Boyes Transcription Factors Flashcards
1
Q
How many DNA contacts do TFs make?
A
10-20 H bonds into Major groove
2
Q
What DNA contacts do TFs make?
A
R-Guanine
D-Adenine
3
Q
What are the 4 types of DNA binding motif?
A
Helix-turn-helix
Zinc finger (2C2C/2C2H)
Leucine Zipper
Helix-loop-helix
4
Q
What is the structure of Plant homeobox TFs?
A
60 residues. 3 helices
1&2 for context,
3 and N-extension (minor) for DNA
5
Q
Which organisms have plant homeobox TFs?
A
mammals, insects, amphibians
6
Q
What does the HTH C terminal helix do?
A
Binds DNA
7
Q
What does the HTH N terminal helix do?
A
Position units 1 turn apart to form dimers
8
Q
What benefit does dimerisation have?
A
Squares the affinity constant
9
Q
What are the 2 types of Zinc Finger?
A
2cys-2cys
2cys-2his
10
Q
How to 2Cys-2His fingers bind?
A
2Cys in beta-sheet
2His in alpha helix
continuous alpha helix
3bp contacts
11
Q
Do TFs have to use all their zinc fingers?
A
No
12
Q
How do 2Cys-2Cys fingers bind?
A
Dimers separated by 1 turn of helix
2 fingers x 2 = 4Zn
N finger binds DNA
C finger controls dimer spacing of palindromic sequence
13
Q
What motif do steroid hormones have?
A
Zinc fingers
14
Q
What is the general structure of transcription factors?
A
Modular with domains for DNA, Ligand and activation
15
Q
What action do Leucine Zippers have?
A
Mostly repressive
16
Q
What is the structure of a Leucine Zipper?
A
dimer of 2 alpha helices with hydophobe every 7 repeats
both helices have DNA binding domain
17
Q
Which TFs have leucine zippers?
A
Myc:Max activates, Mad:Max represses
Fos:Jun represses with 30x affinity of Jun:Jun for API site
18
Q
When do HLH and Leucine zippers become repressive?
A
Heterodimerisation in absence of a DBD
19
Q
Which helix contacts DNA in HLH?
A
Long/C terminal
20
Q
What is the function of the loop in HLH?
A
Allows packing between N/short helix dimers
21
Q
Which TFs have HLH?
A
MyoD:MyoD activation of B cell myosin
Myc:Max:Mad
E2A:E12/E47
22
Q
How is expression of a gene varied between cells?
A
Homo/hetero/inactivation of TFs
Epigenetics
23
Q
What are 2 types of designer TFs?
A
Zinc fingers hooked to Fok endonuclease
Transcription Activator Like Elements
24
Q
What are the disadvantages of Zinc finger designer TFs?
A
Palindromic sequences required for dimerisation
Non -specific triplet binding of 2Cys-2His
25
How are TALEs produced?
Developed from Xanthomas
34residues
12&13 residue pinpoint 16 residue sequence
26
What are the trans domains of Tfs?
Activation domains
27
What are the 3 types of activation domains?
Acidic, Glu-rich, Pro-rich
28
What are the 6 TF activation domains?
```
TF11D
TF11B
Mediator for Pol 2 CTD and TF11H
Coactivators: HAT, Basal Machinery
Remodelling complexes
```
29
Which TF11D component do Acididc domains bind?
TAF11 31
30
Which TF11D component do Glu domains bind?
TAF11 110
31
Which TF11D component do Pro domains bind?
TAF11 55
32
What affects do the activation domains have on TF11B/TF11D?
Recruitment or modify conformation to recruit other components more effectively.
33
What motif do steroid hormones have?
HOMODIMERS of Zinc fingers
34
Which TAF component of TF11D has HAT activity?
250
35
Which type of activation domain recruit remodelling complexes?
Acidic
36
What are the advantages of TF regulation?
Rapid,
Response to signalling
Fine control
Tissue specific
37
What are the 4 main mechanism of TF activation?
Ligand binding
Cleavage from precursor
Release of inhibitor
Covalent modification
38
What type of activation do nuclear hormone receptors have?
Ligand binding cause dimerisation (steroid hormones are zinc finger homodimers)
39
What are the examples of Ligand binding regulation?
Thyriod hormone binds to TF receptor bound to DNA, activating HAT domain
Glucocorticiod hormone binds to TF receptor to release receptor-hormone from HSP 90 to migrate to nucleus to activate HAT
40
What are examples of cleavage from precursor?
Notch delta-epsilon intercellular interaction releases N for cleavage and migration to nucleus
Cholesterol synthesis from SREBPp
41
How is HSF1 activated?
Released from HSP90, interacts with eEF1A for trimerisation and phosphorylation to bind to HSE
42
What are 3 examples of covalent modification?
Phosphorylation,
Acetylation,
Ubiquination
43
How do cytokine stimulate transcription?
JAKs phosphorylate STAT TF for dimerisation
44
How do MAPK cascades stimulate transcription?
RTKs stimulate MAPK to phosphorylate TFs
45
How have the Early Response genes been studied?
Starvation then addition of growth factors
Phosphorylation and interaction of TCF and SRF
Activates SRE in early response genes
46
How are TFs acetylated?
By HATs
47
How is ubiquination activated?
By other modifications:
Srb10 kinase acts on GCN4 as part of holoenzyme
ScFcdc34 E3 ligase recruited
48
What is the function of ubiquination?
Prevents overactivation of the gene- 1 time/de novo firing
| TF activation and degradation domains overlap- unstable
49
How is ubiquitination observed?
ChIP detects cmyc E3 ligase, 19S proteasome at the promoter
| oestrogen receptor S2 promoter and degradation overlap
50
How are tissue specific TFs degraded?
eg Acetylated GATA1 is preferentially MAPK phosphorylated, promoting ubiquitin degradation
51
What is the structure of an enhancer?
100bp long
positive control elements
forward or reverse orientation
1-1000kb upstream or downstream
52
What is the structure of an insulator?
promoter specific/position dependant
| hypersensitive site
53
What is the structure of LCRs?
```
Strong enhancers
Upstream or downstream
Position independant,
Copy number dependant
Tissue specific
```
54
What is the function of LCRs?
open >100kb chromatin, promoting euchromatin
55
What are the 4 potential mechanisms of enchancers?
Looping,
Tracking,
Faciliatated Tracking,
Linking
56
How was looping of enhancers observed?
Chromosome conformation capture crosslinks, digestd, religates and PCR of spatially distant but related sequences.
Different globin genes during different stages of development
Kissing chromosomes in naive T cells to produce interleukins or interferons as Th1/Th2 differentiation
57
What are the caveats of looping?
Inflexibility of looping,
| Effects of insulators would be reduced
58
Which mechanism of enhancers has most evidence?
Facilitated tracking
59
What is the evidence for facilitated tracking?
Moving non-coding transcripts of B-globin gene
| HAT activity from tracking and associated H3K4me3 increasing flexibility
60
What evidence is there for linking?
Chip proteins indentified in Drosophilia cut locus
| LIM homologues in vertebrates
61
What is the main caveat of the linking theory?
Enhancers can lie in introns of other genes
62
What are the 2 main functions of insulators?
Barrier to enhancers
| Stop spreading of heterochromatin
63
How was barrier function observed?
Insertion of enhancer before/after HS4 insulator in B-globin with neomycin reporter
Flanking of IL2R gene maintained expression for 100 days, observed by FACS
64
How is barrier function produced in B-globin?
USF1/2 proteins bind HS4 site to recruit HATs
| Acetylation blocks H3K9 repression
65
How can enhancer function be observed?
Using quantifiable reporters transfected either stabley or transiently
66
What function do CTCF proteins have?
Enhancer blocking
Binds cohesin to prevent external enhancers acting on loops (e.g. If2 blocked but H19 expressed)
Adjacent LAD domains tether to nucloephosmin in laminae.
67
What are the 3 models for enhancer blocking?
Promoter decor,
Physical blocking of tracking
Organising chromatin into loops to separate external enhancers
68
What is the Transcription factory theory?
High concentration of immobile Pol2. DNA loops to the factory
69
What is the unifying/relaxed factory model?
Regions of high [TF] and [Pol2] that is mobile. Genes loop to the factory
70
What are the caveats of the Factory model?
2-10 polymerases per gene, too small for multiple gene loci, no colocalisation of TFs
71
What are the potential downfalls of the relaxed factories?
Trans-splicing and chromosome translocations
72
What is the evidence for factories?
Pulses of biotin-CTP show transcripts localised
Antibodies localise Pol2
Genes leave chromosome domains when active
73
Which vectors are suitable for studies of Long range control?
BACs and YACs
74
How are BACs generated?
Stored in DY380 E.coli cells that have 42'C recombineering by c1857 repressor release from lamda phage.
50bp homologous flanking region
75
How is a transgene inserted into an embryo?
Injection into male pronucleus before nuclear fission of embryo.
Insertion is random, copy number varies and head-to-tail
76
Outline expression of transgenic mice?
```
Isolation of gene from vector.
Linearisation
Screen by PCR to identify gene fragment
Injection into nucleus
Implant into pseudo-pregnant mother
Analyse genome of offspring by PCR
```
77
What is the function of knock-out mice?
To delete whole genes, regions of genes or regulatory elements
78
How are knockout mice produced?
Vector produced with flanks homologous to chromosome. Gene replaced by observable phenotype selection cassette. TK identifies random insertion.
Introduce gene into dominant cells, culture growth and neomycin resistance
Injection into recessive blastocyst 3-5 days old
Inject into 2-5 day pseudopregnant mother
Chimeric offspring can be mated with recessive to get heterozygotes
79
What is the main caveat of knock out mice?
15% embryonicaly lethal
80
What are the caveats of knock out mice?
15% embryonicaly lethal, some show no effect, some mutate other locations
81
How are conditional knockout produced?
Cre recombinase expressed in specific tissues to generate knockout of transgene between 34bp LoxP sites
82
How is Flp Recombinase used?
To remove negative selection cassette from within LoxP/Cre
83
What placement of LoxP sites excise a transgene?
Cis (outside) in same direction
84
What does LoxP cis, opposite placement do?
Invert gene
85
What does trans placement of LoxP do?
In between genes for chromosome translocation
86
What do knock-in mice do?
Introduce point mutations in a gene or regulatory element
87
What can Cre be used for?
To inactivate a gene - deleting a stop codon or gene
| Generate inducible changes in DNA
88
What does the HS4 site of chicken B-globin do?
Binds USF1,2 to prevent heterochromatin spreading
| Binds CTCF to hold cohesin sister chromatids together and looping to prevent enhancers
89
What does the downstream HS1 site do in chicken B-globin?
Binds CTCF to tether 100kb regions to laminae.
90
What is the structure of the chicken B-globin locus?
```
Folate
16kb condensed
5' HS4
LCR
Genes
3' HS1
Olfactory
```
