Lectures 1-4: transcriptional control & chromatin Flashcards
1
Q
What is the main purpose of DNA?
A
To be decoded into protien
2
Q
What are the main components of transcription in prokaryotes?
A
Closed complex - σ70 recognition and holoenzyme bind upstream
Open complex (initiation) - Single stranded DNA allowing access to complex
Elongation - transcription bubble closes behind, after ~10 nucleotides RNA polymerase is released
3
Q
What are promoters?
A
Cis acting DNA regulatory element through which transcription is initiated and controlled
4
Q
Where are prokaryotic promotors located?
A
-35 and -10 upstream
5
Q
What is a consensus sequence?
A
It is a sequence of bases that allows the transcription of prokaryotic genes, the closer the sequence the more efficient the transcription
6
Q
What where are eukaryotic promotors located?
A
In the regulatory and core region
7
Q
What are the names of the core promotor elements?
A
TATA box - upstream
Initiator (Inr) - start-site
MTE - downstream
DPE - downstream
BRE - upstream
8
Q
What are CpG islands?
A
They are regions where transcription occurs with a high frequency of CG sequences
9
Q
What is associated with the methylation of CpG islands?
A
With silencing of transcription
10
Q
What are the different eukaryotic promotors called?
A
UAS & enhancers - activator binding sites
URS & silencer - repressor binding sites
11
Q
What are the different tools for identifying promotor elements?
A
Sequence comparison
Reporter analysis
12
Q
How was the TATA box identified?
A
Using sequence comparison by finding the percentage frequency of different sequences (does not provide information about function)
13
Q
How does reporter analysis work to identify promotor elements?
A
Reporter genes encode proteins allowing levels to be easily measured, amount of reporter proteins allows a measure (can show how active that gene expression is with the regulatory sequence)
14
Q
How was reporter analysis used on a mouse embryos?
A
Expressed a ure2 gene which drives the expression of a lacZ gene which turned blue in the embryo allowing the quantification of expression
15
Q
How many RNA polymerases are in eukaryotes?
A
At least 3
16
Q
What are the target genes of RNA polymerase I?
A
rRNA (28S 18S 5.8S)
17
Q
What are the target genes of RNA polymerase II?
A
mRNA
snRNA
miRNA
18
Q
What are the target genes of RNA polymerase III?
A
tRNA
5S RNA
U6 RNA
7S RNA
19
Q
What are the similarities and differences between prokaryotic and eukaryotic polymerases?
A
They have the same overall shape
Different subunits
Some of the subunits are homologues of the different polymerases
Eukaryotes have more subunits
Core RNA polymerase needs a σ factor in prokaryotes
20
Q
What are the components of general transcription factors?
A
RNA polymerase specific
Multi component factors
Form a complex on the TATA box
Recruit RNA polymerase II to the promotor
Direct initiation at start-site
21
Q
What are the different types of general transcription factors (GTFs) in eukaryotes?
A
TFIIA, TFIIB, TFIID, TFIIE, TFIIIF, TFIIH
22
Q
What is the pre initiation complex of general transcription factors (GTFs)?
A
IID binds to TATA box
IIA binds to IID to stabilise it
IIB binds to IID directing RNA polymerase II to bind with IIF
IIE then binds which helps IIH bind
23
Q
What is the pre initiation complex (PIC) assembly equivalent to?
A
To the closed complex in prokaryotes
24
Q
How is transcription initiated by RNA polymerase II?
A
IIH separates the strands forming an open complex
25
What happens when RNA polymerase II begins transcribing?
It is extensively phosphorylated on the C-terminal domain (CTD)
26
What is the C-terminal domain?
It is a series of repeats located at the c-terminal end of the largest subunit of RNA polymerase II
27
What are the properties of TFIID?
Binds to TATA box
Recruits TFIIB
28
What are the properties of TFIIA?
Stablises TFIID binding
Anti repression function
29
What are the properties of TFIIB?
Recruits RNA polymerase II - TFIIF
Important for start site selection
30
What are the properties of TFIIF?
Assists TFIIB recruits RNA polymerase II
Stimulates RNA polymerase II elongation
31
What are the properties of TFIIE?
Helps recruit TFIIH and modulates TFIIH activity
32
What are the properties of TFIIH?
Promoter melting and clearance
CTD kinase activity
DNA repair coupling
33
How does TFIIH start promoter melting?
It contains an ATPase called XPB (Ssl2)
34
What are the 2 different parts TFIIH can be divided into?
Core and CAK
CAK - kinases that phosphorylates CTD of RNA polymerase II
35
How does the ATPase Ssl2 (XPB) work?
It uses energy from ATP hydrolysis and pushes DNA into cleft where polymerisation is catalysed, causes torsional stress that contributes to transcription bubble formation
36
What is TFIID composed of?
TATA binding protein and TBP associated factors (TAFs)
37
What are the properties of TBP in comparison to TFIID?
TBP directs assembly of PIC on TATA containing promoter (NO TAFs)
TBP alone cannot direct PIC assembly on a TATA-less promoter
TBP cannot support activated transcription
38
What is the function of TAFs?
To promote the interaction of TFIID with basal promoter elements
TAFs interact with activators to promote transcription initiation
39
What are the functions of enhancer elements?
Basal transcription - low/ no transcription
Activated transcription - High
40
What are the different classes of enhancer elements?
Common sequence elements
Response elements
41
What are the properties of common sequence elements?
Often located close to the core promoter
Bind activators that are relatively abundant in the cell and constitutively active
42
What are the properties of response elements?
They bind factors whose activity is controlled in response to specific stimuli
43
How do common and response elements control transcription?
Common (only) increases basal and activated transcription level
Response (only) allows normal basal and an increased activated transcription level
Common + response increases both basal and activated transcription level
44
How does enhancer location impact activation?
It doesn't, enhancers work irrespective to orientation and location
45
How do enhancers continue to work when far from start site?
They work because the DNA is flexible
46
How do activation and DNA binding domains work?
DNA binding is singular but different/ multiple activation domains are still functional
47
How are eukaryotic activators modular?
Still functional when separate
Often are separate
48
How are activation domains characterised?
According to their amino acid composition
49
What are the properties of activation domains?
Lack sequence conservation and structural information (cannot be determined just from protein)
Generally thought to be unstructured
Contain multiple short segments that work together in an additive fashion
Interact with other proteins in transcriptional machinery (help PICs)
50
What are the different in vitro approaches to analysing activators?
DNA footprinting
Electrophoretic Mobility Shift Assays (gel shift)
Transcription Assays
51
How do electrophoretic mobility shift assays work?
They measure the ability of an activator to bind to a specific sequence
Activator + radiolabelled probe DNA → run on non denaturing acrylamide gel
52
How do transcription assays work?
It is a functional assay as it enhances transcription
RNA polymerase II + GTFs + DNA template + Radiolabelled rNTPs
53
What is required in a transcription assay?
An activator has to have both functional DNA binding domain and a functional activation domain
54
What are the in vivo approaches to analysing activators?
Reporter assays
Chromatin Immunoprecipitation (ChIP)
55
How do reporter assays work to analyse activators?
A reporter gene and binding site for a specific protein are inserted into a cell as well as the gene to encode the protein which allows the protein to bind and produce reporter-gene transcripts
56
How does chromatin immunoprecipitation work to analyse activators?
Cross links bond protein to DNA
Chromatin is isolated and DNA sheared using sonication
Precipitate chromatin is bond with protein-specific antibody (purification)
Cross links are reversed and protein is digested forming activator binding sites
57
How is chromatin immunoprecipitation analysed?
Using PCR and Sequencing
Sequencing identifies all the binding sites for the activator
58
What are the mechanisms of activators?
Cooperative binding of an additional activator to stimulate transcription
Stimulate complex assembly by adding components of the PIC complex
Release stalled RNA polymerase
59
Which components of the PIC complex interact to allow activators to work?
TFIID (TAFs)
TFIIB (RNA Polymerase II)
Mediator
60
What is the mediator protein?
It is an additional factor added to allow transcription
61
What is the composition of the mediator protein?
It is a large complex of ~22 polypeptides
Exists on its own or associated with RNA polymerase II
3 domains: Head, middle and tail
62
What is the function of the mediator?
Interacts with many activators with specific subunits
Provides a bridge between activators and RNA pol II
Interactions with activators aid recruitment of RNA pol II and enhance PIC formation
63
How is RNA pol II stalled using activators?
It can stall at or near the promoter
Active activator proteins release stalled RNA pol II by interacting with it
64
How is DNA compacted into a cell?
Using chromatin
65
What is the composition of chromatin?
Small basic proteins called histones
2 types: core and linker histones
66
What are the 2 different parts of core histones?
N-terminal tail - rich in lysine + arginine
Globular domain - α helicies and loops
67
How do core histones bind to DNA?
They form repeating units called nucleosomes
68
What is the nucleosome made up of?
~147 bp of DNA wrapped twice around an octamer of histone proteins
69
What is the nucleosome octamer made up of?
A central H3-H4 tetramer + 2 flanking H2A-H2B dimers
70
How are nucleosomes organised?
DNA passes directly from one nucleosome to the next
Linker histones such as histone H1 bind to the DNA between nucleosomes
In vitro linker histones result in the formation of a thicker 30nm fibre
71
How does the nucleosome fold in vitro?
Into a 30nm fibre which then forms a chromosome
72
What experiments were conducted to find evidence that chromatin inhibits transcription?
RNA pol II + transcription factors + naked DNA → transcription
RNA pol II + transcription factors + chromatin → no transcription
73
What genetic studies were conducted to find evidence that chromatin inhibits transcription?
Chromosomal copies of the H4 (histone) gene were deleted and plasmid under the control of a regulated promoter was added (GAL4)
On when galactose present off when glucose present
Nucleosome depleted but genes were switched on
74
What are the 3 major mechanisms for modulating the structure of chromatin?
Histone variants
Post-translational modification of histones
ATP dependent chromatin remodelling
75
How do histone variants modulate the structure of chromatin?
They are encoded from genes that differ from the conserved types
Expressed at low levels
All the conventional histones have variants (H2A, H2B, H3 and newly H4)
They confer novel structural and functional properties of the nucleosome which affect the dynamics
76
What are the post-translational modifications of histones that modulate the structure of chromatin?
Acetylation, methylation, ubiquitylation and phosphorylation
77
How do post-translational modifications of histones modulate a transcriptional state?
Directly altering the chromatin folding/ structure
Control the recruitment of non histone proteins to chromatin
78
What is histone lysine acetylation?
Histone acetyl transferases (HATs) are added to the lysine residue (ONLY) which changes the charge on the lysine meaning it changes the chromatin structure
79
What is the evolution of histone acetyl transferases (HATs)?
1960s - Correlation between high levels of acetylation and transcription
1990s - The first nuclear HAT was shown to be homologous to yeast GCN5
Important because GCN5 was known to function as a transcriptional activatorConfirmed that acetylation is a key component of transcriptional activation
Nuclear HATs are now known to function in large multisubunit complexes of 2 major types
80
What are the 2 major types of histone acetyl transferases (HATs)?
GNAT family & MYST family
81
How are histone acetyl transferases (HATs) recruited?
Activators recruit HATs to specific promoters
HATs contain specific subunits that interact with activators
Some HATs are part of the general transcription machinery
82
How does acetylation mediate transcriptional activation?
Direct influence on chromatin structure - removes the charge causing a more open complex
Directs the recruitment of BROMODOMAIN proteins - which allow recognition of HATs and are able to promote transcription
83
How does histone methylation occur?
It can occur on lysine and arginine (more often on lysine residues)
Histone lysine methyl transferases (HKMTs) are used to add methyl groups
84
What are the properties of histone methylation?
Lysines may be mono, di or tri methylated by HKMTs
Not readily reversible but demethylases do exist
Methylation does not affect charge so only has a minor influence
85
How is lysine methylation controlled?
Many different domains are involved which recognise specific proteins
Depending on context methyl-lysine residues can function as either activating or repressing marks
86
What is the histone code?
It allows control of chromatin structure in writing, erasing and reading
87
What is ATP-dependent chromatin remodelling?
SWI2/SNF2 ATPase superfamily allows cells to have different remodelling complexes
All have a Snf-related ATPase
At least 4 distinct families, characterised by additional domains and architecture of the ATPase domain
88
What are the different SWI2/SNF2 ATPase subfamilies?
SWI2/SNF2
ISWI
CHD/Mi2
Ino80
89
What are the different chromatin remodelling structures?
Sliding
Unwrapping
Eviction
Spacing
Histone variant exchange
90
How does SWI2/SNF2 remodel chromatin?
Catalytic subunit hydrolyses 1000 ATP molecules per minute in presence of DNA or nucleosomes
Acts as a molecular motor tracking along DNA and inducing torsion
Torsion causes stress which promotes movement of DNA in the nucleosome
91
How do HATs and ATP-dependent complexes cooperate?
SWI/SNF and DCN5 HAT regulate the same genes in yeast
They are commonly recruited to the same promoters
Bromodomains in Snf2 help tether it to acetylated nucelosomes
92
What are the SWI/SNF complexes in humans and what are their roles?
PBAF, cBAF and ncBAF
Needed for a number of TFs
Roles in cell cycle control, interaction with Rb (retinoblastoma) and cyclin E
Roles in development, deletion in mice results in embryonic lethality
Role in tumour suppressor pathways, mutations are associated with a variety of tumour types
93
How do mutations in SWI/SNF correlate with cancer?
Mutations are enriched in particular cancer types
Different gene mutations confer distinct cancer vulnerabilities in mouse models
The tumour-suppressor activity of the SWI/SNF complexes are most likely due roles in facilitating transcription factor function
94
What are the different mutations that arise in SWI/SNF subunits that can contribute to cancers?
Nonsense, frameshift and deletion which may cause loss of function
95
How can transcription be repressed?
By exploiting chromatin structure using chromatin modifying factors
e.g. Histone deacetylases (HDACs) ATP-dependent remodellers and Histone methylases (heterochromatin)
96
How do histone deacetylases act as co-repressors?
They function as a large multi-subunit complex
Recruited to promoters by interaction with site-specific DNA binding proteins
Deacetylate the lysine residue on the histone which causes a tighter complex repressing transcription
97
What are the major groups of histone deacetylases?
Class I, Class II, Class IV (classical HDACs)
Class III Sir2 family (SIRTUINS) require NAD as co-factor
98
How do ATP-dependent remodelling complexes work?
They are used to mediate transcriptional repression by containing HDACs so they deacetylate the lysine residue on the histone which causes a tighter complex repressing transcription
99
What are the biochemical features of heterochromatin?
Hypoacetylation
Specific histone H3 methylation
Association of specific silencing factors
100
How is heterochromatin (H3Lys9me) assembled?
HDACs deacetylate the lysine residue on the histone then Suvar39 adds a methyl group onto the same lysine which is then recognised by heterochromatin protein 1
101
How does HP1 and heterochromatin act as a repressor of transcription?
It compacts nucleosomal arrays
Acts as a platform for the recruitment of further activities that prevent the recruitment/ activity of RNA polymerase II
102
How does heterochromatin use reporter proteins to respress?
heterochromatic centromeric repeats are used to silence euchromatin as they are removed from the normal locus
103
How does heterochromatin act on X-chromosomes?
It inactivates them by containing a condensed structure (Barr body) which is an assembled form of heterochromatin
104
How do Xist and Tsix non coding RNAs work?
Xist is up regulated in later development and Tsix is down regulated which causes a coating to form on the chromosome which recruits H3K27 causing silencing of the X chromosone
