CMB2001- gene expression Flashcards
1
Q
CpG islands
A
areas with high freq of CG sequences
- associated with lower rate of Txn initiation
2
Q
CpG island methylation =
A
silencing
3
Q
UAS + enhancer =
A
activator binding sites
4
Q
URS+ silencer =
A
repressor binding sites
5
Q
Tools for identifying promotor elements
A
Sequence comparison
- identifying TATA box
Reporter analysis
- measure levels of proteins encoded by reporter genes
6
Q
RNA pol I target and location
A
rRNA
Nucleolus
7
Q
RNA pol II target and location
A
mRNA, snRNAs, miRNAs
nucleus
8
Q
RNA pol III target and location
A
tRNA, 5S/ U6/7S RNAs
nucleus
9
Q
GTF =
A
General transcription factor
10
Q
PIC =
A
Pre initiation complex
11
Q
sigma factor recognising promotor DNA (bacterial RNApol)
A
- RNA pol specific
- multicomponent factors
- form a complex on TATA box
- recruit RNA pol II to the promoter
- Direct initiation at start-site
12
Q
CTD =
A
C-terminal domain
- series of repeats located at the C-terminal end of the largest pol II subunit
13
Q
Transcription initiation by RNA pol II
A
helicase activity of TFIIH separates template strand at start site (requires ATP)
-> open complex
-> pol II is phosphorylated on CTD as pol begins transcribing
14
Q
properties of TFIID
A
binds tata box (core promoter)
recruits TFIID
15
Q
TFIID structure
A
central RNA pol II transcription factor
TBP (TATA Binding Protein) + TAFs (TBP associated factors = TFIID
16
Q
TFIIH properties
A
promoter melting and clearance
CTD kinase activity
DNA repair coupling
17
Q
TFIIH stucture
A
2 subunits: CORE + CAK
CAK contains kinase -> phosphorylates CTD of RNApol2
18
Q
TBP properties
A
- can directly assemble PIC on TATA containing promoter
- cannot act alone without TATA
cannot support activated transcription
19
Q
TAF properties
A
promote interaction of TFIID with basal promoter
interaction with activators
20
Q
GC box: sequence and factor
A
GGGCGG
Sp1
21
Q
Octamer: sequence and factor
A
ATTTGCAT
Oct-1
22
Q
CAAT box: sequence and factor
A
GGCCAATCT
NFY
23
Q
common sequence elements
A
promoter proximal
constantly (constitutively) active
24
Q
SRE
A
Response element
binds: serum response factor (SRF)
inducers: growth factors
25
HSE
Response element
binds: heat shock factor
inducer: heat shock
26
activation domains
lack of sequence conservation/structural information
multiple short segments that work in an additive way
interact with other proteins in the transcriptional machinery (e.g. TAFs)
27
characterising activation domains
by amino acid composition:
acidic patch (VP16)
glutamine rich (SP1)
proline rich (Jun)
28
in vitro analysis of activators
DNA footprinting, Electrophoretic Mobility Shift Assays (gel shift), Transcription assay
29
in vivo analysis of activators
reporter assays, chromatin immunoprecipitation
30
chromatin immunoprecipitation
1. cross-link bound proteins to DNA
2. isolate chromatin and shear DNA
3. precipitate chromatin with protein-specific antibody
4. reverse cross-link and digest protein
5. analyse the DNA with PCR and ChiP-Seq
31
how activators work
1. promote binding of an additional activator
2. stimulate complex assembly (recruitment)
32
discovery of mediators
- many activators cannot activate in vitro transcription
- suggests that another factor is needed
-> disovered mediators
33
composition of mediators
- large complex (~22 polypeptides)
- can exist alone or associated with RNA pol II
- three domains: head, middle, tail
34
function of mediators
- many interact with specific mediator subunits
- provides bridge between activators and RNA pol II
- mediator-activator interactions aid recruitment of RNA Pol II -> enhance PIC formation
35
how activators control transciption
1. promote binding of additional activator
2. stimulate complex assembly
3. release stalled RNA pol II
36
chromatin
protein complex that packs DNA
37
Chromatin is primarily composed of:
histones
38
two basic histone types
core histones and linker histones
39
4 highly conserved core types of histones
H2A, H2B, H3, H4
40
N-terminal tail of core histones
highly basic
rich in Lys and Arg
41
globular domain of core histones
alpha helices and loops
42
Repeating unit of chromatin
Nucleosomes
43
composition of histone octamer
central H3-H4 tetramer + 2 flanking H2A-H2B dimers
44
organisation of nucleosomes
- DNA passes directly from one nucleosome to next -> 10nm fibre
- linker histones (e.g. H1) bind to DNA between nucleosomes
- 30nm fibre is formed in vivo
45
In vitro experimental evidence that chromatin inhibits transcription
Experimentally:
RNA Pol II + transcription factors + naked DNA template -> transcription
RNA Pol II + transcription factors + chromatin template -> NO transciption
46
Chromatin inhibits...
transcription
47
Histone variants control chromatin structure
Histone Variants
- encode by genes that differ from highly conserved major types
- expressed at lower levels than conventional counterparts
- all but H4 have variants
- variants have novel structural and functional properties -> affects chromatin dynamics
48
post transcriptional modification of histones
- could directly alter chromatin folding/structure
- could control recruitment of non-histone proteins to chromatin
49
Enzymes for histone acetylation
acetylation mediated by HATs (Histone Acetyle Transferases)
acetylation readily reversed by HDACs (Histone Deacetylases)
50
Histone acetylation and transcriptional activation
High levels of acetylation = high levels of transcription
- direct influence on chromatin structure
- directs recruitment of bromodomain proteins
51
Histone methylation enzymes
methylation: Histone lysine methyl transferases
demethylation: lysine demethylation
- can add up to 3 methyl groups
- can't be readily reversed by hydrolysis
52
histone methylation
doesn't affect charge -> prob only has a minor effect on chromatin structure
53
the histone code
code telling the transcriptional state of DNA
54
ATP-dependent chromatin remodelling
- cells have multiple remodelling complexes
- all have SNF2-related ATPase
-
55
bromodomains bind...
acetylated lysines
56
what can ATPase do to chromatin remodelling
- sliding
- unwrapping
- eviction
- spacing
- histone variant exchange
57
catalytic subunit of SWI/SNF
Snf2 or Swi2
58
Snf2
related structurally to DNA helicase
- molecular motor: uses ATP to track alon DNA -> induce torsion -> disruption of histone-DNA interactions
- pushes DNA from a place where it isn't accessible on the nucleosome to a place where it is
59
ATP-dependent and HAT complexes co-operate
- commonly are recruited to the same promoters
- bromodomains in Snf2 help hold it onto acetylated nucleosomes
60
Classical HDACs
class 1, 2, 4 enzymes
Zinc Dependent
61
Class 3 HDACs
Sir2 family
NAD co-factor required
62
NuRD complex
- belongs to Mi2/CHD family
- spaces out nucleosomes v regularly + tightly together -> stops transcription
63
biochemical features of heterochromatin
- hypoacetylation
- specific H3 methylation
- association of specific silencing factors
- if acetylated, cannot be methylated (HP1 recognises methylation)
64
HP1
chromodomain protein
- often recognises and binds to methylated lysine residues
65
NF-kB =
nuclear factor of the kappa immunoglobin light chain in B cells
66
NF-kB transcription factor pathway
- allows response to external threats
- regulating gene expression helps program responses to these threats -> call can survive + recover or induce death
67
precursor of p50
p105
68
precursor of p52
p100
69
RHD
Rel homology domain
- encodes DNA binding and dimerization of NFkB
70
E3 ubiquitin ligase
protein that facilitates the attachment of ubiquitin chains to a target protein
71
NF-kB induced by
- inflammatory cytokines
- bacterial products
- viral proteins and infection
- DNA damage
- cell stress
72
NF-kB regulates
- stress response
- cell survival/cell death
- cell adhesion
- proliferation
73
IkB
NF-kB inhibitor
IkB is phosphorylated upon activation
74
IKK
phosphorylates IkB -> IkB is ubiquitinated and degraded -> NF-kB can translocate to the nucleus
75
abberant activation of NF-kB pathway ->
many human dieases and inflammation
76
conditions associated with hypoxia
high altitude disease
stroke/ischaemia
diabetes
chronic kidney diesaese
GI diseases
schizophernia
neurodegenerative dieseases
ageing
RA
cancer
77
cell's reaction to low O2
- restoration of oxygen homeostasis
- cell survival
- cell death
78
HIF =
Hypoxia Inducible Factor
79
pathways controlled by HIF
- oxygen supply
- transcription
- cellular metabolism
- cell growth
- HIF control
- cell death
80
p53
tumour suppressor and transcription factor
81
p53 structure
typical domain structure: distinct binding and multimerisation domains
82
negative regulator of p53 activity
mdm2
83
DNA damage or stress ->
Mdm2 dissociation -> p53 activation -> cell cycle arrest -> allow for repair or apoptosis
84
mechanism of mdm2 dissociation
DNA damage -> p53 phosphorylated at ser15 by ATM/ATR kinases
mdm2 is also phosphorylated -> disrupts interaction between p53 and Mdm2
85
P14ARF=
tumour suppressor, induced by oncogenes when cell proliferation is increased
86
role of ARF
disrupts interaction between p53 and mdm2
can inhibit mdm2 -> increased levels of active p53
87
pre-mRNA
precursor to mRNA
88
primary transcript processing
events coupled to transcription:
- capping
- splicing
- polyadenylation
- editing
via RNA pol 2 CTD
89
cap and polyA tail
added post-transcriptionally
- not encoded in the genome
90
m7G cap functions
- protects mRNA from degredation by 5'->3' nucleases
- facilitates splicing
- facilitates export from nucleus
- critical for translation of most mRNAs
- functions mediated through protein binding
CBP80 in nucleus for processing/export
eIF4 complex in cytoplasm for translating
91
conserved sequences in introns
5' and 3' splice sites, branch sites
- sequences define the limits of exon and intron
- sequences recruit the splicing machinery needed to remove intron/join exons
92
2 steps of intron splicing
2 trans-esterefication reactions
1. cut at 5' splice site, create bond between 5' end of intron and branch site
2. cut at 3' splice sites -> release intron -> ligation of two exons
93
spliceosome
- enzymatic complex for intron removal
- requires ATP
94
snRNPs
= small nuclear ribonucleo-protein particles
- non coding RNA
- splicing is catalysed by snRNAs
95
alternative splicing
expands the proteome - number of proteins is greater than the number of genes in the genome
96
splicing is a ..... catalysed event
RNA
97
polyadenylation
Nascent RNA -> endonuclease cleavage -> addition of As by polyA polymerase -> polyadenylated mRNA
98
functional significance of the polyA tail
enhances RNA export
stabilised 3' end of mRNA
enhances mRNA translation
99
proteins required for polyadenylation bind sequences
CPSF: cleavage and polyadenylation specificity factor
CstF: cleavage stimulatory factor
polyA polymerase
100
RNA editing
nucleotide alterations -> different/additional nucleotides in the mature RNA
- changes coding sequence/proterties of mRNA
101
two classes of RNA editing
insertion/deletion
modification
102
effects of mRNA editing
-creation of start (AUG) codons
-new open reading frames
-creation of stop codons
103
N6-methyladenosine
- most prevalent internal euk mRNA modifyer
- regulated by writers/readers/erasers
104
APOBEC-1 enzyme
edits mRNA-> cytidine deamination
- linked to cholesterol control, cancer dev, inhibits viral replication
105
A to I editing in the Q/R site of glutamate receptors
- L-glutamate = maj excitory NT
- editing -> decrease in Ca2+ permeability of channels containing R version
- editing carried out by ADAR2
106
purpose of mRNA localisation
- generate cell polarity
- prevents expression in wrong place
- promotes efficience of protein targeting
- local control of translation
107
prokaryote ribosome
70S
108
eukaryote ribosome
80S
109
ribosome catalyses...
peptide bond formation
110
kozac sequence
ACCACCAUGG
- sequence before and after start codon
111
eIF2B
regulator of translation
- subunit of eIF2, governs eIF2-GTP levels
- down regulated in response to stress
112
eIF2 phosphorylation (stress)
essential amino acids/ER stress/ heavy metals ->eIF2-P -> eIF2B -> protein synthesis
113
where is Fe found in the cell
heme and iron-sulfur clusters
114
cannonical IRE
TfR1B
115
IREs with additional 5' or 3' unpaired nucleotides
L-ferritin
DMT1
HIF-2a
116
IRE =
Iron Response Element
117
iron levels regulate...
production of iron binding proteins -> switch between iron import and storage
118
IRP! binding to mRNA ->
blocks or activates translation
- also affects mRNA stability
119
Reasons for degrading RNA
- damaged RNA
- badly processed mRNA
- to control gene expression: rapidly alters expression by just turning it off
120
casein mRNA
mRNA increases 70-fold when stimulated by prolactin (transcription only increases 2-fold)
- half-life increases dramtically in response to prolactin
121
phase 1 of mRNA degradation
remove cap and polyA tail with decapping enzymes and deadenylases
break up the strand so exonucleases can work
122
phase 2 of mRNA degradation
open ends -> very vulnerable to exonucleases
exonucleases break mRNA down from either end
123
decapping enzymes
DCP1, DCP2
124
endonucleases
argonaute, Swt1, Smg6
125
deadenylases
Ccr/Not complex
126
Exosome
main 3' to 5' exonuclease
multiple nuclease activities: RRP6, RRP44
- all other subunits function in RNA binding and unwinding
127
XRN1
5' to 3' exonuclease
- also involved in transcription termination
- can't work unless mRNA cap is removed
128
NMD
= nonsense mediated decay
mistakes in RNA detected -> RNA targeted for degradation
- premature stop codons -> errors
129
RNAi
RNA interference
130
siRNA
small inhibitory RNA
- viral defense mechanism
- leads to degradation of the target RNA
131
miRNA
micro RNA
- key gene regulatory mechanism
- leads to block in translation
132
RISC
RNA induced silencing complex
