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
Name and briefly describe the three best-known MAP kinase modules.
the mitogenic module :RAS-> RAF-> MKK1,2 -> ERK
the JNK stressmodule = Rac cdc42-> MEKK or MLK -> MKK4,7->JNK (SAPK)
the p38 stress module = MEKK or MLK -> MKK3,6 ->p38
Name and discuss the function two families of transcription factors which are standard substrates of MAP kinases.
All MAP kinases catalyze (directly or nearly directly) the
phosphorylation of transcription factors (TFs). Standard TF
substrates for MAP kinases include:
1. The Ets family (characterized by an Ets domain)
* Critically important for the formation, maintenance, and
function of tissues.
* Controlled by ERK1,2, JNK, and p38 modules.
* Upon phosphorylation, they form complexes with other
transcription factors to control early response genes.
2. The AP1 (activator protein 1) family
* Homo- and heterodimers of cJus, cFos, ATF2, and MAF
* They are controlled by stress modules, and they
regulate the activity of a wide variety of gene
Describe the structure and mechanism of action of the Ets family of transcription factors.
- Ets family transcription factor action
1. Phosphorylation
by MAP kinase
2. Ets domain is
exposed & binds
3. A ternary complex
forms with another
transcription factor
4. TAD domain is exposed
5. Transcription may now
begin
Describe the basic construction of NFκB modules.
NFkB Module Construction:
1. A protein kinase senses inputs
2. A transcription factor transmits
outputs
* Transcription factor has a
regulatory subunit
and a catalytic subunit
– No signal = inhibition of the
“catalytic” subunit.
– With signal, the protein kinase
modifies the regulatory subunit
(IkB) so it is degraded and
outputs are generated.
Describe the basic function of NFκB modules in the presence or absence of input signals.
- NFkB Module Construction:
1. A protein kinase senses inputs
2. A transcription factor transmits
outputs - Transcription factor has a
regulatory subunit
and a catalytic subunit
– No signal = inhibition of the
“catalytic” subunit.
– With signal, the protein kinase
modifies the regulatory subunit
(IkB) so it is degraded and
outputs are generated.
Briefly describe the typical domain structure and function of a catalytic subunit from an NFκB module.
- All catalytic subunits have in common N-terminal
Rel homology domains (RHD) which allow:
1. Dimerization
2. Nuclear targeting
3. DNA binding
Be able to list 3 common input signals for NFκB modules.
` Signals: NFkB inputs include pro-inflammatory
cytokines, antigens, bacterial components, and
stress factors (such as UV light, hypoxia, oxidative
stress, radiation damage, etc.).
* Protein kinases: the IK kinases IKK1 & IKK2 (held
together by scaffold protein NEMO) or alone
phosphorylate the regulatory subunit.
* Transcription Factor Regulatory subunits: IkBs
* Transcription Factor Catalytic subunits: cRel, RelA,
RelB, p50 (NFkB1), and p52 (NFkB2)
Name an example of an NFκB module protein kinase.
the “IK kinases” (PK) IKK1 & IKK2 (held
together by scaffold protein NEMO) or alone
phosphorylate the regulatory subunit
Name 3 examples of an NFκB module catalytic subunit.
cRel, RelA,
RelB, p50 (NFkB1), and p52 (NFkB2)
Discuss how the basic function of NFκB modules is illustrated in both canonical & non-canonical pathways.
look at pic on phone
Discuss the outputs of NFκB modules.
Outputs: NFkB outputs control genes involved in
1. the activation of innate immunity (inflammatory reaction)
2. adaptive immunity
3. growth and differentiation
4. cell adhesion molecules
* In most tissues, NFkB activates genes that promote the
survival of cells by promoting expression of anti-apoptotic
and pro-mitogenic proteins.
* Pathogenic overexpression of NFkB module outputs can
lead to cancer, inflammatory diseases, and enhanced
virulence of certain viruses
Explain the functions of the basic machinery of translation: ribosomes, large and small ribosome subunits, rRNA, tRNA, and mRNA & mRNA modification. How much cellular energy is expended in translation?
- Ribosomes are protein complexes with
catalytic RNA (rRNA) cores that translate
mRNA code into amino acid sequence. - The large (60S) and small (40S) subunits
complex around mRNA. Additional
requirements include tRNAs which match
their anticodons to mRNA codons so that
appropriate amino acids are attached to
the growing polypeptide. - Translation is responsible for up to
50% of ATP/GTP hydrolysis in a cell
Briefly discuss the three phases of translation and the types of factors which control them.
- Translation has 3 phases
1. Initiation: controlled by initiation factors
2. Elongation: controlled by elongation factors
3. Termination: controlled by termination factors
Discuss in detail the events of translation initiation.
- Initiation
1. 43S complex assembly
– (Dissociation of the ribosomal subunits by eIF1A and eIF3)
– 40S binds eIF1, eIF1A, eIF5, and eIF2 ( a GTPase which binds Met-tRNA) (this GTP is required to find the start codon in step 3)
eIF2 & eIF5B GTP hydrolysis provides energy for initiation
complex formation and subsequent disassembly.
2. 48S complex assembly
– The mRNA m7GpppXcap is bound by eIF4F (eIF4A, -G, and -E) which positions the mRNA in the 43S complex.
– The poly(A)-binding protein (PABP) binds the polyadenylated 3’ end (the mRNA tail) and anchors it to the eIF4F complex (MNK is an activator of eIF4E)
3.80S complex forms after scanning to start codon (AUG)
~5% of mRNAs contain internal ribosomal entry
sites (IRES) in their 5’ UTRs rather than a 5’ cap.
Discuss in detail the events of elongation.
- Aminoacyl tRNAs are transferred to the A site by eEF1A
- Ribosome catalyzes peptide bond formation
- eEF2 catalyzes 80S translocation (deacylated tRNA (the tRN without the amino acid on it)→ E site; the peptidyl-tRNA → P site; re-exposing the A site)
this continues as we add amino acids top the poly peptide
-uses peptide bond
Termination
- eRF1 recognizes the stop codon in the mRNA triggering 80S arrest
and polypeptide release - eRF3 releases eRF1 from the ribosome
- eIF3 & eIF1A (+ other proteins) induce 80S dissociation
Discuss in detail the events of translation termination.
Briefly discuss the source of energy for initiation complex formation. Energy for initiation complex formation comes from GTP hydrolysis by which two translation factors?
Explain the function of the following four eukaryotic translation factors: eIF2, eIF2B, eIF4E, and eEF2.
eIF4E = is the translation factor with bi8nds to the five primer cap of mRNA
EEf2 = is the translation factor which used the energy of GTP hydrolysis to translocate the ribosome along the RNA
eIF2 = the translocation factor GTPase which attaches to the methionyl tRNA which will bind across from the start codon to become the starting points and order
eIF2b = is a GEF of eIF2, since eIF2 employs the GTP hydrolysis switch tere is a GEF and a GAP for it so they can control the operations like initiation of the process which consumes up to half of your cellular energy
Briefly discuss the source of energy for elongation complex formation. Energy for elongation complex translocation comes from GTP hydrolysis by which translation factor?
eIF2
Describe the mechanism by which proteins with signal peptides are translated into the ER membrane or into the ER lumen.
membrane = SRPR
lumen = signal peptide
Discuss the GEFs and GAPs of the SRP and SRPR; and explain how the energy from GTP hydrolysis is used.
SRP & SRPR are both regulatory GTPases
* SRP GEF: translating ribosome
* SRP GAP: SRPR-GTP
* SRPR GEF: free translocon
* SRPR GAP: SRP-GTP
GTP hydrolysis provides energy for
1. Transportation of the ribosome to
the translocon
2. Threading of the polypeptide chain
through the translocon
Discuss in detail how signals influence three specific initiation factors to control translation at the level of translation initiation.
Control of Initiation
– Mitogens and Insulin trigger
initiation by inhibiting GSK3
(glycogen synthase kinase 3)
– Mitogens promote initiation
by stimulating activators of
cap-binding protein eIF4E
and by stimulating the
activity of mTOR, a repressor
of eIF4E activity.
– Stress factors stimulate eIF2
kinases (repressors of eIF2
activity) to slow initiation
Describe in the control of protein biosynthesis in a reticulocyte under the following conditions: low iron, viral infection, amino acid deficiency, and the presence of excessive misfolded protein. Is this specific to reticulocytes or a common theme for all cell types?
ron deficiency, viral infection, amino acid deficiency, and the
unfolded-protein response all stimulate eIF2 phosphorylation.
pic on phone
Discuss the inhibition of protein biosynthesis via the four isomers of eIF2 kinase.
- HRI (heme-regulated
inhibitor)
– Adjusts protein synthesis
to iron, heme, and energy
availability
– expressed primarily in
reticulocytes - PKR (protein kinase R)
– Has an interaction domain
for dsRNA and suppresses
protein synthesis when
dsRNA is present - GCN2 (named for gene in
flies)
– Dimeric transmembrane
protein of the ER
– Activated by unloaded tRNA
and slows protein synthesis
when amino acids are low - PERK (PKR-related ER kinase)
– Transmembrane ER protein
– Slows protein synthesis as
part of the unfolded-protein
response (UPR)
Describe the details of each of the four events of the unfolded protein response.
IIIA. The Unfolded-Protein Response (UPR)
* UPR events
1. Recognition of
defective ER
proteins.
2. Stoppage of
protein
synthesis.
3. Activation of
stress-
responsive
genes
4. Elimination of
defective
proteins or
apoptosis if
that fails
Discuss three mechanisms which lead to the production of transcription factors that recognize genes with UPR response elements. How does a cell ensure the resulting mRNA transcripts are translated into protein?
- Recognition of
defective ER proteins.
* BiP is an HSP70-type
ATPase (a chaperone)
which binds misfolded
proteins.
* [BiP] is sensed by 3
proteins: - PERK
- IRE1
- the ATF6 precursor
- Stoppage of protein
synthesis.
– PERK is an eIF2
kinase and promotes
transcription factor
ATF4 expression
– ATF6 is also a
transcription factor
(it is released by
precursor
proteolysis)
– IRE1 is an
endoribonuclease
which releases the
mRNA of XBP1. - Activation of stress-
responsive genes
– ATF4, XBP1, and
ATF6: transcription
factors which bind
to genes with UPR
response elements. - Elimination of
defective proteins
(or apoptosis if that
fails).
– Defective proteins
are shuttled to
cytoplasm for
ubiquitinylation
Discuss in detail how signals influence eEF2 to control translation at the level of translation elongation.
- Control of Elongation
– Mitogens and Insulin trigger
elongation through increased
PI3K-PKB signaling which
results in activation of mTOR
which inhibits the activity of
eEF2 kinases which inactivate
eEF2 by phosphorylation.
– Stress factors stimulate
AMPK (5’-AMP-dependent
protein kinase) to stimulate
eEF2 kinases and slow
elongation
therefore mTOR promotes eEF2 phosphorylation through inhibition of eEF2 kinases which inhibits eEF2
Discuss the functions of AMPKs in the human cell.
AMPKs are monitors of cellular energy status and reserves. In
response to low [ATP] or low [fuel], you get activation of AMPK and they turn down ATP-consuming anabolic metabolism (synthesis of macromolecules,
fatty acids, cholesterol, etc.) and instead stimulate ATP-delivering
catabolic reactions (glycolysis, fatty acid oxidation, etc.).
AMPK is a universal energy sensor, a
master regulator of energy metabolism
Describe how PKB is activated by survival and insulin signals, and describe how PKB promotes insulin action.
- PKB transduces
survival signals
and insulin signals,
with mTOR being a
major target/effector
Discuss the two major functions of mTOR.
mTOR is activated
by Rheb-GTP (Ras
homology enriched
in brain)
mTOR activation
– Rheb-GEFs are
downstream of
growth factor and
hormone receptors
– Rheb-GAPs include
TSC proteins (tumor
suppressor proteins
inhibited by PKB and
activated by AMPK)
- mTOR is
1. a key regulator of cell
growth
2. encoded by one gene
3. connection btwn PI3K
signaling & regulation
of protein synthesis
Explain how the three major targets of mTOR affect translation.
IIIB. Mitogenic Signaling Cascades Controlling
Translation: Ser/Thr Kinases PKB & mTOR
* PKB transduces
survival signals
and insulin signals,
with mTOR being a
major target/effector.
* mTOR is
1. a key regulator of cell
growth
2. encoded by one gene
3. connection btwn PI3K
signaling & regulation
of protein synthesis
- eEF2 kinases: these
phosphorylate and
inhibit translocation
due to eEF2 - 4E-BP (eIF4E binding
protein) binds to eIF4E - S6Ks
Explain the negative feedback of S6K on the insulin receptor.
About ribosomal S6 kinases
– Two families: kinases p70S6K (S6Ks)
and kinases p90RSK (RSKs)
– Named for one of their substrates (S6) –
a component of small ribosomal subunits
S6Ks inhibit IRS1, a
major insulin
receptor substrate:
– This suppresses
insulin signaling.
– Insulin receptors are
down-regulated.
– Cells become less
sensitive to insulin.
Briefly discuss the activation and inactivation of mTOR.
mTOR activation
– Rheb-GEFs are
downstream of
growth factor and
hormone receptors
– Rheb-GAPs include
TSC proteins (tumor
suppressor proteins
inhibited by PKB and
activated by AMPK
For S6Ks, briefly discuss their function, mechanism of activation, and specific mechanism of up-regulation of translation.
- Kinases p70S6K
– up-regulate protein synthesis (positive
regulators of cell size and body growth).
– Mechanism of activation: 1. N-terminal
domain phosphorylation by mTOR, 2.
activation loop phosphorylation by PDK1
– S6K Actions to up-regulate translation - by inhibiting eEF2 kinases
- by activating eIF4B
(this protein activates eIF4E)
S6K also inhibit IRS1 a major insulin receptor substrate
Mechanism of how cell growth is stimulated by S6K is still a mystery
For RSKs, briefly discuss their functions, mechanism activation, and specific mechanism of up-regulation of translation.
- Kinases p90RSK
– up-regulate protein synthesis by S6 phosphorylation
– act primarily to stimulate transcription
– inactivate the pro-apoptotic protein Bad
– inactivate MYT1 to promote cell division
– activate mTOR signaling
– Activation: 1. phosphorylation by
MAPKs (e.g. ERK or p38), 2. activation
loop phosphorylation by PDK1
– RSK Actions to up-regulate
transcription - by activating transcription factors (such as CREB and the
estrogen receptor) - by activating transcriptional co-activators (such as CBP/p300)
MAP kinases and PDK-1 promote RASK p90 to promote transcription, cell proliferation and inhibition of apoptosis
Explain how mTOR and AMPK signaling interact.
AMPK stimulation and inhibition of protein kinase B battle for the TSC complex, because the more activation of AMPK the more inactivation of mTOR if you have more protein kinase B by insulin factor growth hormones then you have more activation of mTOR because you have activated RHEB
Explain how mTOR overactivation can lead to insulin insensitivity.
s6Ks inhibit IRS1, a
major insulin
receptor substrate:
– This suppresses
insulin signaling.
– Insulin receptors are
down-regulated.
– Cells become less
sensitive to insulin.
-S6K feeding back to phosphorylate IRs on serines, which serine phosphorylation on insulin receptor and insulin receptor substrate can lead to less effective signaling downstream of insulin. The fewer serines that are phosphorylated on the RTK, the insulin receptor, and the IRS docking protein the more effective insulin will be at turning on Pisk and activating protein kinase B which leads to negative feedback because the more activation you get the more activation of S6K activation leads to inhibition of IRs1 which will suppress insulin signaling
Take-home
summary of mTOR
involvement in
insulin resistance:
1. mTOR → S6K –|IRS
(less signaling)
2. high [glucose]blood
→ more insulin
(more signal)
3. adaptation of
overstimulated
insulin receptors
Explain AMPK functions, and explain how AMPK can “save insulin.”
- LKB1 is a constitutively-active kinase that phosphorylates AMPK
when 5’-AMP is bound. LKB1 is a tumor-suppressor (inactive in
GI tumors of those with Peutz-Jeghers syndrome). - Insufficient activation of AMPK (low physical
activity characteristic of obesity, etc) leads to:
1. Greater activation of mTOR
2. Activation of anabolic enzymes
3. Inhibition of glucose transport
NOTE: sufficient AMPK activation can
“save insulin”, preventing insulin resistance
Explain how aberrant AMPK signaling can lead to cancer.
Explain how obesity can contribute to the development of diabetes.
because obesity can lead to greater mTOR activation which will lead to greater activation of S6K activation and you are gonna become desensitized to IRs and become resistant to insulin signaling which will increase insulin signaling and increase insulin levels in the bloodstream and overactivation of insulin receptors and tis combined with large amounts of carb intake it will lead to type two diabetes
Acetylation of Lys residues in histones is expected to lead to activation of transcription.
True
Which histone protein’s role in the unfolding of chromatin is controlled primarily by phosphorylation?
H1
Which region, upon phosphorylation, becomes a scaffold for recruitment of elongation factors and mRNA processing enzymes?
CTD
Retinoblastoma (Rb) protein forms a complex with histone deacetylase 1 (HDAC1) at sites of genes encoding DNA-replication proteins
so under-expression might favor development of cancer.
Identify all of the co-activators of transcription in the list below.
CBP and p300
Which of the following is not true regarding transcription?
Transcription of a given gene requires a unique transcription factor designed for that individual gene only.
What is the name for a transcription factor that binds to non-promoter gene regions to encourage formation of a transcription-initiation complex?
Activator
Which of the following is a histone modification which always encourages the activation of transcription?
Acetylation
What is the interaction partner for bromo interaction domains?
acetylated Lys
Which of the following modifications is made to both DNA and histone proteins in humans?
methylation
Which component of the chaperone complex of HSP70 and HSP90 operates as a true ATP hydrolysis switch (hydrolyzing ATP in order to create greater order in itself for later transfer to client proteins)?
HSP90
Which of the following receptors is the least common in our cells?
guanylate cyclase (GC)-coupled receptors
Which of the following is a receptor anion channel whose subunit interacts with steroids?
GABAA
The two most common structural motifs in proteins for interaction with DNA sequences are helix-turn-helix motifs and
Zn2+ finger motifs
Which of the following is a receptor for toxins such as dioxin?
ArH
Which receptor, when activated, promotes the production of proteins responsible for metabolism of more than 60% of drugs?
PXR
Immunophilins are widely distributed chaperone assistants which catalyze changes in protein structure. Their enzymatic activity categorizes them as __________.
peptidyl-prolyl cis-trans isomerases (PPIases)
Steroids are __________ chemical messengers which can directly modify the genetic output of a cell.
lipophilic (hydrophobic)
Peptidyl-prolyl cis-trans isomerases catalyse rotation about which type of bond?
peptide
PPIases catalyze isomerization of peptide bonds next to which amino acid?
proline
Which of the following is identical to the hypoxia-inducible transcription factor HIFβ?
ArNT
CRE stands for
cAMP response element.
Which of the following is a gene-specific transcription factor and happens to also be a very abundant co-activator thanks to its versatility as a scaffold protein?
CBP
One effect from the binding of glucocorticoids to glucocorticoid receptors is the sequestration and inhibition of _____. This at least partially explains the anti-inflammatory effect of glucocorticoids.
NFκB
Modification of HIFα in which way promotes its action as a transcription factor?
S-nitrosylation
VHL is which type of enzyme?
an E3 ubiquitin ligase
With regard to our discussion of alcohol addiction, greater expression of NMDA receptors leads to __________ for a nerve cell, and that action balances out (adapts the nerve cell to) the __________ induced by alcohol-activation of GABAA receptors.
depolarization; hyperpolarization
Theoretically, what type of mutation in NFκB signaling could lead to cancer?
Loss of Rel-inhibiting function by IκB.
What other mutations in NFκB signaling could lead to cancer?
mutations resulting in a hyperactive IKK isoform
A hyperactive IKK isoform is an oncogenic factor for breast cancer.
How can a virus’s virulence be enhanced by over-activation of NFκB signaling?
It can lead to tumor development.
This happens with HIV, hepatitis C virus and herpes simplex virus.
Which of the following is not a direct sensor of free BiP concentration in the lumen of the ER?
XPB1
Mark the way(s) in which a cell reacts to the situation of ER stress.
by destroying misfolded proteins.
by making more chaperone proteins.
Match the following eukaryotic translation factors with their function.
__ eIF1__
This translation factor recognizes the start codon in mRNA (AUG).
__ eRF1_
This translation factor recognizes the stop codon.
__ eIF3__
This translation factor cooperates with eIF1A to induce dissociation of the ribosomal subunits so that mRNA can bind to the ribosomal translation apparatus.
__ eEF2__
This is a GTPase which uses the energy of GTP hydrolysis to translocate mRNA through the ribosome (or as described classically, to translocate the ribosome along the mRNA).
__ eIF4F__
This translation factor binds to the m7GpppX cap and is composed of eIF4E, eIF4A, and eIF4G.
Which of the following kinases directly phosphorylates and inhibits eEF2 kinase?
mTOR
In contrast to the other kinases in the following list, which is a lipid kinase rather than a protein kinase?
PI3K
Which of the following is a lipid anchor for the activation of PKC by PDK1?
DAG
Which of the following is a lipid anchor for the activation of PKB by PDK1?
PIP3
Which of the following kinases directly phosphorylates and activates eEF2 kinase?
AMPK
There is a ubiquitous enzyme of ATP metabolism named Adenylate Kinase (ADK) which reversibly-catalyzes the following reaction: ADP + ADP ↔ ATP + AMP. The action of this enzyme is essential for ATP metabolism, especially when ADP levels rise. Signaling by which of the following enzymes is directly connected to the action of ADK?
AMPK
Which of the following is a lipid anchor for the activation of mTOR by PDK1?
Rheb-GTP
Which enzymes are inhibitors of IRS function (and thus potential contributors to insulin resistance in cells)?
S6Ks
TSC proteins are activated when phosphorylated by _____, and they are inhibited when phosphorylated by _____.
AMPK; PKB
Kinases p90RSK are activated 1) by phosphorylation by MAPKs, and 2) by activation loop phosphorylation by PDK1. Which MAP Kinase modules are involved in the first process?
p38 and ERK
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
