L1-4: Control of transcription and chromatin Flashcards
1
Q
gene expression
A
process by which information in DNA is decoded into protein
2
Q
promoters
A
cis-acting DNA regulatory element initiating and controlling transcription.
3
Q
prokaryotic promoters
A
involves consensus sequences
4
Q
eukaryotic promoters
A
has a regulatory and core region prior to open reading frame
5
Q
core promoter elements
A
TATA box -31–26
initiator -2-+4
CpG islands
6
Q
CpG islands
A
CG sequence high frequency
~100-1000 bp
C res escapes methylation (hypomethylation)
CpG methylation associated with silencing
7
Q
regulatory promoter elements
A
UAS and enhancer
URS and silencer
8
Q
identification of promoter elements tools
A
sequence comparison
reporter analysis
9
Q
sequence comparison to identify promoter elements
A
TATA box identification
10
Q
reporter analysis to identify promoter elements
A
reporter genes encoding easily measurable proteins (e.g. GFP/lacZ/luciferase)
reporters identify when, where, quantity and signal response
11
Q
luciferase
A
enzymes that emit light when they oxidise their substrate
12
Q
RNA pol I/ II/ III location
A
pol I> nucleolus
pol II/III > nucleus
13
Q
bacterial RNA polymerase structure
A
B / B’ unit
2 alpha sub-units
w sub-unit
14
Q
eukaryotic RNA polymerase structure
A
12 sub-units
15
Q
general transcription factors
A
TFII A/B/D/E/F/H
sigma factor role in eukaryotes
16
Q
Bacterial RNA polymerase requirements
A
sigma factor to recognise promoter DNA
17
Q
general transcription factor characteristics
A
RNA pol specific
multi-component factors
complex on TATA box
recruit RNA pol II to promoter
direct initiation at start-site
18
Q
PIC
A
Pre initiation complex
19
Q
RNA polymerase II transcription initiation of prokaryotes
A
- helicase of TFIIH at start-site> open complex
- promoter clearance with extensive phosphorylation on CTD
- TFIIA/D may stay
TFIIB/E/H released
TFIIF moves down template w pol II
20
Q
CTD
A
C terminal domain
repeat series at C-terminal of largest B’ homologous sub-unit of RNA pol II
21
Q
TFIID function
structure?
A
TATA box binding
TFIIB recruitment
central RNA pol II txn factor
associated w TBP
trilobular > molecular saddle?
22
Q
TBP
A
TATA binding protein
23
Q
TFIIA function
A
stabilizes TFIID binding
anti-repression
24
Q
TFIIB function
A
recruits RNA pol II-TFIIF
IMPT for site selection start
25
TFIIH function
promoter melting and clearance
CTD kinase activity
DNA repair coupling
contains ATPase for promoter melting
26
TFIIH structure
9-10 sub-units
core + CAK
27
CAK
contains kinase phosphorylating CTD of RNAPII
28
TBP vs TFIID
TBP can direct assembly of PIC on TATA promoter but TFIID can direct PIC assembly on TTA-less assembly, supporting activated txn
TFIID require TAFs
29
TAF
promote interaction of TFIID w basal promoter and interact w activators
30
UAS / enhancer element function
increases basal transcription from low and inactive to activated transcription which is high
31
classes of UAS
Common
response
32
common UAS
close to core promoter/ promoter proximal
bind relatively abundant activators in cell> constitutively active
33
common UAS examples
GC box
octamer
CAAT box
34
response UAS
bind factors induced by specific stimuli
35
response UAS examples
SRE (binds serum response factor/ induced by growth factors)
HSE (binds heat shock factor and induced by heat shock)
36
combinatorial control of transcription
type and combination of elements dictates when and at what level the gene is transcribed
37
UAS location
adjacently upstream to TATA box
38
methods of activator basal transcription contact
tracking
looping
39
euk activators characteristics
modular
40
DNA binding domains
homeodomain
helix loop helix
leucine zipper
zinc finger
41
activation domains example
acidic patch (clusters of negative residues > asp/glu)
glutamine rich
proline rich
42
activation domain characteristics
low sequence conservation/ structural info
unstructured
short segments working additively
interact w other proteins like TAFs
43
activator analysis tools
in vitro
DNA footprinting
electrophoretic mobility assays
transcription assays
44
electrophoretic mobility assays/ gel shifts
ability of activator to bind to specific sequence
*radiolabelled probe DNA
45
transcription assays
activator has functional DNA binding domain and activation domain
46
in vivo activator analysis
reporter assays
chromatin immunoprecipitation
47
chromatin immunoprecipitation
1. cross-link bound proteins to DNA
2. isolate chromatin and shear DNA
3. precipitate chromatin w protein-specific antibody
4. reverse cross-link and digest protein
5. analyse DNA w PCR/ sequencing
48
activator functions
1. promote binding of additional activator
2. recruitment/ stimulate complex assembly
3. release stalled RNA polymerase
4. chromatin modulation
49
mediator structure
22 polypeptides
associated w RNA pol II via C-terminal domain / on own
3 domains: head/ middle/ tail
50
mediator function
provides bridge between activators and RNA pol II
aids recruitment of RNA pol II and increases PIC function
51
chromatin basic function
DNA compaction
histone units
52
2 types of histones
core
linker
53
core histone structure
N-terminal tail (v basic / Arg and Lys rich)
globular domain (alpha helices and loops)
54
nucleosome
core histone repeating unit
~147 bp 2* wrapped around octamer of histone proteins
55
core histone
highly conserved
v basic > rich in Lys/ Arg
nucleosome repeating unit
56
histone structure
N-terminal tail
globular domain
2* form 'handshake' interaction
57
nucleosome octamer
central H3-H4 tetramer + 2 flanking H2A-H2B dimers
58
what type of histone is H1
linker
59
nucleosome organisation
DNA transfer between nucleosomes via a 10nm fibre
linker histones bind to DNA between histones resulting in formation of a thicker fibre
60
evidence that chromatin inhibits transcription
1. in vivo reconstitution experiments
2. in vivo nucleosome positioning experiments
3. genetic studies in Saccharomyces cerevisiae
61
in vivo reconstitution studies
RNA pol II + txn factors only caused txn w DNA template not chromatin template
62
in vivo nucleosome positioning experiments
nucleosomes are disrupted/ lost in txn activation
63
saccharomyces cerevisiae
engineered yeast strain
64
genetic studies in saccharomyces cerevisiae
plasmid expressing H4 replaces chr H4 genes with a GAL4 regulatable promoter (turns off w glucose and on w galactose)
glucose presence> nucleosome depletion/ inducible gene expression
65
characteristic of chromatin due to its inhibition of txn
dynamic
66
3 chromatin structure modulation mechanisms
1. histone variants
2. PTM
3. ATP-dependent chromatin remodelling
67
histone w no variants
H4
68
histone variants
encoded by genes different to conserved major types exp at low levels
confers structural/ functional properties of nucleosome
69
PTM of chromatin effects
folding/ structural alteration
control of recruitment of non-histone proteins to chromatin
70
chromatin PTM examples
lysine acetylation
acetylation
methylation
71
histone lysine acetylation
mediated by HATs (histone acetyl transferases)
reversible by HDACs (histone deacetylases)
v dynamic
72
HATs
correlate w high levels of acetylation/ txn
yeast GCN5 homologue (Txn activator) in 1st nuclear HAT
recruitment via activators to specific promoters/ part of txn machinery
73
nuclear HAT 2 large sub-unit complexes
GNAT/ MYST
74
acetylation chromatin structure mediation
1. charged N+ terminal binding DNA
2. bromodomain protein direct recruitment
75
bromodomain proteins
recognise specific acetylated Lysine residues/ promote txn
76
where does histone methylation take place
lys
Arg
77
histone methylation
via histone lysine methyl transferases (HKMTs) and lysine demethylases
HKMTs mono/di/tri methylate w SET domain
not readily reversible
78
does histone methylation affect charge
no
therefore minor influence on structure
79
Snf-2 related ATPase
found in cells of ATP-dependent chromatin remodellers
helicase/ NTP driven nucleic acid translocase super family 2
snf-2=swi2
80
DNA-dependent processes on nucleosome
sliding
unwrapping
eviction
spacing
variant exchange
81
SWI/SNF
snf2 is catalytic sub-unit
hydrolyzes 1000 ATP/min (in DNA/nucleosome presence)
82
Snf2
DNA helicases
molecular motor tracks and induces torsion
therefore histone-DNA disruption and nucleosome movement
83
bromo-domain
recognize acetyl lysines
84
cooperation between ATP-dependent remodellers and histone acetylases (HATs)
recruited to same promoters
bromodomains tether to acetylated nucleosomes
PIC recruitment
85
PIC
Pre Initiation Complex
86
yeast swi/snf
reg expression of 5% yeast genes
reg 25% of genes expressed at end of mitosis
87
human PBAF/cBAF/ncBAF
3 complexes w shared sub-units
required for txn factors/ glucocorticoid/ retinoid receptors/ heat shock factor/ MyoD
interacts w Rb/ cyclin E
embryonic lethality in mice deletion
role in tumour suppressor pathways
88
swi/snf relation to Cancer
mutations associated w tumour variety
role in txn factor function
89
txn repression chromatin-modifying factors
HDACs
ATP-dependent remodellers
histone methylases
90
HDAC vs HATs
HDAC co-repressor of hypoacetylated regions
HAT co-activator of hyperacetylated regions
91
4 groups of HDAC
1/2/4 > classical, Zn dependent HDACs
3> Sir2 family (NAD required as co-factor)
92
HDAC characteristics
multisubunit complexes
promoter recruitment via interaction w site-specific DNA-binding proteins (co-repression)
93
ATP-dependent remodellers in txn repression
conserved in plants/ animals
broad expression in normal differentiation/ tumourogenesis
multi-subunit containing HDACs
94
NuRD action
closes chromatin to turn off txn of tumour suppression genes
95
2 chromatin types
euchromatin (gene-rich/ transcribable)> light
heterochromatin (gene poor/ repetitive regions/txn silencing) dark
96
biochemical features of heterochromatin
hypoacetylated
associated w specific histone 3 methylation
associated w silencing factors
97
heterochromatin protein 1
chromodomain protein
recognize / bind to methylated Lys
chromodomain specific to Lys9me2/3
98
HP1 binding function
compacts nucleosomal arrays
base of recruitment for activities preventing RNA pol II activity
99
heterochromatin assembly
1. HDAC deacetylases Lys9
2. histone methyl transferase (suvar39) methylates Lys9 on H3 (docking site for HP1)
3.HP1 recognizes methylated lysine and binds
100
reporter silencing assays to analyse heterochromatin
Ade6 gene expression forms white colonies on Adenine-limiting medium in NORMAL
Ade6 moved to heterochromatin and switched off > RED COLONIES (silencing)
101
X chromosome inactivation
1/2 X genes inactivated in females to equalize X-linked genes in M/F (Barr body of heterochromatin)
102
Barr body formation
controlled by non-coding RNA's Xist and Tsix
1 Chr inhibits Tsix and upregulates Xist> coats chr and recruits methylase via H3K27 therefore inactive
other becomes active, repressing Xist
103
Xist and Tsix relation
antisense transcript
Tsix prevents Xist
104
