila Ubx gene cause?

Answer 31

It prevents Ubx expression in the third thoracic segment. This transforms the segment, which normally has a balancing organ (haltere) into wings. (double wings)

Question 32

Approximately ____ of eukaryotic protein-coding genes (trans-acting element) are transcriptional regulators.

Answer 32

10%

Question 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

Answer 33

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

Question 34

Chromatin condensation __________ gene transcription.

Answer 34

Inactivates

*It blocks RNA polymerase and general TFs from interacting with gene promoters.

Question 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.

Answer 35

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.

Question 36

Which genes have a TATA box starting 35 bp upstream of the start site? What do these boxes do?

Answer 36

Genes transcribed at HIGH levels (strong promoters).

The TATA box functions similarly to an E.coli promoter, positioning RNA polymerase for transcription initiation.

Question 37

What are promoters?

Answer 37

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

Question 38

What are promoter-proximal elements?

Answer 38

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.

Question 39

What kind of mutations can be used to find promoter-proximal elements?

Answer 39

Linker-scanning mutations.

*this approach is good for detailed analysis of a short stretch of DNA.

Question 40

(T/F) Most eukaryotic genes are regulated only by one transcriptional-control element.

Answer 40

False!

Most are regulated by multiple transcription-control elements.

Question 41

(T/F) Mammalian genes with a TATA box promoter are regulated by promoter-proximal elements and enhancers.

Answer 41

True.

Question 42

Where are enhancers located?

What do they do?

Answer 42

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.

Question 43

Most S. cerevisiae genes contain ____ upstream activating sequence (UAS) and a ______ box, ~90 bp upstream from the transcription start site.

Answer 43

one; TATA

*UAS in yeast = enhancer in a mammalian gene
*mammalian gens have multiple enhancers.

Question 44

Monomers (transcription regulator) typically bind _____ nucleotide pairs of DNA.

These nucleotide sequence would occur every ______ nucleotides.

Answer 44

6-8

6-nucleotide sequence would occur once every 4,096 nucleotides (4^6).

*transcription regulators = transcription factors

Question 45

What does dimerization of monomers (transcription regulators) do? What kinds are there?

Answer 45

It increases AFFINITY and SPECIFICITY.

There are HOMODIMERS and HETERODIMERS.

Question 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

Answer 46

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

Question 47

What is a reporter gene?

Answer 47

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.

Question 48

Transcription activators and repressors are modular PROTEINS that contain a single _________ domain and one or a few ______ or ________ domains + intrinsically disordered protein domain.

Answer 48

DNA binding domain; activation; repression

Question 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

Answer 49

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

Question 50

(T/F) There are several classes of DNA binding domains.

Answer 50

True!

*Transcription factors (activators/repressors) usually contain ONE DNA binding domain.

Question 51

What are the 4 major classes of activation (transactivation) domains?

Answer 51

1) Acidic (lots of D and E residues)
2) Glutamine-rich
3) Proline-rich
4) Isoleucine-rich

Question 52

What are intrinsically disordered domains and what can they do?

Answer 52

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

Question 53

(T/F) Repressors are the functional converse of activators and therefore with a DNA binding domain, they have a repression domain.

Answer 53

True!

Question 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.

Answer 54

alpha helix; major

positively; minor

Question 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.

Answer 55

DIMERIC; helix-turn helix; 1.5

major

Question 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.

Answer 56

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.

Question 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.

Answer 57

True!

Question 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.

Answer 58

phosphorylation; serine

amphipathic alpha

*mutation of serine leads to a broken CREB

Question 59

In an estrogen receptor ligand-binding activation domain, what happens when estrogen is present and what happens when estrogen is absent?

Answer 59

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.

Question 60

Transcriptional factor interactions ________ gene-control options.

Answer 60

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.

Question 61

What are the three different types of heterodimeric TFs?

Answer 61

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

Question 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.

Answer 62

True!

Question 63

How is combinatorial transcription regulation achieved of two unrelated TFs?

Give examples.

Answer 63

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

Question 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.

Answer 64

Monomeric; heterodimeric

Low

Stability

Question 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.

Answer 65

Dimeric; monomeric; 5; 30

inverted; flexible linker

Question 66

What is beta-interferon enhancer?

What is it composed of?

What complex forms on the enhancer?

Answer 66

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.

Question 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.

Answer 67

minor; transcription factors

Question 68

What are the two ways for regulation of transcription factor activity?

Answer 68

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.

Question 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.

Answer 69

4! Nuclear receptors are regulated by extracellular hormone signals.

Question 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.

Answer 70

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)

Question 71

What happens when there is no hormone (cortisol) to bind to the nuclear receptor (glucocorticoid receptor)?

Answer 71

Receptor complex is retained in the cytoplasm by interaction between its ligand-binding domain (LBD) and chaperone proteins.

Question 72

What happens when there is a hormone (cortisol) to bind to the nuclear receptor (glucocorticoid receptor)?

Answer 72

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

Question 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.

Answer 73

Inverted; direct

Symmetric dimers; three

Question 74

Heterodimeric nuclear receptor contains one ______ subunit associated with another nuclear-receptor subunit that defines the hormone response.

Answer 74

RXR

Question 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)

Answer 75

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)

Question 76

(T/F) Tissue specificities come from transcription factor specific combinations in cell types.

Answer 76

True!

Question 77

What’s the difference between class I and class II nuclear receptors?

Answer 77

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!

Question 78

What are the four different families of receptors involved in the four different signal transduction pathways?

Answer 78

Pathway A: GPCR (G-protein coupled receptors)

Pathway B: TGF-β

Pathway C: Notch/Delta

Pathway D: Wnt, Hedgehog, IL-1, TNF α

Question 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.

Answer 79

GTP; kinase

Cytosolic; serine-threonine; Smad

Extracellular; proteolytic cleavage; cytosolic

Ubiquitination; degradation

Question 80

What is the key signaling molecule in the canonical Wnt pathway?

How is its stability controlled?

Answer 80

The cytoplasmic protein, β-catenin.

Its stability is controlled by a large multiprotein, β-catenin destruction complex.

Question 81

Briefly describe the Wnt signaling pathway in THE ABSENCE OF WNT.

Answer 81

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.

Question 82

Briefly describe the canonical Wnt signaling pathway.

Answer 82

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.

Question 83

What are the seven forms of regulation of TF activity?

Answer 83

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!

Question 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.

Answer 84

Downstream TFs

Specific; tissue

Gene Regulatory Networks (GRN)

Question 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?

Answer 85

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.

Question 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?

Answer 86

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!

Question 87

What is the Pax6 protien?

Answer 87

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.

Question 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.

Answer 88

True!

Question 89

(T/F) There are several alternative Pax6 promoters and these regulate expression of the gene at different embryogenesis times in different tissues.

Answer 89

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

Question 90

What is eyeless?

Answer 90

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)!

Question 91

How does the UAS/GAL4 system work?

Answer 91

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.

Question 92

GAL4 driven ectopic expression of ey induces the formation of _____ structures with _____________.

Answer 92

eye; photoreceptors

*whether it is being expressed in the wing, the distal antennae, leg, etc.

Question 93

(T/F) The pax6 gene is highly conserved across animals.

Answer 93

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.

Question 94

What are the two logic circuits for gene expression? Describe.

Answer 94

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.

Question 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.

Answer 95

True!

*not just about where it is expressed, but also about when it is expressed.