Chapter 11 Flashcards
1
Q
Which statement(s) about Class II general transcription factors in eukaryotes is/are
true?
A) The polymerase will bind to DNA only if TFIIB is already bound at the promoter.
B) TFIIH has DNA helicase/ATPase activity for unwinding double-stranded DNA.
C) Electrophoretic mobility shift assay (EMSA) can be used to identify whether a transcription factor binds to DNA at the promoter.
D) Only two of the above statements are true.
E) All of the above statements are true.
A
D (A and B)
2
Q
T/F: Only Class II polymerases rely on transcription factors to bind to promoters.
A
False, all three classes rely on transcription factors.
3
Q
T/F: Gene-specific transcription factors are also known as activators.
A
True
4
Q
How many types of transcription factors are there and what are the types?
A
Two, general factors and gene specific factors (activators).
5
Q
What are the components of a preinitiation complex?
A
General transcription factors and polymerase. These bind at the TSS.
6
Q
What are the protein factors associated with a Class II polymerase?
A
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH.
7
Q
Which of the following is the correct order of binding for the Class II preinitiation complex?
a. DAPolBFEH
b. BADPolHEF
c. DABPolFEH
d. DABPolHEH
e. None of the above
A
C
8
Q
Describe the steps of the Class II preinitiation complex formation.
A
1) TFIID binds to DNA at the TATA box with the help of TFIIA.
2) TFIIB binds, at or near the TATA box.
3) TFIIF helps RNA Pol II to bind, extending the complex footprint to +17.
4) TFIIE binds.
5) TFIIH binds to complete the complex.
9
Q
What does EMSA stand for?
A
Electrophoretic Mobility Shift Assay. AKA Gel Mobility Shift Assay.
10
Q
Describe the process of a gel mobility shift assay
A
- Strands (either single or double strands) of DNA are radioactively labelled.
- Proteins are mixed with this DNA.
- Electrophoresis is performed with lanes for DNA alone, and DNA with protein(s). The lowest bands will be lone DNA while any DNA bound to proteins will be higher on the gel. Each lane can have one or more proteins to determine if proteins also bind to each other in the presence of the DNA.
11
Q
T/F: Gel Shift can be used to detect DNA-protein interactions.
A
True
12
Q
Is there a difference between class II promoters in vivo vs. in vitro?
A
Yes, in vitro we see stepwise formation of the Class II preinitiation complex. In vivo everything happens simultaneously.
13
Q
Which assays were used to determine the behaviour of Class II promoters?
A
Footprinting (established where factors bound to DNA and the sequence)
Gel Shift (established whether a protein bound to DNA at all).
14
Q
What does TFIIF bind to?
A
The polymerase.
15
Q
What does TFIIE do?
A
It guides TFIIH to the preinitiation complex.
16
Q
T/F: Gel shift assay cannot explain the order of protein binding.
A
False
17
Q
T/F: TFIID is a large but simple protein.
A
False, it is a complex of 14 components, the TBD and 13 TAFs
18
Q
T/F: TAF stands for TATA-Associated Factors
A
False, its TBP-Associated Factors
19
Q
T/F: TBP stands for TATA-box Binding Protein
A
True
20
Q
T/F: TFIID is highly conserved with 90% amino acid identity similarity between yeasts, humans, and plants.
A
False, it is 80%.
21
Q
T/F: Yeast TBP can substitute for human TBP
A
True
22
Q
T/F: The CTD of TFIID is 180 amino acids long and highly basic.
A
True
23
Q
T/F: The CTD of TFIID can bind to the TATA box without the rest of the protein
A
True
24
Q
T/F: TBP binds to the major groove of DNA
A
False, it binds to the minor groove (and this is highly unusual).
25
Describe how it was shown that TBP binds to the DNA, and where it does so.
It binds to the minor groove.
DNA was made but in the TATA box inosine was swapped for A, and C was swapped for T. Inosine is similar to A, but has a double bonded O where A has an amino group (and C has an amino group where T has a double bonded O). These changes mean the minor groove stays the same as in a TATA sequence, but the major groove was different.
A gel shift assay was then used to compare TATA DNA with CICI DNA. It showed that there was no difference in binding between the two.
26
T/F: TBP distorts DNA when it binds.
True, it bends DNA at an 80degree angle, opening the minor groove.
27
T/F: When TBP binds to DNA it binds when flanking prolines interchelate with DNA, locking the TBP in place.
False, it is phenylalanine (F, Phe)
28
Why does TBP bend DNA?
Likely to help melt the DNA to form the open promoter complex.
29
How do we know there are 13 TBP associated factors?
Immunoprecipitation assays.
30
How many functions do TAFs perform and what are they?
Three.
1) Interaction with promoter DNA
2) Interaction with activators (gene-specific TFs)
3) Enzymatic activity
31
Describe how it was determined that TAFs help with extended binding.
Researchers performed in vitro transcription with either TBP or TFIID and two different DNA templates, DNA with only a TATA box or DNA with a TATA box, initiator, and a downstream element.
RNA production was then measured for each using primer extension (but could have used radio labelling).
Results showed a significant increase when the 13 TAFs were present (complete TFIID).
Footprinting was then completed to confirm binding specificity.
32
T/F: Sp1 associates with TAF4.
True
33
T/F: TBP is a universal transcription factor.
True, usually.
34
Give reasons why TBP can be considered a universal transcription factor.
- It functions with Pol II promoters with a TATA box.
- It functions with Pol II promoters without a TATA box.
- It functions with TATA-less Pol III promoters.
- It functions with TATA-less Pol I promoters.
35
T/F: TBP will not bind to DNA without a TATA box, even when an initiator is present.
False. TAF1 and TAF2 bind to the initiator, and TBP will bind to them.
36
T/F: TBP will not bind to DAN without a TATA box if a GC box is present.
False, Sp1 will bind to TAF4, which binds to TAF1 which binds to TAF2. TBP binds to TAF1 and TAF2.
37
T/F: Sp1 is a general transcription factor
False, it is an activator.
38
How do we test the level of transcription with TBF is combined with different TAFs?
Mix in vitro and measure transcription levels.
39
T/F: TFIID + Sp1 is not sufficient to participate in activation.
False, but TBP + Sp1 is not.
40
T/F: Activation will occur if Sp1, TAF4, TAF1, and TAF2 are present.
True
41
Describe the experiment used to show Sp1 binds to TAF4.
Affinity chromatography was used. Beads were loaded with GC boxes and Sp1. TAFs were added and then it was determined which TAFs eluted. TAF4 did not elute.
42
T/F: TFIID can interact with several activators at once.
True. This can happen because there are multiple TAFs which can interact with different activators, especially since DNA is flexible.
43
What is the benefit of TFIID being able to interact with multiple activators at the same time?
Increased transcriptional control.
44
What are the enzymatic properties of TAF1?
1. HIstone acetylation (adds acetyl groups to histones which activates transcription).
2. Protein kinase (self-phosphorylates and phosphorylates TFIIF).
45
T/F: TBP is found in all preinitiation complexes.
False. Some organisms have TRF1 (TBP Related Factor 1)
46
T/F: If a gene uses a TRF, it will not have a TATA region.
False. It can have both.
47
T/F: TRF1 binds to a TC-rich region.
True.
48
T/F: TFIIB has no associated factors.
True.
49
T/F: TFIIB is not necessary for the TFIIF-Pol complex to bind to TFIID.
False. The TFIIF-Pol complex will bind if DAB is present, but not if only DA is present.
50
T/F: The CTD of TFIIB binds TBP, and the NTD binds to TFIIH-Pol.
False, the CTD of TFIIB binds to TBP, btu the NTD binds to the TFIIF-Pol complex.
51
T/F: TFIIB is instrumental in positioning the TFIIF-Pol complex to the TSS.
True
52
What is the main function of TFIIH?
Phosphorylation of Pol II.
53
T/F: The O form of Pol II is the result of phosphorylation by TFIIH.
True
54
Why is the O form of Pol II phosphorylated?
It binds less tightly to TBP, allowing elongation to commence.
55
How did we get evidence that TFIIH phoshphorylates Pol II?
Incubated purified Pol IIA with TFIIH and [gamma-32P] ATP. Added other factors, and then measured phosphorylation.
Results - Phosphorylation only occurred when TFIIH was present, and it was more pronounced when other factors were present too.
56
T/F: The CTD of Pol II is needed for phosphorylation.
True. Shown by incubating A, B and O forms with [gamma-32P] ATP. A was phosphorylated, B was not, and O showed some due to equilibrium.
57
How do we know TFIIH phosphorylates only when bound to DNA?
Incubated various DNA templates with Pol II and TFIIH. Phosphorylation occurred better when the TATA box or initiator was present than when no promoter sequence was present.
58
T/F: TFIIH has 9 subunits in 2 conmplexes.
True. 4 subunits have kinase activity, 5 have helicase activity.
59
In what organism did researchers establish helicase activity of TFIIH?
Yeast.
60
How do you establish helicase activity is present?
Radiolabel one strand of a dsDNA segment.
Electrophorese alone and incubated with the suspected helicase.
A difference in band lengths will indicate DNA unwinding if helicase is present. dsDNA will not migrate as far as ssDNA.
Adding or not adding ATP establishes ATP dependence.
This experiment is best done with increasing amounts of the suspected helicase.
61
What is the basic model for initiation, promoter clearance, and elongation?
TFIID attaches to DNA with the help of TFIIA.
TFIIB binds, forming DAB.
TFIIF + Pol II complex forms, binds to DAB.
TFIIE guides TFIIH to the DNA and the complex is formed.
TFIIH phosphorylates CTD using ATP.
Also using ATP, TFIIH melts DNA to form bubble.
CTD is more phosphoylated so the polymerase clears the promoter and elongates.
TFIID and TFIIB stay at promoter.
TFIIE and TFIIH dissociate.
Polymerase does its thing.
62
What are elongation factors?
Proteins that stimulate elongation of the polymerase.
63
What are elongation factors and how do we know what they do?
Elongation factors stimulate elongation of the polymerase and we determined their function by:
- Incubating Pol II + DNA template + NTPs and allowing initiation to occur.
- Heparin was added to block new initiation since heparin is a stronger competitor.
- Add either TFIIS (elongation factor) or buffer.
- Measure rate of incorporation of radio-labelled GMP into RNA.
Results - When TFIIS was present, there was an increase in GMP incorporation. With buffer the amount of incorporation stayed steady.
64
T/F: TFIIS is an elongation factor
True
65
What is the main role(s) of TFIIS?
It minimizes transcription arrest by stimulating the RNase activity of Pol II, causing it to cut off extruded DNA on the wrong side caused by backtracking. This makes a 3' -OH group available again.
It also promotes proofreading by stimulating RNase activity of Pol II to cleave misincorporated nucleotides.
66
T/F: TFIIS is always bound to Pol II.
False, it binds when Pol II stalls.
67
What is included in Class I pre-initiation complexes?
Pol I with two factors. SL1 and UBF (humans and mammals, respectively; TIF-IB and UAF (others, yeast. We don't really discuss these).
68
What is UBF?
Upstream binding factor. It binds to the upstream promoter element (UPE).
69
What is SL1?
Pol I factor that binds to the core promoter.
70
T/F: SL1 binds to the upstream promoter element (UPE), UBF binds to core.
False, SL1 binds to the core element, UBF binds to the UPE.
71
T/F: Core binding factor is sufficient to recruit Pol I in all organisms.
False, it is not sufficient in humans, who need UBF to assist with binding of SL1.
72
T/F: UBF is made of two polypeptides and both are required for activity.
False, it is made of two polypeptides, but only the larger is required for activity.
73
What does UBF stand for?
Upstream Binding Factor.
74
What does UBF do in vitro?
Stimulates transcription of the rRNA gene.
75
How was UBF's function shown?
Pol I + NTP +/- UBF +/- SL1 --> rRNA?
DNA with a wild-type rRNA promoter and DNA with a mutant promoter missing UPE were each tested. They were mixed with Pol I and various combos of UBF and SL1.
Transcription was then measured by S1 assay (but you could use a radiolabel assay instead).
The amount of rRNA produced in each mixture was measured.
Results: SL1 was needed for basal activity.
UBF enhances activity in the presence or absence of the UPE.
n.b. SL1 without UBF showing transcription is a contradiction of both being needed in humans. Probably due to in vitro assay.
76
T/F: UBF requires UPE to function.
False. UBF may bind to the core element if there is no UPE present. This is a theory based on experimental results but has not been proven.
77
T/F: SL1 is made of TBP and 4 TAFs
False, it is TBP and 3 TAFs.
78
How did we determine the composition of SL1?
Immunoblotting. SL1 was isolated using two different methods, ion-exchange chromatography and glycerol gradient.
Ion-exchange fractions were collected and tested for SL1 activity (was RNA made). If it was, then it was tested on a Western blot with anti-TBP antibody.
Fractions were then taken using glycerol gradient centrifugation and tested for SL1 activity. The ones that showed activity were tested with Western blot, with anti-TBP as above.
Results - TBP was present in both types of assay.
79
T/F: After addition of an anti-TBP antibody, SL1 activity is inhibited.
True
80
T/F: If you inhibit SL1 activity with anti-TBP and then add excess SL1, activity resumes.
True
81
T/F: If you inhibit SL1 activity with anti-TBP and then add excess TBP, activity resumes.
False. Therefore other factors are necessary, forming SL1.
82
How were the components of SL1 determined?
SDS-PAGE of immunoprecipitation, then stained with coomassie.
83
How many TAFs are associated with SL1?
Three (TAF(I)110, TAF(I)63, TAF(I)48). I in parens is a subscript.
84
How do we know the TAFs in SL1 are different from TFIID TAFs?
Immunoblotting of each after denaturing shows a different number of bands with different molecular weights.
85
T/F: TBP is the same in SL1 and in TFIID
True, but the TFs are different.
86
Which transcription factors are required by Class III genes?
TFIIIB and TFIIIC are required for all.
87
Which transcription factors are required by Class III 5S RNA genes?
TFIIIA, TFIIIB, TFIIIC
88
Where do TFIIIA proteins bind?
The internal promoter of the 5S RNA gene.
89
Describe the shape of TFIIIA.
Transcription factor of the 9 (zinc) fingers.
90
Describe zinc fingers.
Finger-shaped protein domain.
Contain 4 conserved amino acids that bind to 1 Zn molecule.
Usually the 4 amino acids are 2 His and 2 Cys (2C2H)
91
How do we know 5S rRNA requires 3 transcription factors?
Antibodies against TFIIIA halt transcription against 5S rRNA, but not against tRNA.
92
Describe the experiment used to show TFIIIA is required for 5S rRNA but not tRNA.
5S rRNA and tRNA genes were added to germ cells and somatic cells, along with radiolabeled nucleotides.
Each was run with:
no antibody
irrelevant antibody
anti-TFIIIA antibody.
Results - transcription of tRNA happened in all three scenarios. There was no 5S transcription when anti-TFIIIA was added.
93
T/F: Footprinting can be used to show polymerase migration.
True
94
T/F: TFIIIC binds to the upstream promoter.
False, TFIIIC binds to the internal promoter, specifically Box B. It does not bind upstream of the TSS.
95
Where does TFIIIB bind?
TFIIIB binds upstream of the TSS, but only if TFIIIC has bound to Box B (and A?) first. It will also remain after TFIIIC dissociates.
96
T/F: It is useful that TFIIIB stays bound to DNA because it can then promote further rounds of transcription.
True
97
T/F: TFIIIB does not associate with TBF.
False. TFIIIB is TBF + 2 TAFs.
98
T/F: Pol III requires the binding of TFIIIB + TFIIIC to bind to DNA.
False, Pol III requires TFIIIB only, but TFIIIB requires TFIIIC to bind to DNA in the first place.
99
What is the composition of TFIIIB?
TBP + 2 TAFs.
100
T/F: TFIIIB TAFs overlap with TFIID TAFs.
False, each has unique TAFs.
101
T/F: Classical Pol III genes have TATA boxes.
False, they do not have TATA boxes, they have A and B boxes, which are downstream of the TSS.
102
T/F: Non-classical Pol III genes always have TATA boxes.
False, but they may have TATA boxes (this is kinda unclear actually).
103
T/F: If a non-classical Pol III gene has a TATA box, it will not have B and C boxes.
True.
104
T/F: If a Pol III gene has a TATA box, it still requires TFIIIC.
False, TBP can bind to the TATA box without the assistance of TFIIIC (which won't have anywhere to bind anyway).
105
T/F: All three classes of Pol can bind without a TATA box.
True.
Class I can bind using UBF to bind SL1 to core.
Class II can bind Sp1 to GC boxes, binding TFIID and thus general factors to the initiator.
Class III uses TFIIIC to bind to box A and B, then binding TFIIIB upstream of the TSS.
106
What are the unifying principles for all classes of Pol?
1) Pre-initiation complex always starts when a transcription factor binds to a promoter region, then it recruits other components.
2) TBP is an organizer that brings other factors and polymerase to the complex.
3) TAFs give specificity to TBP.
107
Match the following:
1. Pol I a. TFIID x. 2 TAFs
2. Pol II b. SL1 y. 3 TAFs
3. Pol III c. TFIIIB z. 13 TAFs
1-b-y
2-a-z
3-c-x
108
What part of TFIID binds to DNA, as shown by a footprinting assay?
The 180 aa CTD.
109
Where does the TBP in TFIID bind?
To the minor groove of the TATA box.
110
How does TBP interact with the minor grove of DNA in a Class II gene?
It bends it 80 degrees, inserting phelylalanines into the DNA on either side.
111
Besides TBP-TATA box binding stabilization, what else does TFIIA do?
Stimulated TFIID binding through antirepression.
112
What do TAF1 and TAF2 do?
Cooperate in binding to the initiator alone and to the initiator plus a DPE. These type of initiators can be used as TATA-less promoters
113
What does TFIIB do?
Positions polymerase. Has a CTD and NTD which act as a bridge between TFIID and Pol II.
114
What are the major jobs of TFIIH?
1) Phosphorylating the CTD of Pol II
2) Uses helicase function to form bubble at TSS.
