Control of transcription and chromatin Flashcards
(152 cards)
1
Q
What is gene expression?
A
Process by which information in genes (DNA) is decoded into protein, it is a multistep process which is highly regulated.
2
Q
What is gene expression regulated by?
A
Mostly transcription.
3
Q
What is the function of a promoter?
A
RNA polymerase binds here.
It dictates how efficient transcription will be.
4
Q
What is the promoter in prokaryotes (bacteria)?
A
Sigma 70 subunit/factor
5
Q
What is a promoter?
A
Cis acting DNA regulatory element through which transcription is initiated and controlled.
6
Q
What is the structure of the promoter in prokaryotes?
A
Hexametric
35 sequence long upstream
A simple molecule
7
Q
What are the regions within the eukaryotic promoters?
A
Regulatory region
Core (basal) region
ORE
8
Q
What is within the regulatory region in prokaryotic promoters?
A
Enhancer
x2 UAS
9
Q
What is within the core (basal) region in prokaryotic promoters?
A
TATA box
Initiator (Inr)
DPE
BRE
10
Q
What is the DPE?
A
Downstrean core promoter element
11
Q
What is BRE?
A
TFIIB recognition element
12
Q
What is the location of the TATA box?
A
-31 to -26
13
Q
Why are some gene locations negative?
A
Upstream of the transcription site.
14
Q
What is the location of the initiator?
A
-2 to +4
15
Q
What is the location of DPE?
A
+28 to +32
16
Q
What is the location of BRE?
A
-37 to -32
17
Q
What is a TATA box?
A
The TATA box is a sequence of DNA, consisting of nucleobases TATAAA, located in the promoter region about 25 base pairs before the site of transcription.
18
Q
What are the eukaryotic promoters?
A
Associated with regions with a high frequency of CG sequences: “CpG islands”
19
Q
What are the specifics of CpG islands within acting as a promoter?
A
In mammals most C residues followed by G a are methylated (to 5-methyl C).
However generally C residues in CpG islands escape methylation (hypomethylated).
20
Q
What is the function of CpG islands within silencing?
A
Methylation of CpG islands is associated with silencing (txn “switched off”).
21
Q
What within the eukaryotic promoters is the regulatory region?
A
Silencer
Enhancer
URS
X2 UAS
22
Q
What are the activator binding sites within the eukaryotic promoters?
A
UAS & Enhancer
23
Q
What are the repressor binding sites within the eukaryotic promoters?
A
URS & Silencer
24
Q
What within the eukaryotic promoter is inside the core (basal) region?
A
Txn Start-site
25
What are the Tools for Identifying Promoter Elements?
1. Sequence comparison
2. Reporter Analysis
26
What is reporter analysis?
Fuse promoter to reporter.
Reporter genes encode proteins whose levels can easily be measured.
Allows for a measure of gene expression.
27
What can reporter analysis identify?
When a gene is expressed?
Where it is expressed?
What signals it responds to?
What factors and sequences control its expression?
28
What are the target genes for RNA polymerase I?
rRNA
29
What is the location of RNA polymerase I?
Nucleolus
30
What are the target genes for RNA polymerase II?
mRNA
snRNAs
miRNAs
31
What is the location of RNA polymerase II?
Nucleus
32
What are the target genes for RNA polymerase III?
tRNA
5S RNAs
U6 RNA
7S RNA
33
What is the location of RNA polymerase III?
Nucleus
34
What is the structure of Bacterial RNA polymerase?
ß subunit
ß’ subunit
α subunit (x2)
ω subunit
35
What is the structure of Yeast RNA polymerase II (Eukaryotic)?
12 subunits
36
What are the similarities between RNA polymerases?
All DNA dependent
37
What are General Transcription Factors (GTF’s)?
Bacterial RNA polymerase requires σ-factor to recognise promoter DNA.
38
What are the functions of General Transcription Factors (GTF’s)?
(1) RNA pol specific
(2) Multi component factors
(3) Form a complex on TATA box
(4) Recruit RNA pol II to the promoter
(5) Direct initiation at start-site
39
What is the order of addition for Pre Initiation Complex (PIC) Assembly?
Recognition and binding TF2 d to TATA.
Then TF2A followed by TF2B.
TFIF already attached to RNA polymerase II.
TF2E the TF2H.
Not the only order.
40
How is transcription initiated by RNA polymerase II?
Helicase activity of TFIIH separates the strands at the start site.
As Pol II begins transcribing it is phosphorylated on the C-terminal domain (CTD)
The CTD is a series of repeats located at the C-terminal end
of the largest β’ homologous subunit of Pol II.
41
What happens to the rest of the transcription factors when transcription begins?
TFIID and TFIIA may stay behind
TFIIB, TFIIE and TFIIH are released
TFIIF moves down the template with Pol II
42
How many subunits does the TFIID have?
13
43
How many subunits does the TFIIA have?
3
44
How many subunits does the TFIIB have?
1
45
How many subunits does the TFIIF have?
2
46
How many subunits does the TFIIE have?
2
47
How many subunits does the TFIIH have?
9
48
What is the function of TFIID?
BINDS TO THE TATA BOX (CORE PROMOTER).
RECRUITS TFIIB.
49
What is the function of TFIIA?
STABILIZES TFIID BINDING
ANTI REPRESSION FUNCTION
50
What is the function of TFIIB?
RECRUITS RNA pol II-TFIIF
IMPT FOR START SITE SELECTION
51
What is the function of TFIIF?
STIMULATES ELONGATION
DESTABILIZES NON SPECIFIC RNA pol II-DNA INTERACTIONS
52
What is the function of TFIIE?
RECRUITS TFIIH AND MODULATES TFIIH ACTIVITY
53
What is the function of TFIIH?
PROMOTER MELTING AND CLEARANCE
CTD KINASE ACTIVITY
DNA REPAIR COUPLING
54
What is the structure of TFIIH?
TFIIH is composed of 9-10 subunits
It can be divided into two parts, CORE + CAK
Contains ATPase.
55
What is the CAK molecule?
The CAK module contains one of the kinases that phosphorylates the CTD of RNAP II
56
What is the function of the ATPase within the TFIIH?
TFIIH contains a ATPase called XPB (or Ssl2) that plays a key role in PROMOTER MELTING
57
What is TFIID?
TFIID is the Central RNA pol II Transcription Factor
58
What is the structure of the TFIID?
TATA binding protein (TBP)
TBP associated factors (TAFs)
59
What is TBP?
Molecular saddle
60
What is the advantage function of TBP?
TBP can direct the assembly of the PIC on a TATA-containing
promoter (in vitro).
61
What are the limitations of TBP?
TBP alone can NOT direct PIC assembly on a TATA-less promoter.
TBP can NOT support “activated” Transcription
62
What is the PIC?
Pre initiation complex
63
What is the function of TAFs?
Promote the interaction of TFIID with the basal promoter
Interact with activators
64
Define the core (basal) promoter.
Consists of the region around the transcription start-site
Associated with elements such as the TATA Box and the Initiator (Inr) element.
65
Define UAS?
Upstream activating sequence. An activator binding site.
--or--
Upstream repressing sequence. An a repressor binding site.
66
Define enhancer.
DNA regions close or far from the start-site. Binding sites for activator proteins.
Often composed of multiple UAS elements.
67
Define silencer.
DNA regions close or far from the start-site. Binding sites for repressor proteins.
68
Define general transcription factors (GFTs).
A set of factors that recruit RNA pol II to the promoter & direct initiation at the start-site.
69
Define basal transcription machinery.
Another name for GTFs and RNA pol II.
70
Define preinitiation complex (PIC).
Assembly of the basal machinery at the core promoter.
71
Define activator.
A factor that binds to gene specific regulatory sequences and stimulates transcription initiation.
72
Define basal transcription activated TXN.
The level of transcription from a core promoter
Increased levels of transcription mediated by an Activator protein.
73
Define carboxy-terminal domain (CTD).
The CTD of the largest subunit of RNA pol II. Binds the Mediator complex and becomes extensively phosphorylated during initiation of transcription.
74
What is the function of TF2B?
Directs RNA polymerase II.
75
What are the common sequence elements of UAS?
* Often located close to the core promoter (promoter proximal)
* Bind activators that are relatively abundant in the cell and constitutively active
76
What is the factor associated with the GC box common sequence?
Sp1
77
What is the factor associated with the octamer common sequence?
Oct-1
78
What is the factor associated with the CAAT box common sequence?
NFY
79
What are the response element class of UAS?
Bind factors whose activity is controlled (induced) in response to specific stimuli
80
What does the response element SRE bind?
Serum Response Factor (SRF)
81
What is the inducer of response element SRE?
Growth factors
82
What does the response element HSE bind?
Heat Shock Factor
83
What induces the action of response element HSE?
Heat Shock
84
What are the characteristics of enhancer location?
The binding site can be at any location and the activation will still take place.
This means the enhancer works at any orientation.
85
What is required for activator to work?
Must be bound to DNA to work.
86
Examples of DNA binding domains.
Leucine Zipper
ZincFinger
Homeodomain
Helix Loop Helix
87
What are activation domains characterised by?
Often characterised according to their amino acid composition
88
What is the structure of activation domains?
* Lack of sequence conservation and structural information
* Generally thought to be unstructured
* Contain multiple short segments that work together in an additive fashion
89
What are the In vitro Approaches to the Analysis of Activators?
1. DNA footprinting
2. Electrophoretic Mobility Shift Assays (Gel Shift)
90
What is the simple outline of Electrophoretic Mobility Shift Assays (Gel Shift)?
1. Activator bound to Radiolabelled probe DNA
2. Run on non denaturing acrylamide gel
3. DNA protein complex is large so it moves more slowly.
91
What do invitro approaches tell us about activators?
Simply measures the ability of an activator to bind to a specific sequence
92
What are the in vitro approaches to the analysis of activators?
Transcription Assays
93
What are the characteristics required for transcription assays?
Requires the activator to both have a functional DNA binding domain (DBD) and a functional activation domain (AD).
94
What are the components of a transcription assay?
RNA pol II
GTFs
DNA template
Radiolabelled rNTPs
95
What are the steps to the in-vivo approach to analysing activators, chromatin immunoprecipitation (ChIP)?
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 using
i. PCR
ii. Sequencing (ChiP-Seq)
96
How do activators work?
An activator can promote the binding of a second activator.
Stimulate recruitment of complex assembly to promote.
Release stalled RNA polymerase.
97
What components of the PIC complex do activators interact with to promote assembly?
1. TFIID (via TAFs)
2. TFIIB
3. Mediator
98
What is the structure of mediator?
Mediator is a very large complex of approximately 22 polypeptides
Can exist on its own or associated with RNA pol II (through the C-terminal domain)
EM studies have revealed that MEDIATOR is composed of three domains
99
What are the 3 domains of the mediator?
1. Head
2. Middle
3.Tail
100
What is the function of the mediator?
Many activators interact with specific mediator subunits
Mediator provides a BRIDGE between Activators and RNA pol II
Mediator-activator interactions aid recruitment of RNA pol II and therefore enhance PIC formation
Modulation of chromatin
101
How do Activators increase the rate of PIC formation?
increasing TFIID binding
increasing TFIIB binding
increasing RNA pol II recruitment
102
What is the function of chromatin?
compact DNA
103
What is the structure of chromatin?
CHROMATIN is composed primarily of small BASIC proteins called HISTONES.
104
What are the two types of histones are there?
Core Histones
Linker Histones
105
What is the structure of core histones?
N-terminal tail
Globular domain
106
What is the N-terminal tail?
Highly basic: rich in Lys & Arg
107
What is the globular domain?
α-helices & loops
108
What are nucleosomes?
The core histones form repeating units called nucleosomes
109
What is the Nucleosome structure?
~147 bp of DNA wrapped twice around an octamer of histone proteins
110
What is an octamer?
central H3-H4 tetramer
2 flanking H2A-H2B dimers
111
How are Nucleosomes Organised?
1. DNA passes directly from one nucleosome to the next (10 nm fibre)
2. Linker histones such as histone H1 bind to the DNA between nucleosomes.
3. The 30 nm fibre is formed
112
What are the In vitro reconstitution experiments to test chromatin?
Naked DNA is transcribed and chromatin template is not.
113
What are the In vivo nucleosome positioning experiments to test chromatin?
Numerous experiments have shown that nucleosomes are disrupted or lost during transcriptional activation
114
How are Genetic studies in Saccharomyces cerevisiae, Evidence that Chromatin Inhibits Transcription?
Chromosomal copies of H4 genes were deleted and a plasmid expressing H4 under the control of a ‘regulatable’ promoter is present.
The promoter is the GAL4 promoter.
This is ON when there is GALACTOSE in the medium BUT OFF when GLUCOSE is present.
Therefore when GLUCOSE was added to the medium the expression of H4 was rapidly shut off.
This resulted in nucleosome depletion and the expression of many inducible genes.
115
What are the three major mechanisms for modulating the structure of chromatin?
1. Histone Variants
2. Post-Translational Modification of Histones
3. ATP dependent chromatin remodelling
116
What are histone variants?
Histone variants confers novel structural and functional properties of the nucleosome which affect the chromatin dynamics.
117
What is Post-Translational Modification of Histones?
Histone modification state has been proposed to constitute a code that sets its transcriptional state
118
What happens after Post-Translational Modification of Histones?
1. Could directly alter chromatin folding/structure
2. Could control the recruitment of non histone proteins to
chromatin
119
What is Histone lysine acetylation mediated by?
Histone Acetyl Transferases (HATs).
Histone Deacetylases (HDACs).
120
How is the histone lysine acetylation mediated?
Acetylation is mediated by HATs and is readily reversible by HDACs
Therefore the acetylation state of histones is highly dynamic
121
How are HATs recruited?
Activators recruit HATs to specific promoters.
Also some HATs are part of the general transcription machinery
122
What is tra1?
Attaches to promoters.
123
How does acetylation mediate transcriptional activation?
Direct influence on chromatin structure
Directs the recruitment of BROMODOMAIN proteins
124
What are bromodomains?
Specific acetylated lysine residues are recognised by proteins with bromodomains
Bromodomain proteins often promote transcription
125
What is the effect of Histone Methylation?
Methylation is not readily reversible but demethylases do exist
Methylation does not affect charge so probably has only minor if any influence on chromatin structure.
126
What is the histone code?
Writing
Erasing
Reading
127
What is ATP-dependent remodelling?
All have a Snf2-related ATPase
Characterised by additional domains and the architecture of the ATPase domain
128
What kinds of ATP-dependent Chromatin Remodelling exists?
Sliding
Unwrapping
Eviction
Spacing
Histone variant exchange
129
How does SWI/SNF remodel chromatin?
It hydrolyses 1000 ATP molecules per minute in the presence of DNA or nucleosomes.
Snf2 is a related to DNA helicases
Thought to be a molecular motor that that uses the energy from ATP hydrolysis to track along DNA and induce torsion
This results in disruption of histone-DNA interactions and movement of the nucleosome.
130
What is SWI/SNF?
The catalytic subunit of SWI/SNF is called Snf2 (or Swi2)
131
What are the simple known things about the SWI/SNF remodelling?
It causes change in shape and coiling.
It is ATP dependent .
Pushes around the nucleotide, disrupts contacts.
132
How do ATP-dependent (Swi/Snf) and HAT complexes co-operate?
* Regulate the same genes in yeast.
* Re-modellers are commonly recruited to the same promoters
* Bromodomains in Snf2 help tether it to acetylated nucleosomes
* HATs & ATP-dependent re-modellers function co-operatively
133
What is the function of SWI/SNF Complexes in Humans?
Cell cycle control via interaction with Rb & cyclin E.
Development, deletion results in embryonic lethality.
Tumour suppressor pathways
134
What is the major way Cells commonly exploit chromatin structure?
Cells commonly exploit chromatin structure to bring about transcriptional repression
Mediated by the recruitment of chromatin modifying factors
135
What kinds of chromatin modifying factors are there?
Histone Deacetylases (HDACs)
ATP-dependent Remodellers
Histone Methylases (heterochromatin)
136
What is the function of histone deacetylase?
Whereas ACTIVE regions of the genome are HYPERACETYLATED
REPRESSED regions are HYPOACETYLATED
Deacetylation is mediated by HISTONE DEACETYLASES (HDACs)
Just as HATs were shown to be transcriptional co-activators, HDACs function as co-repressors
137
What are the 4 major groups of Histone Deacetylases?
Class I
Class II
Class IV
Class III
138
What are classes I, II AND IV Histone Deacetylases?
Classical HDACs (zinc dependent)
139
What is the class III Histone Deacetylases?
Sir2 family (SIRTUINS).
Require NAD as a co-factor
140
What are HDAC Co-Repressor Complexes?
* HDACs commonly function in the context of large multi-subunit complexes
* Recruited to promoters by interaction with site-specific DNA binding proteins
141
What are the two basic types of chromatin?
Euchromatin
Heterochromatin
142
What are the characteristics of Euchromatin?
Gene rich
Potential to be transcribed
143
What are the characteristics of heterochromatin?
Gene poor
Repetitive regions
Transcriptional silencing
144
What are the Biochemical Features of Heterochromatin?
(1) HYPOACETYLATION
(2) SPECIFIC HISTONE H3 METHYLATION
(3) ASSOCIATION OF SPECIFIC SILENCING FACTORS
145
What is the function of CHROMODOMAIN Protein
Chromodomains often recognise and bind to methylated lysine residues
The chromodomain of HP1 is specific for H3 Lys9me2/3
146
What is the function of HP1?
* Binding of HP1 is thought to compact nucleosomal arrays
* And act as platform form the recruitment of further activities that prevent recruitment/activity of RNA pol II
147
What is the result of analysis of the wild type heterochromatin using reporter assays?
ade6 gene expressed
white colonies
148
What is the result of analysis of the Silencing reporter strain heterochromatin using reporter assays?
ade6 silenced
red colonies
149
What is the result of analysis of the Silencing reporter strain with a mutation in gene encoding a component of
heterochromatin heterochromatin using reporter assays?
ade6 silencing alleviated
white/pink colonies
150
How do we analyse heterochromatin?
Reporter silencing assays
151
Why do we get red colonies in reporter silencing assays?
Red colonies result from build up of a red pigment which is an adenine biosynthetic intermediate
152
How is the X chromosome inactivated via heterochromatin?
* The inactivated X-chrm is seen in the nucleus as a condensed structure (Barr body) that is assembled into a specific form of heterochromatin
* Formation of the Barr body is controlled by non coding RNAs Xist and Tsix
