PSIO 404 Exam 4 Flashcards

1
Q

In complex with stimulatory
transcription factors,

A

histone
acetyltransferases (HATs) catalyze the
acetylation of histone Lysines to
“relax” the nucleosome and recruit
proteins with bromo domains

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2
Q

In complex with inhibitory
transcription factors

A

histone
deacetylase complexes (HDACs)
deacetylate histone Lysines to
“compact” the nucleosomal structure
and inhibit gene transcription.

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3
Q

V. Two Routes for Chromatin Activation

A
  • 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

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4
Q

inactive versus active chromosome

A

Condensed chromatin
(DNA + histones) is
called “inactive”

Unfolded chromatin is
called “active”

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5
Q

Gene-Specific Transcription Factors

A

binds to specific DNA
sequences called response elements.

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6
Q

Design of a Typical Gene-Specific
Transcription Factor

A
  • DBD = DNA-binding domain
  • RD = regulatory domain
  • TAD = transactivating domain
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7
Q

Activators

A

are signal-activated, gene-specific transcription factors which recognize (bind to) specific response elements.

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8
Q

The phosphorylation of many Ser & Thr residues is required to

A

remove H1 from the linker DNA region between nucleosomes and generate active (unfolded) chromatin.

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9
Q

H1

A

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

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10
Q

the five types of histones

A

H1 = linker
H2A, H2B, H3, H4 = core histones (these wrap 1.8 turns of DNA into a nucleosome)

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11
Q

Briefly explain how the cell integrates information.

A

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

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12
Q

How does nearly every cellular signal physically change the cell?

A

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

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13
Q

Define transcription factors, and explain how they work.

A

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

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14
Q

Compare and contrast hydrophilic vs. lipophilic signaling with regard to transcription.

A

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

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15
Q

combinatorial strategy

A

Genes are regulated by combinatorial strategy. A gene is generally controlled by several response
elements, each of which interacts with an individual transcription factor.

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16
Q

Termination

A
  • 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.
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17
Q

Initiation

A

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

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18
Q

Elongation

A
  • 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.
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19
Q

Name and briefly describe the three phases of gene transcription.

A

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

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20
Q

Describe the components of the transcription initiation complex.

A

-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

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21
Q

Describe how general and specific transcription factors are alike and how they are different with regard to their functions.

A

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.

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22
Q

Describe response elements. How is the expression of 25,000 human genes controlled by only 707 human response elements?

A

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)

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23
Q

Histones & Nucleosomes

A
  • 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
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24
Q

Describe two routes for how chromatin is activated.

A

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)

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25
Give at least 3 examples of post-translational modifications to histones which control the availability of DNA to the transcriptional machinery.
1. Acetylation 2. Methylation, Arg (R) modification: methylation. 3. Phosphorylation, Ser (S) and Thr (T) modification: phosphorylation 4. Ubiquitinylation 5. Poly(ADP-ribosylation) Lys (K) modifications: methylation, acetylation, ubiquitinylation, ADP-ribosylation, sumoylation (ubiquitin like)
26
Describe the way in which acetylation is used to control chromatin activation.
1. TFs work with HATs (histone acetyltransferases) to catalyze acetylation of histone Lys 2. This relaxes nucleosome and recruits proteins with bromo domains 3. Inhibitory TFs work with HDACs (histone deacetylase complexes) to undo it and compact nucleosome to inhibit gene transcription
27
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
28
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
29
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.
30
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)
31
Describe the non-genomic effects of anesthetics.
2. 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
32
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:
33
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?
1. The ligand-binding domain (a specific type of regulatory domain, RD) which binds to hydrophobic ligands like steroid hormones 2. A DNA binding domain (DBD) 3. 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
34
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:
35
Response Elements for Nuclear Receptors
Orientation may be parallel or anti-parallel (in which case the response element is palindromic).
36
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
37
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)
38
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
39
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
40
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
41
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.
42
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.
43
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!).
44
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
45
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
46
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
47
What is CREs, and where are they found?
cAMP response element (CRE). CRE is found in the promoters of more than 100 different genes
48
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
49
Be able to list at least 7 proteins which interact with CBP/p300.
1. CREB 2. RXR 3. TBP 4. TFIIF 5. TFIIEB 6. Steroid hormone receptor 7. SMAD 8. GATA 1 9. p53 10. TWIST 11. Junb
50
Describe an example of signaling through CREB, from the binding of a signal to gene transcription.
1. upon activation CREB recruits the transcriptional coactivator CBP/p300 to CRE containing genes (only were it is interacting with the DNA) 2.
51
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
52
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)
53
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)
54
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
55
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
56
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
57
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
58
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.
59
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.
60
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
61
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)
62
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
63
Name 3 examples of an NFκB module catalytic subunit.
cRel, RelA, RelB, p50 (NFkB1), and p52 (NFkB2)
64
Discuss how the basic function of NFκB modules is illustrated in both canonical & non-canonical pathways.
look at pic on phone
65
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
66
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
67
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
68
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.
69
Discuss in detail the events of elongation.
1. Aminoacyl tRNAs are transferred to the A site by eEF1A 2. Ribosome catalyzes peptide bond formation 3. 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
70
Termination
1. eRF1 recognizes the stop codon in the mRNA triggering 80S arrest and polypeptide release 2. eRF3 releases eRF1 from the ribosome 3. eIF3 & eIF1A (+ other proteins) induce 80S dissociation Discuss in detail the events of translation termination.
71
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
72
Briefly discuss the source of energy for elongation complex formation. Energy for elongation complex translocation comes from GTP hydrolysis by which translation factor?
eIF2
73
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
74
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
75
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
76
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
77
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)
78
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
79
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?
1. Recognition of defective ER proteins. * BiP is an HSP70-type ATPase (a chaperone) which binds misfolded proteins. * [BiP] is sensed by 3 proteins: 1. PERK 2. IRE1 3. the ATF6 precursor 2. 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. 3. Activation of stress- responsive genes – ATF4, XBP1, and ATF6: transcription factors which bind to genes with UPR response elements. 4. Elimination of defective proteins (or apoptosis if that fails). – Defective proteins are shuttled to cytoplasm for ubiquitinylation
80
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
81
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
82
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
83
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
84
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 1. eEF2 kinases: these phosphorylate and inhibit translocation due to eEF2 2. 4E-BP (eIF4E binding protein) binds to eIF4E 3. S6Ks
85
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.
86
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
87
For S6Ks, briefly discuss their function, mechanism of activation, and specific mechanism of up-regulation of translation.
1. 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 1. by inhibiting eEF2 kinases 2. 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
88
For RSKs, briefly discuss their functions, mechanism activation, and specific mechanism of up-regulation of translation.
2. 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 1. by activating transcription factors (such as CREB and the estrogen receptor) 2. 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
89
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
90
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
91
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
92
Explain how aberrant AMPK signaling can lead to cancer.
93
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
94
Acetylation of Lys residues in histones is expected to lead to activation of transcription.
True
95
Which histone protein's role in the unfolding of chromatin is controlled primarily by phosphorylation?
H1
96
Which region, upon phosphorylation, becomes a scaffold for recruitment of elongation factors and mRNA processing enzymes?
CTD
97
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.
98
Identify all of the co-activators of transcription in the list below.
CBP and p300
99
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.
100
What is the name for a transcription factor that binds to non-promoter gene regions to encourage formation of a transcription-initiation complex?
Activator
101
Which of the following is a histone modification which always encourages the activation of transcription?
Acetylation
102
What is the interaction partner for bromo interaction domains?
acetylated Lys
103
Which of the following modifications is made to both DNA and histone proteins in humans?
methylation
104
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
105
Which of the following receptors is the least common in our cells?
guanylate cyclase (GC)-coupled receptors
106
Which of the following is a receptor anion channel whose subunit interacts with steroids?
GABAA
107
The two most common structural motifs in proteins for interaction with DNA sequences are helix-turn-helix motifs and
Zn2+ finger motifs
108
Which of the following is a receptor for toxins such as dioxin?
ArH
109
Which receptor, when activated, promotes the production of proteins responsible for metabolism of more than 60% of drugs?
PXR
110
Immunophilins are widely distributed chaperone assistants which catalyze changes in protein structure. Their enzymatic activity categorizes them as __________.
peptidyl-prolyl cis-trans isomerases (PPIases)
111
Steroids are __________ chemical messengers which can directly modify the genetic output of a cell.
lipophilic (hydrophobic)
112
Peptidyl-prolyl cis-trans isomerases catalyse rotation about which type of bond?
peptide
113
PPIases catalyze isomerization of peptide bonds next to which amino acid?
proline
114
Which of the following is identical to the hypoxia-inducible transcription factor HIFβ?
ArNT
115
CRE stands for
cAMP response element.
116
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
117
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
118
Modification of HIFα in which way promotes its action as a transcription factor?
S-nitrosylation
119
VHL is which type of enzyme?
an E3 ubiquitin ligase
120
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
121
Theoretically, what type of mutation in NFκB signaling could lead to cancer?
Loss of Rel-inhibiting function by IκB.
122
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.
123
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.
124
Which of the following is not a direct sensor of free BiP concentration in the lumen of the ER?
XPB1
125
Mark the way(s) in which a cell reacts to the situation of ER stress.
by destroying misfolded proteins. by making more chaperone proteins.
126
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.
127
Which of the following kinases directly phosphorylates and inhibits eEF2 kinase?
mTOR
128
In contrast to the other kinases in the following list, which is a lipid kinase rather than a protein kinase?
PI3K
129
Which of the following is a lipid anchor for the activation of PKC by PDK1?
DAG
130
Which of the following is a lipid anchor for the activation of PKB by PDK1?
PIP3
131
Which of the following kinases directly phosphorylates and activates eEF2 kinase?
AMPK
132
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
133
Which of the following is a lipid anchor for the activation of mTOR by PDK1?
Rheb-GTP
134
Which enzymes are inhibitors of IRS function (and thus potential contributors to insulin resistance in cells)?
S6Ks
135
TSC proteins are activated when phosphorylated by _____, and they are inhibited when phosphorylated by _____.
AMPK; PKB
136
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
137
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