PSIO 404 Exam 4 Flashcards
In complex with stimulatory
transcription factors,
histone
acetyltransferases (HATs) catalyze the
acetylation of histone Lysines to
“relax” the nucleosome and recruit
proteins with bromo domains
In complex with inhibitory
transcription factors
histone
deacetylase complexes (HDACs)
deacetylate histone Lysines to
“compact” the nucleosomal structure
and inhibit gene transcription.
V. Two Routes for Chromatin Activation
- The association of histones for DNA can be weakened by
mechanical force (Swi/Snf ATPases) - The association are also be weakened by covalent
modifications to histone proteins.
1. Acetylation = Lys
2. Methylation = Arg, Lys
3. Phosphorylation= Ser and Thr
4. Ubiquitinylation= Lys
5. Poly(ADP-ribosylation) = Lys
*lys also uses sumoylation
inactive versus active chromosome
Condensed chromatin
(DNA + histones) is
called “inactive”
Unfolded chromatin is
called “active”
Gene-Specific Transcription Factors
binds to specific DNA
sequences called response elements.
Design of a Typical Gene-Specific
Transcription Factor
- DBD = DNA-binding domain
- RD = regulatory domain
- TAD = transactivating domain
Activators
are signal-activated, gene-specific transcription factors which recognize (bind to) specific response elements.
The phosphorylation of many Ser & Thr residues is required to
remove H1 from the linker DNA region between nucleosomes and generate active (unfolded) chromatin.
H1
H1 kinase and enzymes catalyzing core histones modification
H1 phosphates and enzyme eliminate core histone modifications
Unfolding of chromatin
starts w/ phosphorylation
of H1 Ser/Thr residues by
H1 kinases
the five types of histones
H1 = linker
H2A, H2B, H3, H4 = core histones (these wrap 1.8 turns of DNA into a nucleosome)
Briefly explain how the cell integrates information.
Signals bind receptors which induce intracellular signaling. All meaningful (non-filtered
out) intracellular signaling has an effect on the genome, be it minor (altering proteins such
as histones slightly) or major (inducing major changes in gene expression). In the latter
case, the result is an altering of the makeup of the data-processing protein network (brain)
of the cell
How does nearly every cellular signal physically change the cell?
The moment-to-moment summation of signaling effects on the genome influences
immediate or future behavior of the cell, and in this way a cell has 1. memory and 2. learns
from its environment as well as it is able. This is cellular comprehension of the world
around it, and this comprehension is the basis for a cell’s exhibition of “responsiveness”
a cell does this to alter it DPPN to become less error prone
Define transcription factors, and explain how they work.
a transcription factor are proteins that control gene transcription by interacting with DNA motif (promoters enhancers, and silencers) to favor or prevent the binding and progression of RNA polymerase which will form RNA and DNA templates
Compare and contrast hydrophilic vs. lipophilic signaling with regard to transcription.
hydrophilic (loves water) = involves binding to membrane receptors and employing second messengers that lead to covalent modifications of TF
lipophilic(loves fat)= involves binding to nuclear receptors that lead to non covalent modifications of TF
combinatorial strategy
Genes are regulated by combinatorial strategy. A gene is generally controlled by several response
elements, each of which interacts with an individual transcription factor.
Termination
- At the end of the gene, changes in phosphorylation in RNAPII’s CTD
recruits 3’-end processing factors and leads to dissociation. - A dissociated RNAPII is reset for reinitiation of transcription by CTD
phosphatases.
Initiation
Activator binding triggers assembly of pre-initiation complex.
* Phosphorylation of RNA polymerase II’s repeating heptapeptide C-terminal
domain (CTD) at Ser5 by TFIIH forms a scaffold.
TFIIH = CAK = CDK7/cyclinH
Elongation
- Thanks to Ser5 phosphorylation in RNA polymerase II’s (RNAPII’s) CTD, the
mRNA is 5’ capped. - CDK9/cyclinT(K) phosphorylation at Ser2 in RNAPII’s CTD recruits
elongation factors and mRNA processing enzymes.
Name and briefly describe the three phases of gene transcription.
initiation = begins with the formation of an initiation complex at a gene region as the promoter this region is marked in advance by TF that recognize the nucleotide sequence of the promoter DNA motif
Elongation = initiation complex is replaced by an elongation complex
termination = transcription ends with dissociation of an elongation complex upon reaching the terminator gene region
Describe the components of the transcription initiation complex.
-the initiation complex is formed by RNA polymerase II and the TFII family general TF
-TFIID binds to DNA at the promoter and nearby regions
Describe how general and specific transcription factors are alike and how they are different with regard to their functions.
general TF – In combination with RNA polymerase II, these constitute the “basic machinery” required for transcription.
– Assemble at promoters bound by a Gene-Specific TF
Specific TF binds to specific DNA
sequences called response elements.
-Response elements are in promoters, enhancers, and silencers.
Describe response elements. How is the expression of 25,000 human genes controlled by only 707 human response elements?
A gene is generally controlled by several response elements, each of which interacts with an individual transcription factor. In this way, genes are regulated by a combinatorial strategy
-Via a combinatorial strategy, the 6% of our genome (genes) which encodes
transcription factors can control all gene activity.
* Additional proteins, called co-activators and co-repressors are involved as well (enhanceosomes)
Histones & Nucleosomes
- Five major types
– 1 linker histone: H1
– 4 core histones: H2A, H2B, H3, and H4. - The 4 core histones wrap 1.8 turns of DNA into a nucleosome
Describe two routes for how chromatin is activated.
To activate H1 kinases and enzymes catalyzing core histone modifications that allow availability of the linker region of DNA to allow gene specific TF can interact with them
1. The association of histones for DNA can be weakened by mechanical force (Swi/Snf ATPases) by constantly pushing on DNA to move them off histones make them available quicker
2. The association are also be weakened by covalent modifications to histone proteins
To inactive H1 phosphatase and enzymes eliminating core histones modifications and encourage a region of DNA to become inactive (condensed)
Give at least 3 examples of post-translational modifications to histones which control the availability of DNA to the transcriptional machinery.
- Acetylation
- Methylation, Arg (R) modification: methylation.
- Phosphorylation, Ser (S) and Thr (T) modification: phosphorylation
- Ubiquitinylation
- Poly(ADP-ribosylation)
Lys (K) modifications: methylation, acetylation, ubiquitinylation, ADP-ribosylation, sumoylation (ubiquitin like)
Describe the way in which acetylation is used to control chromatin activation.
- TFs work with HATs (histone acetyltransferases) to catalyze acetylation of histone Lys
- This relaxes nucleosome and recruits proteins with bromo domains
- Inhibitory TFs work with HDACs (histone deacetylase complexes) to undo it and compact nucleosome to inhibit gene transcription
Define ‘activators’ and explain their importance in transcription.
- In complex with stimulatory
transcription factors, histone
acetyltransferases (HATs) catalyze the
acetylation of histone Lysines to
“relax” the nucleosome and recruit
proteins with bromo domains.
-“activators” are gene-specific TFs bind to identify promoters where transcription will take place - triggers assembly of pre-initiation complex
-In complex with inhibitory
transcription factors, histone
deacetylase complexes (HDACs)
deacetylate histone Lysines to
“compact” the nucleosomal structure
and inhibit gene transcription
Describe the role of the CTD region of RNA polymerase II in signaling during the transcription cycle.
The CTD region is phosphorylated (at Ser5) by TFIIH to form a scaffold for the elongation proteins.
The p-lation at CTD also provides a 5’ cap!
At the end of the gene, changes in p’lation at CTD recruit 3’ end processing factors to lead to dissociation/termination.
RNAPII’s CTD is reset by phosphatases
Steroids
are lipophilic chemical messengers which can
directly modify the genetic output of a cell (estrogen,
progesterone, cortisol, aldosterone, etc.). However, it
can take up to hours to see the results of changes in
gene transcription and subsequent protein synthesis.
* Steroids can do more than control gene transcription
and can do so quickly.
1. Progesterone also triggers the acrosomal reaction within
seconds.
2. Steroids also rapidly dampen excitation of nerve cells.
Describe the non-genomic effects of progesterone.
these are non genomic because they cannot be mediated by gene expression
progesterone has the ability to trigger the acrosomal reaction within seconds
1. Progesterone induces a Ca2+
influx into a sperm cell within
seconds of binding to a non classical GPCR to trigger
the acrosomal reaction. This reaction generates the ability for a sperm cell to effectively digest through the layers of the oocyte
Progesterone likely binds to a
non-classical GPCR in sperm in
order to accomplish this task.
*progenstion can also help active hyperactivation of the sperm tail through the same second messenger (calcium influx)
Describe the non-genomic effects of anesthetics.
- Steroids interact specifically
with a subunit of the
g-aminobutyric acid (GABA A)
receptor anion channel,
stimulating an influx of
chloride ions leading to
hyperpolarization. By this, the
excitation of nerve cells in
dampened to produce
anesthesia
*this is also like gaba flood from a brain injury when the brain is impacted the neuroglia release large amounts of GABA to inhibit neural activity
Discuss three ways in which transcription factors can be activated: directly by Ca2+, by covalent modification by other proteins (mainly phosphorylation), and by noncovalent binding of lipophilic messengers (in the case of nuclear receptors).
directly by Ca2+: sensory stimuli to allow ion channels to allow Ca influx which Ca activates calmodulin dependent kinases and they target transcription factors
Covalent modification by other proteins:
noncovalent binding of lipophilic messengers:
List and very briefly describe the function of the three general domains of steroid receptors. How is the structure of steroid receptors different from that of other transcription factors?
- The ligand-binding
domain (a specific
type of regulatory
domain, RD) which
binds to hydrophobic
ligands like steroid hormones - A DNA binding domain (DBD)
- A transactivating domain (TAD)
steroid hormone receptors (most common) require maintenance of structure due to interaction with heat shock proteins (chapernes) they are different because they have complexes with HSP90 homodimerization, which is when two glucocorticoid attach to the dimer unlike non steroid nuclear receptors which use retinoid x RXR, where only one hormone is required which are heterodimers
Name 2 common structural motifs of DNA binding domains in transcription factors used to bind specific DNA sequences.
helix - turn - helix motifs =
Zinc finger motifs =
“consensus sequences” are an
attempt to define a single target
sequence of DNA for a
transcription factor’s DBD, but in
truth, variation exists in what DBDs
can bind to, and the variation is
important for DPPN function.
– Variations between response
elements confers additional
possibilities for fine tuning when a
transcription factor can bind to
multiple response elements.
– Not all transcription factors can bind
more than 1 response element though
The DNA-binding domain of the
Glucocorticoid receptor (GR) binds
to all of the following “consensus-
containing” response elements:
Response Elements for Nuclear
Receptors
Orientation may be parallel or anti-parallel (in which
case the response element is palindromic).
Describe the mechanism of activation of nuclear receptors which bind to steroid hormones.
Monomers each
bind lipophilic ligand
* Bound monomers
homo- or hetero-
dimerize
* Dimers translocate
to nucleus to act as
gene-specific
transcription factors
Describe the role of chaperones steroid receptor function.
maintain the function state of nuclear receptors (steroids) because they are continue collapsing into the low energy non active stable state so chaperone help them regain
there active state help by using energy (ATP)
Chaperones form complexes with other proteins (chaperone
assistants) that support their effects on protein conformation.
* Chaperone assistants include immunophilins, cyclophilins, and
FK-binding proteins. They are widely distributed (~30 isoforms)
Describe the actions of HSP70 and HSP90 in the activation of steroid receptors. Highlight two steps involving the delivery of energy from ATP hydrolysis to the receptor.
HSP70 and HSP90 along with other
proteins, transfer
the energy of ATP to
the receptor in two
steps to maintain
the active receptor
conformation
step 1:HSP40 binds HSP70 and they bind to a collaples stable state of a receptor, then HSP70 will hydrolyze ATP where the energy will be delivered to the receptor
step 2: ADP loaded HSP90 with high order interacts with the receptor that has been particle activated where and ADP ATP exchange will occur and cause the site to expand to its full extent (ATP hydrolyze switch) and this just repeats
Describe the action of peptidyl-prolyl isomerases (PPIases) on a specific type of bond in proteins. Why is their action needed?
Immunophilins are
peptidyl-prolyl
cis-trans isomerases
(PPIases) which
catalyze isomerization
of peptide bonds at the
imide nitrogen of prolyl
residues (proline amino
acids) in proteins.
they use peptide bonds
think of them like co chaperons
these many only help a a small amount each but when you add all of the thousands up kit can lead to noticeable effects of the effectiveness of the DPPN
having too few lead to neurodegenerative disease because of the accumulation of trans or cis in many proteins leading to greater likelihood of aggregation of proteins
having to many is associated with cancer because th4ere is a resistance in going back to the state before
Non-Steroid Nuclear Receptors and
the Toxic Stress Response
III. Non-Steroid Nuclear Receptors and
the Toxic Stress Response
* Upon dimerization with RXR, non-steroid nuclear receptors
act as transcription factors to carry out signals involving
thyroid hormone, vitamin D, cholesterol metabolism, etc.
* Some receptors in this second family of nuclear receptors are
capable of responding strongly to foreign compounds such as
pharmaceutical drugs and poisons. Such compounds are called xenobiotics, and the receptors which are bound by
them are thus called xenosensors. When this happens, the
cell initiates the toxic stress response.
* The toxic stress response ideally uses p450-dependent
oxygenases to oxidize dangerous substances into products
that are generally less toxic or biologically inactive
Define xenobiotics, xenobiotic receptors, and the toxic stress response.
- Xenobiotic receptors bind to, and enable metabolism of,
drugs and toxins, and there are 3 types:
– PXR (SXR in humans, and its endogenous ligands are
glucocorticoids) induces production of enzymes of
detoxification (such as cytochrome P450 type 3A responsible
for more than 60% of drug metabolism)
– CXR (Constitutive Androstane Receptor) binds barbiturates and
targets genes with a phenobarbitol response element in them.
(Phenobarbitol is a barbiturate, a very large family of drugs.)
– FXR binds derivatives of farnesol, vitamin A and bile acids and
targets genes with a bile acid response element in them. A
related receptor, LXR, binds oxysterols which are products of
oxidative cholesterol metabolism.
Discuss the functions of cytochrome p450.
Cytochrome p450 enzymes add oxygen atoms
to targets. This usually makes the molecule
more soluble in water and therefore easier to
eliminate. Added oxygen atoms also provides a
foundation for other detoxifying enzymes to
further modify and destroy toxic molecules.
Cytochrome p450 enzymes use heme prosthetic
groups (orange) to add oxygen to a molecule.
* Cytochrome p450 enzymes also have an
essential role in synthesis of normal
biomolecules. For example, some cytochrome
p450 isoforms perform chemical steps in the
construction of steroids, vitamins A and D,
eicosanoids, and cholesterol.
Discuss the functions of the arylhydrocarbon (ArH) receptor.
- The arylhydrocarbon receptor (ArH) is not a member of the nuclear receptor family, but it acts like one.
- ArH binds to lipophilic xenobiotics (like dioxin or
benzopyrene) thanks to its association with Hsp70 &
Hsp90. - Upon binding xenobiotics, ArH heterodimerized with
ArNT (ArH Nuclear Translocator) which activates
genes carrying a dioxin or xenobiotics response
element (more than 100 genes are directly
controlled by dioxin!).
Explain how phosphorylation can be either activate or inhibit a transcription factor.
activated: In the transactivating domains (AF1/AF2) phosphorylation
usually activates the transcription factor (TF)
inhibited: In the DNA-binding domain (DBD) or nuclear-localization
signal (NLS), phosphorylation usually inactivates the TF
*Simple repulsion! Negatively-charged phosphates added by
phosphorylation in the DBD repel the negatively-charged
phosphates in DNA
List at least 4 processes involving transcription factors which are controlled by phosphorylation.
Phosphorylation of transcription factors controls
1. nuclear translocation, 2. oligomerization, 3. interaction
with cofactors, 4. DNA binding, 5. transactivating activity, and 6. lifespan via ubiquitinylation.
* Example: cAMP response element binding protein
(CREB) and its isoform CREM (cAMP response
element modulator)
– CREB is a transcription factor which targets genes
containing the cAMP response element (CRE). CRE is found in the promoters of more than 100 different genes
What is CREB, what is its function, and how is it activated?
CREB is a transcription factor which targets genes
containing the cAMP response element (CRE). CRE is found
in the promoters of more than 100 different gene
* CREB is activated by Ser phosphorylation from a variety of kinases (targets of several signaling cascades)
In this example, signals lead
to activation of CREB by:
1. Binding to a GPCR.
2. GSa activates AC.
3. AC increases [cAMP].
4. PKA is activated.
5. CREB is phosphorylated.
6. CREB dimerizes
7. CREB binds to (~100)
CRE-containing genes
What is CREs, and where are they found?
cAMP response element (CRE). CRE is found
in the promoters of more than 100 different genes
Describe the involvement of CBP/p300 with CREB.
Upon activation, CREB recruits the transcriptional co-activator CBP/p300 to CRE-containing genes.
* CBP (CREB-binding protein) is a very abundant co-activator thanks to its
versatility as a scaffold protein.
* CBP/p300 is also activated by numerous signaling cascades (PKA, MAPK, CaMK, etc.).
* CBP/p300 binds to many general as well as to other gene- specific transcription factors, including the steroid hormone (nuclear) receptors
Be able to list at least 7 proteins which interact with CBP/p300.
- CREB
- RXR
- TBP
- TFIIF
- TFIIEB
- Steroid hormone receptor
- SMAD
- GATA 1
- p53
- TWIST
- Junb
Describe an example of signaling through CREB, from the binding of a signal to gene transcription.
- upon activation CREB recruits the transcriptional coactivator CBP/p300 to CRE containing genes (only were it is interacting with the DNA)
2.
Explain how cAMP and CREB can be involved in addiction.
Cellular adaptation to a non-endogenous signal (in
this case alcohol) creates a long-term change in
excitatory vs. inhibitory signaling in cells.
CRE-containing genes control cell type-specific transcription,
metabolism, cell cycle, and secretion. They also control
memory fixation as well as addiction in the case of cAMP
superactivation
Fully describe the hypoxic stress response.
The key to the hypoxic stress response is HIFa, a
transcription factor which is also an oxygen sensor.
– HIFa is constitutively modified by
hydroxylation (an O2-dependent reaction).
– Hydroxylation of HIFa triggers action by the E3 ubiquitin ligase
VHL, keeping levels of HIFa low by degradation.
– If HIFa levels rise slightly, it heterodimerizes with HIFb
(identical to ArNT!) to transcribe genes necessary for survival
such as Glu transporters, enzymes of anaerobic metabolism,
transporter/exchangers to prevent acidification, transferrin
and its receptor, and factors that increase blood supply to the
tissue (angiogenesis)
Describe the role of HIFα in the activation of the hypoxic stress response.
HIFa is constitutively modified by
hydroxylation (an O2-dependent reaction).
– Hydroxylation of HIFa triggers action by the E3 ubiquitin ligase
VHL, keeping levels of HIFa low by degradation.
– If HIFa levels rise slightly, it heterodimerizes with HIFb
(identical to ArNT!) to transcribe genes necessary for survival
such as Glu transporters, enzymes of anaerobic metabolism,
transporter/exchangers to prevent acidification, transferrin
and its receptor, and factors that increase blood supply to the
tissue (angiogenesis)
Describe the organization of MAP kinase modules.
MAP kinase modules are
“bottleneck” devices that
transform a wide variety of
input signals into a
comparatively large variety
of output signals.
– outputs signals address
metabolic reactions, cell
architecture and mobility,
and gene transcription
