22 - Signaling and Gene Regulation & Control of Gene Transcription Flashcards
1
Q
What is the major takeaway of cell signaling?
A
Signaling pathways lead to changes in gene expression
2
Q
True or false: almost all cells in an organism are genetically identical
A
True: they all have the same DNA
3
Q
What produces differences between cell types?
A
Differences in gene expression
4
Q
What is differential gene expression?
A
The expression of different genes by cells within the same genome
5
Q
True or false: gene expression only refers to translation
A
False: it includes everything from chromatin remodeling to protein degradation
6
Q
Which steps (6) can gene expression be controlled?
A
- Transcriptional control
- RNA processing control
- RNA transport and localization control
- Translation control
- mRNA degradation control
- Protein activity control
7
Q
What characteristics describe signaling that alters protein function/activation state?
A
More rapid, less energy, more quickly reversible
8
Q
What characteristics describe signaling that alters protein levels?
A
Less rapid, more energy, less quickly reversible
9
Q
What is an example of translational control?
A
ERK signaling being compartmentalized within the cell
10
Q
What scaffold localizes ERK at the golgi?
A
Sef
11
Q
What is a polysome?
A
mRNA transcript with multiple ribosomes on it
12
Q
What does MNK1 do?
A
It phosphorylates eIF-4E to initiate protein synthesis
13
Q
How is MNK1 activated?
A
Through phosphorylation by ERK and p38
14
Q
What happens when 4E-BP1 is phosphorylated?
A
It releases eIF4E, which can help form the initiation complex
15
Q
What does MNK stand for?
A
MAP kinase interacting kinase
16
Q
How is 4E activated?
A
Through phosphorylation by MNK
17
Q
What is needed for 4E to initiate protein synthesis?
A
- It needs to be phosphorylated by MNK (through p38/ERK)
2. It needs to be localized by being released from p4E-BP1
18
Q
What does 4E-BP1 stand for?
A
4E binding protein 1
19
Q
What phosphorylates 4E-BP1?
A
the mTORC1
20
Q
What does mTORC1 stand for?
A
mTOR complex 1
21
Q
How is mTOR activated?
A
Through phosphorylation by Akt
22
Q
What is cap-dependent mRNA translation?
A
A 5’ cap is needed on the mRNA to form the proper rings for translation
23
Q
What does IRES stand for?
A
Internal ribosome entry site
24
Q
What can inhibit TOR?
A
Cancer cells, viruses, and stress
25
What happens if mTOR is inhibited?
4E-BP1 cannot be phosphorylated, so it remains bound to 4E
26
What is another name for eIF-4E?
4E
27
What happens in cap-independent mRNA translation?
4E cannot bind to the IRES at the mRNA
28
How come 4E cannot bind to the IRES in cap-independent mRNA translation?
It is not released from 4E-BP1, since mTOR is inhibited
29
Which translation process uses IRESes?
Cap-independent mRNA translation
30
What type of translation do viruses typically use?
Cap-independent mRNA translation
31
How come viruses typically uses cap-independent mRNA translation?
They usually do not have the capping proteins to go through cap-dependent mRNA translation
32
What proteins are produced (in a healthy cell) from a cap-independent mRNA translation process?
Proteins that respond to stress (hypoxia, etc.)
33
What are TSC1 and TSC2?
Tumor suppressor genes
34
Where are TSC1 and TSC2 found?
Upstream of mTOR
35
What do TSC1 and TSC2 do?
Influence and slow down cell growth
36
How are TSC1 and TSC2 regulated?
By phosphorylation due to many different effectors
37
What is the significance of TSC1 and TSC2 having many phosphorylation sites?
It can be phosphorylated by a variety of effectors
38
How can mRNA be targeted to subcellular locations?
Through complexes bound to motor proteins
39
How is localized translation of mRNA achieved at mushroom bodies?
Through transport of mRNA to that specific location
40
How can localization and translation of mRNA lead to axon growth?
Different factors can stimulate mRNA translation to promote filopodial extension or withdrawal
41
How is axon turning mediated?
Through specific translation at the growth cone, leading to filopodial extension or withdrawal
42
How does local translation affect the post-synaptic density?
By altering the function and stabilization of the synapse
43
True or false: all proteins are made in the cell body
False: some mRNA can be transported to be locally translated elsewhere
44
How can signaling (4 ways) be controlled in the nucleus?
1. Transcriptional (initiation, elongation, termination)
2. RNA processing (capping, splicing, poly-A tail)
3. mRNA export
4. Surveillance (degradation)
45
What do gene regulatory proteins do?
Bind to regulatory sequences to influence transcription
46
What do general transcription factors do?
Bind close to the TATA box to promote RNA polymerase II binding
47
What is the shape of DNA?
3D (not linear)
48
Why is DNA having a 3D structure important?
It is important for the DNA to fold in order to transcribe gene properly
49
What is an enhancer?
A DNA region that activators can bind to to promote gene transcription
50
What is an activator?
A protein that binds to an enhancer region to promote gene transcription
51
What is a mediator?
A protein that brings together the general transcription factors and the activators to regulate gene expression
52
What are DNA-bending proteins?
Proteins that help DNA bend into 3D shapes to allow for gene transcription
53
Which parts of transcription are usually the downstream signaling target?
The enhancers (gene regulatory proteins)
54
What is the structure of cohesin?
A ring-type structure
55
What does cohesion do?
Keep the DNA looped together to mediate proper gene expression
56
What was the first discovered DNA binding factors for transcription?
CRE
57
What does CRE stand for?
cAMP response element
58
What question led to the discovery of CRE?
How does cAMP signaling regulate transcription of cAMP-responsive genes?
59
What are some common reporter assays used in molecular biology?
CAT reporters, and luciferase reporters
60
What does CAT reporter stand for?
Chloramphenicol acetyltransferase reporter
61
How does a CAT reporter work?
Higher amounts of CAT leads to higher amounts of acetylated products
62
How does a luciferase reporter work?
Higher amounts of luciferase lead to stronger glows when D-luciferin (substrate) is added
63
What is the importance of the SST gene?
It was known to be stimulated by cAMP
64
What does SST stand for?
Somatostatin gene
65
What was the experimental procedure using CAT and SST?
Parts of SST were cleaved, and the luminescence was recorded
66
What was the result of the experiments using CAT and SST?
There was a significant increase up to a deletion of 48 bp
67
What was the conclusion of the experiments using CAT and SST?
There was a DNA region somewhere in the identified 23 bp that was responsive to cAMP (CRE)
68
What question was asked about the 23 bp found using the CAT reporter?
Are these 23 bp necessary and sufficient to activate a cAMP response?
69
Are the 23 bp necessary and sufficient to activate a cAMP response (in the CRE experiments)?
Yes: it makes other genes cAMP responsive if added upstream
70
What is CRE?
A DNA segment that is responsive to cAMP
71
What is the structure of CRE?
An eight bp palindromic sequence (TGACGTCA)
72
What do native genes that respond to cAMP have in common?
They all have the CRE sequence
73
Where is the CRE usually found?
Within 100 bp of the TATA box
74
What happens if the CRE is moved upstream (more than 100 bp)?
It becomes less active
75
What question was asked after discovering CRE?
What binds to CRE?
76
How was CREB discovered?
Through an affinity column with CRE
77
What was the experimental procedure to discover CREB?
1. Create an affinity column with CRE on beads
2. Run cell extract over it
3. Purify whatever binds
78
What is CREB?
The binding protein that binds to CRE
79
What does CREB stand for?
CRE binding protein
80
What domains are found on CREB?
A DNA binding domain, and an activation domain
81
What does the DNA binding domain on CREB do?
Form dimeric leucine zippers
82
What is the importance of the palindromic CRE sequence?
Needs dimeric CREB to bind and recognize to each strand of the DNA through the leucine zipper
83
What does the activation domain on CREB do?
Contains Ser133 that needs to be phosphorylated for CREB to function
84
What phosphorylates Ser133 on CREB?
PKA
85
How does CREB bind to CRE?
Through a leucine zipper motif
86
What protein negatively regulated CREB?
PP-1
87
What is PP-1?
A phosphatase that dephosphorylated CREB
88
What is the rate limiting step of transcription due to cAMP?
PKA entering the nucleus
89
How does attenuation of the CREB signal work?
Through dephosphorylation by PP-1
90
True or false: CREB can only be activated by PKA
False: it can also be phosphorylated by CaM-KII and MAPK
91
True or false: CREB activation occurs at a constant rate
False: it can happen slowly or quickly
92
What pathway mediates fast activation of CREB?
The CaMKII pathway
93
What pathway mediates slow activation of CREB?
The MAPK/rsk pathway
94
What is the feedforward mechanism of CREB?
Calmodulin can activate both the CamKII and the MAPK/rsk pathway, leading to activation of CREB
95
What is the significance of the feedforward mechanism of CREB?
This leads to branched timing of impact (quick and sustained gene changes)
96
What is another name for MAPK?
ERK
97
What is another name for ERK?
MAPK
98
True or false: CRE is an enhancer
True: it is a segment of DNA where an activator can bind to stimulate gene expression
99
True or false: CREB is a specific transcription factor
True: it is an activator that binds to CRE to stimulate gene expression
100
What does CBP stand for?
CREB binding protein
101
What does CBP do?
Binds CREB to the basal transcription complex
102
What is the purpose of KID/KIX/Q2 domains?
Allow for transcription factors to bind to each other to allow for gene transcription
103
What is the first level of transcriptional control in cAMP signaling?
PKA phosphorylates CREB
104
What is the second level of transcriptional control in cAMP signaling?
CBP has histone acetylation activity to open up the chromatin
105
True or false: transcription complexes only have binding functions
False: they can also have activation aspects as well as binding
106
What are miRNAs?
Short RNA sequences derived from a primary transcript
107
Where are miRNAs created?
From a specific gene in the genome
108
What is the function of miRNAs?
Down-regulate cytoplasmic mRNAs through translational repression and mRNA decay (regulation)
109
What are siRNAs?
Short RNA sequences derived from longer double stranded RNAs
110
What does duplex mean?
Double stranded RNA
111
Where are siRNAs created?
From viruses or other endogenous sources
112
What is the function of siRNAs?
Target RNAs for cleavage in the cytoplasm (defense)
113
What does rasiRNA stand for?
Repeat-associated small interfering RNAs
114
What are rasiRNAs?
Short sequences derived from repetitive regions (centromeres and transposon regions)
115
What are the different types of rasiRNAs?
hcRNAs (yeast) and piRNAs (mammals)
116
What base pairing does miRNA have?
Incomplete base pairing
117
What base pairing does siRNA have?
Perfect matching
118
What process does miRNA mediate?
Inhibition of translation
119
What process does siRNA mediate?
Cleavage
120
What are argonautes?
Proteins that make up the RISC complex
121
What is the RISC complex?
The complex of protein and RNA responsible for RNAi
122
What is the structure of the RISC complex?
Argonaute proteins and miRNA or siRNA
123
What does RISC stand for?
RNA induced silencing complex
124
How are RISCs loaded with miRNA created?
By processing of the pre-miRNA in the nucleus, and loading it onto the argonaute protein
125
How are RISCs loaded with siRNA created?
By cutting dsRNA with dicer proteins, and loading it onto the argonaute protein
126
What do dicers do?
Cut foreign dsRNA to create siRNA
127
What does dsRNA stand for?
Double stranded RNA
128
What is thought to be the origin of siRNA?
Defense against dsRNA viruses
129
What is RNA silencing?
The process of small RNAs inhibiting gene expression in a variety of ways
130
What does RNAi stand for?
RNA interference
131
What is the relationship between RNAi and RNA silencing?
RNAi is a branch of RNA silencing
132
What parts of RNA silencing are not part of RNAi?
RNA silencing at the DNA and chromatin level
133
How does the RISC complex work?
It scans mRNA with the miRNA or siRNA to find and interfere with that particular mRNA
134
How are miRNAs created?
From single stranded precursor RNAs that fold back on themselves (with complementary sequences)
135
What roles (6) do miRNAs play?
1. Regulatory role in development
2. Patterning of the nervous system
3. Control of cell proliferation and cell death
4. Leaf and flower development in plants
5. Differentiation of various cell types
6. Development of cancer
136
True or false: miRNAs are general throughout the body
False: there are specific to different tissues in the body
137
In what part of the genome are miRNAs coded for?
In both introns and exons
138
True or false: miRNAs are only found in coding RNAs
False: they can also be found in noncoding RNAs
139
What is non-extensive base pairing?
Only a few of the base pairs match in the RISC and the target mRNA
140
Which small RNA uses non-extensive base pairing?
miRNA
141
What is the outcome of non-extensive base pairing?
Repression
142
What is extensive base pairing?
All of the base pairs match in the RISC and the target mRNA
143
Which small RNA uses extensive base pairing?
siRNA
144
What is the outcome of extensive base pairing?
Degradation
145
Which type of base pairing leads to a change in the argonaute conformation?
Extensive base pairing
146
Which type of base pairing does not lead to a change in the argonaute conformation?
Non-extensive base pairing
147
How can RNAi be used to study gene function?
It can be used to knockout the gene, and see the effect of removing this gene
148
True or false: RNAi received a Nobel Prize
True: it received the Nobel Prize in 2006 in Physiology/Medicine
149
How can researchers use siRNA to their advantage?
They can introduce siRNA to allow for degradation of a specific mRNA, thus knocking out the gene that mRNA translates for
150
How does miRNA relate to cell signaling?
Cell signaling can lead to regulation of miRNAs
151
What is an example of a cell signaling pathway regulating miRNA?
The MAPK pathway can turn on or turn off specific miRNAs, thus impacting protein levels
152
How are miRNAs and Ras signaling connected?
Through multiple feedback and feedforward loops
153
How can miRNAs be used in a feedback loop?
They can inhibit proteins that inhibit effectors of the pathway that leads to expression of the miRNA
154
How are miRNAs and the ECM connected?
Signaling through the ECM can lead to miRNAs that lead to changing and readjusting the ECM
155
How are miRNAs and the glucose pathway connected?
miRNAs can target insulin mediators (both kinases and phosphatases) to influence glucose levels
156
What effectors can miRNAs target in the glucose pathway?
Both kinases and phosphatases
157
How come miRNAs can be described as dial systems?
They can tune gene regulation up or down, depending on the signaling pathways
