Exam 1 Flashcards
(158 cards)
1
Q
what RNAs does Pol I transcribe and their functs
A
one single pre r-RNA, processed into subunits (28S, 18S, 5.8S rRNAs)
ribosome components e protein synthesis
2
Q
what RNAs does Pol II transcribe and their functs
A
mRNA, snRNAs, miRNAs, ncRNAs, eRNA
encode protein, RNA splicing, post-transcriptional gene control
3
Q
what RNAs does Pol III transcribe and their functs
A
tRNAs, 5s rRNA, snRNA U6, 7SRNA, other stable short RNAs
Protein synthesis, ribosome component, RNA splicing, singal reco particle for insertion of polypeptides into ER
4
Q
describe pol I transcription initiation
A
pr DNA made up of upstream element e core element, UAF (upstream activator factor) binds upstream element, recruits TBP and CF (core factor), Pol I is recruited and starts transcription
transcription of pre-rRNA by pol I causes the formation of the nucleolus bc more ribosomes need to be made to keep up
5
Q
what is the nucleolus
A
area in nucleus where ribosomes are made and assembled, occurs after pol I transcription
6
Q
what is TSS and what does it interact with
A
transcription start site
interacts w TFIID
7
Q
what is PAS
A
poly-A signal
8
Q
what is DPE
A
downstream pr element
interacts w TFIID
9
Q
what is TEF
A
transcription elongation factor
10
Q
what is coding strand
A
5’ to 3’ DNA, upper strand, same code as RNA
11
Q
what is template strand
A
3’ to 5’ DNA, lower strand, used as template for RNA binding
12
Q
cis-regulatory elements
A
promoters, enhancers, and silencers; regions of non-coding DNA, which regulate the transcription of nearby genes.
In contrast, trans-regulatory factors regulate the expression of distant genes by combining with their target sequences
13
Q
TATA box
A
-30 to transcription; important for initiation; strong pr; only 10-15% of genes transcribed by Pol II have TATA box, many of the highly expressed genes (can be w/ or w/o Inr)
facilitates binding of TFIID
14
Q
Initiator (Inr)
A
loose consensus seq at beginning of transcription (A at +1), sufficient for basal transcription, the simplest funct pr that is able to direct transcription initiation without a functional TATA box (can be w/ or w/o TATA box)
facilitates binding of TFIID
15
Q
describe layout of basal pr elements
A
|BRE|TATA|——|Inr|———————-|DPE|
BRE - TFIIB recog element (–37 - –32)
TATA - TATA box (–31 - –26); 10-15% Pol II transcribed genes
Inr - Initator (–2 - +4); 50% Pol II transcribed genes
DPE - downstream pr element (+28 - +32)
16
Q
focused transcription initation
A
more tightly regulated genes/pr; focused transcription; often developmental genes that are important at checkpts; more tissue specific, tends to have basal pr elements (BRE/TATA/Inr/DPE)
TATA in 15-20% of genes transcribed by Pol II
Inr in 50% of genes transcribed by Pol II
can be silenced by presence of nucleosomes
17
Q
dispersed transcription initiation
A
multiple TSSs, not more clear basal promoter element
constitutive expressed genes, housekeeping genes; lower level, but constant, transcription
GC rich, reg by methylation
cannot be silenced by a single nucleosome bc so many TSSs, which leads to the constant low level activation / always accessible by Pol II
18
Q
role of nucleosomes in transcription regulation
A
[presence / removal] causes [suppression / activation] of genes at the area by making the DNA inaccessible to Pol II
19
Q
describe PEAT
A
paired end analysis of TSS; Identify pr of a specific gene (need to have a gene-specific primer)
The RNA fragment is isolated and treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase (BAP and TAP). This removes the 5’ cap; the 5′ ends of uncapped mRNAs are ligated to chimeric linkers containing MmeI restriction endonuclease sites prior to RT. The RT primers also contain an MmeI site. This results in single-stranded cDNA flanked by MmeI sites. The fragments are PCR-amplified and circularized into circular single-stranded cDNA, which is amplified by rolling-circle amplification (RCA). MmeI is used to cut circular cDNA at the 2 MmeI sites to create linear, double-stranded cDNA fragments. The fragments are ligated to paired-end adapters, amplified, and sequenced.
20
Q
describe CAGE
A
cap analysis gene expression; identify all TSS near cap (using random primer)
RNA is reverse-transcribed to generate an RNA/DNA hybrid; the 5’ cap is oxidised and biotinylated, and the duplex digested with RNAse I to release and remove the excess ends of RNA. The biotinylated capped ends are pulled down w streptavidin beads, and cDNAs are isolated. 5’ and 3’ linkers are added. Samples are pooled before low-cycle PCR to generate completed library for seq
21
Q
what is the purpose of PEAT v CAGE
A
PEAT identifies the TSS of a specific gene, CAGE identifies TSS generally, using random primers
PEAT: paired end analysis of transcription start sites
CAGE: cap-analysis of gene expression
22
Q
what is Puffin
A
Puffin: Deep-learning-inspired explainable sequence model of transcription Initiation
Use previously made CAGE libraries to identify TSS; which ones lead to high abundance of transcription
which ones associated w bidirectional transcription.
Identify seq in uni and bi directional promoters, Conservation of pr seq
form prediction model: where does transcription initiate on a gene that the TSS hasn’t been identified yet
23
Q
what is YY1
A
transcription motif that can activate or repress
24
Q
5’ cap
A
guanine nt added at 5’ end, methylated immediately after being added (m7G)
signal for the recruitment of translation machinery
25
what is HARPE
high-throughput analysis of randomized promoter elements: identify downstream prs by inserting random seq at +17 to +35, each one is taken up by a cell, isolate polyA RNA, seq; some seq have higher or lower transcription, align nts, identify consensus seq
26
formation of the RNA Pol II transcription complex
TBP (TATA-binding protein) binds to the TATA-box as part of TFIID; TFIIA stabilizes TBP binding and makes contacts with the upstream DNA backbone; TFIIB further stabilizes the complex and recruits the Pol II/TFIIF complex; TFIIE stimulates recruitment of TFIIH, which harbors a DNA translocase activity that assists in melting the DNA
27
what is the purpose of TFIIA
stabilizes TBP:TATA box binding
28
what is the purpose of TFIIF
binds Pol II, regulates Pol II, which would otherwise bind and initiate at nicks
29
what is the purpose of TFIIH
3 functs: separate/unwind DNA strands, kinase activity (phos CTD of Pol II to initiate) , and nucleotide excision repair (NER)
3 funct activities: helicase, kinase, ATPase
also threads the DNA
contains XPB e Cdk7
30
what is the PIC
pre-initiation complex, assembles at the basal pr region of gene
made up of RNA pol II and GTFs (general transcription factors)
GTFs = TFIID, TFIIB, TFIIF, TFIIE, TFIIH (and TFIIA?)
31
what is TFIID made up of
TBP - TATA binding protein
TAFs - TBP-associated factors
32
funct of TFIID
topological changes in pr DNA
TBP in TFIID binds minor groove and causes near 90' bend
binds DNA (all DNA binding ability)
33
canonical v rearranged TFIID
canonical - not bound to DNA, not activated, lobe A in association w lobe C, TBP close to TAF1 via TAND domain, which competes w binding to DNA
rearranged - bound to DNA, activated can bind DNA, forms after TFIIA stim binding of TBP (e TFIID) to TATA box, causes flexible A loop move closer to lobe B e to bind DNA, TAF4 stabilizes complex e DNA
34
role of TAF1
part of canonical (inactive) TBP (TFIID), TAND competes w lobe A for binding to DNA
must be displaced to allow DNA binding
helps move lobe A closer to lobe B
35
role of TAF4
stabilizes complex between TBP (TFIID) e DNA
interacts w TFIIA e DNA
interacts w transactivating proteins & help recruit complex
36
role of TAF6
stabilizes lobes B and C of TBP (TFIID)
37
mechanisms for initiation e recruitment of transcription machinery
3 main mech:
histone modifications (the +1 histone)
DNA seq for affinity for specific subunits (TBP, TAF1, TAF2)
transcription activators that interact w TAF4
38
what is the role of nucleosomes in the context of transcription
they are repressive, need to be removed in order for transcription to take place
histone tails can be modified to recruit specific TAFs (as part of TFIID)
39
how does TFIID conformation change in the PIC in the absence of TATA box
conformation is similar
40
what are the components of TFIIB
B-ribbon, b-reader, b-linker, b-core N-term, b-core C-term
41
what does b-reader do
part of TFIIB
b-reader interacts w DNA:RNA hybrid, initially stabilizes it, then is eventually disrupted as RNA gets longer
acts as ‘ruler’ of how long after TBP does transcription start
42
what is the XPB domain
part of TFIIH
required for DNA melting during transcription initiation, causes slight change in DNA conformation that allows for opening of the DNA
aka Ssl2
43
steps in RNA pol II transcription complex formation
TFIIA facilitates binding of TBP to minor groove, bends DNA
TFIIB bridges TBP-TATA box complex w RNA pol
TFIIF interacts w RNA pol II (prevents non-pr initiation)y of TFIIH
TFIIE stim helicase e ATPase activity of TFIIH
TFIIH melts the DNA during initiation--> formation of closed pre-initiation complex
phos Ser5 of CTD via TFIIH --> start transcription
NTPs added, ATP hydrolysis, release of general factors (except TBP), e elongation
44
closed v open complex
closed - RNA pol + TFs bound to pr
open - DNA strands have been separated, transcription bubble is formed
45
what is CTD
heptapeptide repeat 52x
must be phos for elongation to take place
early repeats are canonical, later repeats have diff aas, used for regulation e termination
Ser5 phos, then Ser2 phos
after Pol II falls off, all phos on CTD can be removed --> allows for recycling of Pol II
46
what is a puff region
area of unfolded chromatin, open, active, e associated w Pol II CTD phos
47
CTD Ser2 phosphorylation
mediated by PTFB complex, makes it elongation competent, allows binding to elongation factors e splicing elements
2nd, AFTER ser5 phos
48
CTD Ser5 phosphorylation
mediated by Cdk7 (part of TFIIH), disrupts interactions w PIC, allows Pol II to move to elongate
1st, BEFORE ser2 phos
49
what does Cdk7 do
phos Ser5 of CTD
part of TFIIH
disrupts interactions w pre-intiation complex, allows Pol II to move to elongate
**makes complex go from initiation to elongation**
50
what does Cdk9 do
phos Ser2 of CTD
part of pTEFb
makes it elongation competent, allow binding to elongation factors and splicing elements
phosphorylates NELF and DSIF during pausing
**makes complex elongate**
51
steps in RNAPII transcription cycle
initiation > elongation> termination > recycling
different parts of the CTD are phosphorylated or dephosphorylated at different times in the cycle
52
what is the funct of SPT4/5
forms DSIF complex
53
what is PPP
pr proximal pausing
pol II stalls downstream of TSS, where it is bound to NELF e DSIF; NELF, DSIF, e pol II need to be phos by CDK9 subunit of PTEFb to form elongation complex
54
what is NELF
neg elongation factor
55
what is DSIF
DRB-sensitivity-inducing factor, made up of Spt4:Spt5 complex
causes pausing, needs to be phos to cause conformational change e allow for elongation
56
describe pol II transcription elongation
1. initiation: TFIIH mediated phos of CTD Ser5; if abortive transcription, Integrator removes Pol II
2. pausing association w NELF e DSIF > 5' capping > phos of NELF, DSIF, and CTD Ser2 by CDK9 > release of Pol II e CTD Ser7 phos, association w elongation factors
3. termination: PAS destabilies pol II, exonucleases degrade RNA and push towards pol II, phosphatases remove pol II CTD phosphate groups
57
what is integrator PP2A
integrator associated phosphatase, removes phos from CTD e DSIF (Spt5)
removes low processive pol II
The Integrator Complex Attenuates Promoter-Proximal Transcription at Protein-Coding Genes by removing low processivity pol II
terminates snRNA transcription
58
what is spt6
transcription elongation factor, inc processivity
binds to CTD that has been phos
comes in after DSIF has been phos
59
what is ITC
initially transcribing complex
includes abortive initiation e premature transcription termination
60
what is EEC
early elongation complex
pr escape complete, nascent RNA stably associated w transcription complex
61
7 steps of transcription elongation
1. initiation, formation of ITC from PIC, v unstable, abortive initiation
2. pr clearance/escape, formation of EEC, pr escape and stable complex
3. pr proximal pausing
4. productive elongation
5. transcription termination
6. transcription elongation through nucleosomes
7. bidirectional transcription (caused by R loops)
62
what is premature transcription termination (PTT)
accumulation of pol II at 5' end of genes, 99% of transcription initiation events result in PTT
ITC is unstable bc of short DNA/RNA hybrid, TFIIB is needed to stabilize the complex until an 8bp hybrid is made but PTT can still occur
integrator contributes to PTT in unstable Pol II elongation complexes
Release of Pol II into productive elongation is likely regulated by activities that inhibit PTT
strong pr (like TATA e INR) overcome pausing
63
pausing index
Occupancy of Pol II at promoter proximal region compared to gene body
64
travelling ratio
The ratio of Pol II accumulation at gene body versus promoter proximal site
65
pausing index v traveling ratio
pausing index: occupancy of pol II at pr proximal region compared to gene body
traveling ratio: ratio of pol II accumulation at gene body v pr proximal site
66
formation of the transcription bubble
needed for change from ITC to EEC
unwinding begins ~20bp downstream of TBP binding site, TFIIB (upstream edge) stays fixed until pr escape is complete, while downstream edge expands, putting stress on the complex; there are several cycles of abortive initiation, until 4 nts are made, stabilies by TFIIB; when 5 nts are made, the RNA clashes w B-finger of TFIIB, causing stress, which can cause abortive initiation/pausing/slippage if RNA/DNA hybrid is weak; once there are 7nts there is transcription bubble collapse, providing E to remodel transcription complex, at 8-9 nts the RNA can go through exit tunnel --> no more abortive transscription or need for ATP, forming early elongatio complex
67
what do pausing factors do
stabilize pol II, cause pausing, need to be removed for elongation
NELF - needed to get through +1 nucleosome e avoid pr proximal termination, dissociates after phos
DSIF - pausing, after phos helps elongation
PAF1C - prevents formation of super elongation complex
Gdown 1 (subunit of pol II) - blocks recruitment of TFIIF (causes pausing)
68
what is pTEFb
positive transcription elongation factor b
69
what does PAF1C do
CDK9 phos SPT5, PAF1C recruits integrator-PP2A which removes phos from SPT5 --> inc processivity
w/o PAF1C, SPT5 is rapidly phos e integrator-PP2A cannot remove phos well --> low processivity
integrator removes low processivity pol II
70
what is GRO-seq used for
study on-going transcription
in RNA-seq you can only look at RNA already present, GRO is good for active RNA transcription
can also use PRO-seq
71
explain GRO-seq
Polymerases are allowed to run-on ~100 bases in isolated nuclei in the presence of Br-UTP. The RNA is then base hydrolyzed to ~100 bases and bound to agarose beads that are coated with an α-BrdUTP Ab. 5’-7meG caps are then removed, and the ends of the RNA are prepared for adapter ligations. Illumina small RNA adapters are added to the 5’ end followed by the 3’end, with an additional round of immuno-enrichment after each adapter ligation. The RNAs are then reverse transcribed, amplified, and PAGE purified prior to Illumina sequencing
72
experimental: pausing v activity
Pausing: looked at using Pol II ChIP, paused will have accumulation of pol II near pr
activity: look at gro-seq, active will have transcripts all the way through gene
not paused e active: some pol II at pr, Gro-seq activity throughout
paused e active: pol II accumulated at pr (wider area), low gro-seq activity
paused e not active: no pol II, no gro-seq
not paused e not active: no pol II, no gro-seq
lec 2 slide 15
73
Pro-seq v gro-seq
look at same thing, pro-seq uses biotin tagged NTPs, gro-seq uses Br-UTP
74
ATAC-seq
used for looking at accessible chromatin
light digest DNA then seq
75
what is the function of integrator in early transcription events
The Integrator Complex Attenuates Promoter-Proximal Transcription at Protein-Coding Genes by removing low processivity pol II
transcription rapidly decays at beginning with high peak and fall off at beginning (a)
high amount of integrator found at area where pausing is happening w fast decay (e)
dec of integrator --> transcription through gene body
important for pausing e removal of pausing (A, D)
elrod paper, lec 2 slide 16-18
76
what factors remove low processivity pol II
integrator e CRL3ARMC5 E3 ligase
CRL3ARMC5 ub RNA pol II at Ser5
both are needed, depleting one is not sufficient to remove pol II
77
what is the funct of elongin
inc processivity of pol II by making conformational changes in pol II
78
how does pol II speed change over someones lifetime
younger people have slower pol II, allowing for inc abundance of ea transcript compared to fast pol II, which makes less of ea transcript
79
Auxin-inducible degron tagging
rapid degradation system
express protein of interest w mAID tag, add auxin --> auxin binds mAID e TIR, TIR leads to rapid degradation
80
allosteric model of transcription termination
conformational change or factors recruited to or dissociated from pol II caused termination
81
torpedo model of transcription termination
PAS cleavage generates pol II associated RNA that is degraded 5'>3' by exonuclease (XRN2), then leading to termination
82
unified allosteric/torpedo model of transcription termination
PAS cleavage promotes pol II slowing bc of dephos of SPT5 by PNUTS/PP1, causing allosteric switch; PAS cleavage inc threonine4 phos on CTD, makes pol II 'stranded', allowing for exonuclease XRN2 to then remove pol II
83
what is FACT
histone chaperone, facilitates transcription by nucleosomes by altering the H2A/H2B dimer
'guardian' in chromatin structure integrity
84
FACT mediated transcription through nucleosome
FACT facilitates transcription THROUGH nucleosomes by removing a H2A/H2B dimer, and replacing it after RNA pol II has finished transcribing at that region
reminder: H2A/H2B is on the outside of histone, H3/H4 at core
85
4 chromatin states in eukaryotes
silent - chromatin + repressors
ground - default, only chromatin
poised - chromatin + activators
active - chromatin + activators + TFIID e pol II holoenzyme
86
what aas make up the majority of histone tail marks
lys (K) e arg (R)
both have pos charge
87
list types of histone marks
phos
methylation
acetylation
Ub
88
histone subunit structure
H2A e H2B dimerize, form outer-most components
H3 e H4 dimerize, make core
H1 is linker
89
analysis of chromatin by nuclease digestion
use enzyme to cut DNA, enzyme does 'light digestion' (w MNase) so can only cut at linker not at histone, get histone ladder where bottom length is the repeating size (make sure by checking that each band is equal length) ~200bp - this is the size of the linker / H1 region
use stronger enzyme to do more cutting --> 147 bp ladder, bc 147bps around ea histone
90
fundamental nucleosome architecture
two H2A–H2B heterodimers associate on the outside of the core H3–H4 tetramer, e the neg charged backbone phosphates of each DNA strand bind to the positively charged surface of the histone octamer at every ~10 bases. 147bp of DNA is lefthandedly wrapped 1.65 turns around the histone octamer (DNA ends do not meet)
phosphates of DNA backbone binds every ~10bp
histone arg interacts w ea minor groove of the DNA (14 bc 147 / 10)
NO seq specific interactions
has 7 SHL on each side of dyad (center)
nucleosome forms 'disk' not bead, w one side thicker than the other
the H3/H4 core has strongest protein:DNA interactions, tho not fixed
90
SHL
superhelical location
defined as a site where the major groove faces towards the histone surface, numbered -7 – +7 away from dyad (SHL0)
91
ncp
nucleosome core particle
includes histone + DNA
92
nucleosome breathing
ends of DNA furthest from nucleosome core have weakest interaction w histone, can spontaneously dissociate e re-associate
93
how can you change the stability of the nucleosome
modify the histone subunits (eg: H2A for H2A.Z)
modify histone tails, can be deep into core of nucleosome, not always outside
remodeling complexes
DNA methylation
mutations in the histone (eg: H2BE76 disrupts dimer-tetramer interface)
94
what is the beads on a string nucleosomal array
10nm fiber, the open, ACTIVE form of chromatin, compared to the higher order structure (30nm) which is inactive
95
what is the higher order structure of chromatin
30nm fiber, the condensed, INACTIVE form of chromatin, also called a solenoid (beads on a string 10nm is active)
there is might not be a 30nm fiber in vivo
96
levels of chromatin fiber organization
DNA double helix > winding around histones > histone stacking > formation on protein scaffold w loops > winding into chromosome
nucleosomes are found on the loops
97
what internucleosomal interactions are there
H4 tail (pos) interacts w H2A/H2B dimer (neg) of ADJACENT nucleosome --> contributes to tight stacking
98
what is two-start nucleosome geometry
histones do not just stack on top of ea other, but rather form a zig-zag coil
observed by cutting linker DNA, and seeing that half of the nucleosomes are still attached to ea other, chromatin forms a zig-zag ribbon (lec 4 slide 37)
99
how does the nucleosome repeat length affect the 30nm fiber
size of linker changes size of chromatin fiber, longer linker more wide where shorter linker more tall (dont mem)
100
how does histone tail modifications cause changes in the chromatin
most: affect interactions w other proteins (readers)
modify electrostatic interactions between DNA (neg) e histone (pos)
nucleosome stability
one histone mark can recruit complexes that place other histone marks
101
H3K4me3
near TSS / proximal pr --> gene activation
mediated by MLL1/2
102
H4K16ac
disrupts internucleosome interactions, causing open chromatin bc prevents stacking
prevents interaction of H4 w H2A/H2B acidic patch of neighboring histone
gene expression, DNA repair, e chromatin remodeling, can be activating e repressive
103
what do all histone marks have in common
they are all reversible
104
writers
add mark, usually complexes
HATs (aka KATs), histone methyltransferases
105
readers
recog histone mark, have some effect
bromodomains, chromodomains, PHD fingers, etc
106
erasers
remove histone marks
HDACs, lysine demethylases
107
how does acetylation affect the chromatin
- weakens electrostatic interactions between histone e DNA; acetylation neutralizes the pos charge of K
- weakens electrostatic interactions between histones of neighboring nucleosomes, promoting decondensation of chromatin fiber (H4K16ac)
- provide mark for recog by reader proteins w bromo domains
allows SWI/SNF binding
108
Mammalian HAT familes
MYST
GNAT
p300/CBP
some (eg: p300/CBP) have bromodomains --> recog acetylation, and expand it
109
what does the bromodomain recog
histone acetylation
110
HDAC characteristics
usually lack high degree of specificity of a particular ac group
111
HAT characteristics
ususally cause transcription activation
usually ac more than one lys
usually part of multisubunit complexes
some can ac other proteins
usually regulated by availability of metabolites
loose specificity
112
where does acetyl-CoA come from
mostly glucose metabolism (but some lactic acid metabolism)
is needed for ac of histone tails
113
what does chromodomain recog
methylation
114
what does PHD recog
methylation
115
what domain recognizes lys ac
bromodomain
116
what domain recognizes methylation
chromo e PHD
117
H3K27ac
at active enhancers, permits binding of TFIID e BRD4
deposited by KAT3A or KAT3B
118
how does histone acetylation impact DNA repair
H4K16ac limits 53BP1 association with damaged chromatin to promote homologous recombination
119
key domain of SWI/SNF
bromodomain (bind ac)
120
H3K4me
pan-enchancer
mediated by MLL3/4
121
super enhancer histone marks
all enhancers have H3K4me
super enhancers also have H3K27ac
122
role of HDACs in cancer
HDACs can have gene specific effect, in cancerm more HDAC at genes for things like p21
HDAC inhibitors are being researched as cancer treatment, but hard bc they are pan-inhibitors, affecting other important maintenance genes
123
how does methylation affect chromatin
does not change charge
act as specific histone marks depending on position e number of me
can me activating or repressive
124
HMT characteristics
much more specific than HATs
repressive or activating
125
HMT domains
SET (lys)
DOT1L (lys)
PRMT1 (arg)
126
HDMT famiies
LSD (removes H3K4me e me2)
JMJC (remove 1 me at a time; H3K4me3 > H2K4me2 > H2K4me)
can have reader domains (recruited by other histone mark)
127
what does LSD1 do
demethylate H3K4me or me2 (NOT me3)
acts as transcriptional co-repressor in CoREST complex (H3K4me is usually activating)
128
what does CoREST do
demethylate H3K4me or me2
uses LSD1 component of complex
129
facultative heterochromatin
can sometimes be transformed into euchromatin, which may or may not be transcribed
130
constitutive heterochromatin
always condensed, especially at telomeres and centromeres
131
facultative v constitutive heterochromatin
facultative can become euchromatin, constitutive is always condensed
132
translationally phased
the same DNA seq is wrapped around the histone octamer in all cells in the population
population level description
133
how do you map nucleosome positioning
limit digest (light digest) w MNase (cannot cut to mononucleosome, need distribution of nucleosomes), run gel to separate, label w P32 e do northern blot
can also seq (peaks where MNase cut, normal curve bc MNase can cut a little to the right or left)
note if fig has occupancy or availability
134
active v inactive positioning
active has euchromatin shape, w space at linkers
inactive is heterochromatin, many more histones per length of DNA
135
how does occupancy change along a gene
more organized near TSS, with highest degree of organization at the +1 (then +2, then +3) nucleosome
136
what make good organizing centers
TSS (best)
insulators
certain DNA seq have higher affinity to histones
137
persistence length
how hard to bring two ends of DNA e close them together
measures how bendable is a DNA seq
default is 210bp (very hard to do but technically can be done)
138
intrinsic nucleosome organization
some DNA seq have higher affinity for nucleosomes than others
~50% of nucleosome positioning is accounted for by seq specificity IN YEAST
in humans only ~20%
139
rotational positioning
how the DNA seq is positioned around the histone
projected seq = part towards solution / away from histone
bending ability is determined by the seq in minor groove bc the minor groove is smaller, so large aas hinder it more
GC clash more than AT, want AT in minor groove e GC in major groove
140
where does DNAse cut
binds at minor groove e cuts backbone
can cut max every 10bp (cuts where minor groove is accessible)
141
which nts are preferred towards v away from histone
AT preferred at minor groove facing towards histone
GC preferred at minor groove projecting towards solution
142
dyad
technically requires symmetry everywhere, including DNA seq
most histones are pseudo-dyads
143
how do you look at rotational phasing
have to use DNase I not MNase bc MNase prefers linkers, DNase I prefers minor groove
see if cuts line up --> if not, then NOT rotationally phased
need very strong positioning to get rotational phasing
144
how does chromatin remodeling affect transcription
remodeling of higher-order chromatin structure e nucleosome remodeling changes position e occupancy
changes accessibility of DNA to diffusible factors
activation is associated w opening of chromatin
145
what are DHS
DNase I hypersensitivity sites
146
how do you map DHS
DNase I hypersensitivity sites
treat chromatin w DNase I > purify > digest w restriction enzyme > fractionate by agarose gel > southern blot > hybridize w radiolabeled probe > image
will see bands at sites where DNase I cuts (relative to start site)
147
types of binding sites for transcription factors
bind at linker DNA
bind at nucleosomal DNA
- inward site or
- outward site
remember nucleosomal breathing, ends of nucleosomes will be more exposed
148
positioned v random nucleosomes
positioned is a population level term
can be regularly spaced within a single cell, but not aligned within the population --> regularlly spaced but not translationally or rotationally phased (lec 7 slide 22)
149
role of nucleosomes in initiation
inhibit initiation
150
what is the most important thing about chromatin remodelers
they are ATP dependent, and facilitate SLIDING
151
what do all chromatin remodeling complexes have
ATPase sybunit
ATP-dep chromatin perturbation
all have SWI/SNF related domains
152
activities of ATP-dep chromatin remodeling complexes
nucleosome sliding
nucleosome remodeling
nucleosome displacement
nucleosome replacement (w different type of histone eg: H2A.Z)
nucleosome assembly
change chromatin access
nucleosome editing
nucleosome disassembly
153
what is SWI/SNF
ATP dep chromatin remodeler
positions nucleosomes
facailitates binding of transcription factors to DNA in a reconstituted/assembled nucleosome core particle
facilitates nucleosome *sliding* via inchworming
can push histones in either direction
can displace histones
154
what does RSC do
catalyze the transfer of histone octamer form nucleosomes to free DNA
155
what does ISWI e CHD do
make well spaced chromatin
not necessarily translationally phased
156
mechanisms for targeting remodeling complexes to sites of activity
target by DNA-binding factors
target by methylated DNA (silencing)
target by interaction w nuclear matrix and actin-like proteins
target by recog of histone modifications
157
how does SWI/SNF disassemble nucleosomes
requires adjacent nucleosomes, slide until it bump into next nucleosome, bump it off
