1
Q
what is gene expression
A
process by which information in genes is decoded into proteins
DNA transcribed to RNA which is translated into proteins
2
Q
what is transcription
A
transfer of genetic information from dsDNA to ssRNA (mRNA)
3
Q
what are promoters
A
cis acting DNA regulatory element through which transcription is initiated and controlled
4
Q
eukaryotic promoters
A
- core promoter elements - cpg islands
- ten intitation occurs at lower rate and at several start sites
associated with regions with a high frequency of CG sequences cpg islands
5
Q
UAS and enhancer
A
activators binding sites
6
Q
URS and silencer
A
repressor binding sites
7
Q
CpG islands
A
- in mammals most C residues followed by G are methylated
- generally C residues in CpG islands escape methylation in mammals
- methylation of CpG islands is associated with silencing (txn switched off)
8
Q
tools for identifying promoter elements
A
- sequence comparison
- reporter analysis
9
Q
what is sequence comparison
A
- identification of the TATA box
10
Q
what is reporter analysis
A
- reporter genes encode proteins whose levels can easily be measure
- e.g. GFP, luciferase, lacZ
- amount of reporter protein provides a measure of gene expression
11
Q
what can reporters be used to identify
A
- where a gene is expressed
- when it is expressed
- what signal it responds to
- what factors and sequences control its expression
12
Q
general transcription factors
A
- bacterial RNA polymerase requires sigma-factor to recognise promoter DNA
13
Q
what does the job of sigma factor in eukaryotes
A
- RNA pol specific
- multi component factors
- form a complex on TATA box
- recruit RNA pol II to the promoter
- direct initiation at start site
14
Q
steps of transcription initiation by RNA pol II
A
- helicase activity of TFIIH separates the template strand at the start site - requires ATP hydrolysis
- as pol II begins transcribing it is extensively phosphorylated on the C terminal domain
- TFIID TFIIA may stay behind
- TFIIB, TFIIE, TFIIH are release
- TFIIF moves down the template with pol II
15
Q
what is the Carboxyl terminal domain
A
- a series of repeats located at the C terminal end of the largest pol II
16
Q
function of TFIID
A
- binds to the TATA box
recruit TFIIB
17
Q
function of TFIIA
A
- stabilises TFIID binding
- anti repression function
18
Q
function of TFIIB
A
- recruits pol II - TFIIF
- important for start site selection
19
Q
function of TFIIF
A
- stimulates elongation
- destabilises non-specific RNA pol II - DNA interactions
20
Q
function of TFIIE
A
- recruits TFIIH and modulates its activity
21
Q
function of TFIIH
A
- promoter melting and clearance - enzyme XPB
- CTD kinase activity
- DNA repair and coupling
22
Q
2 parts of TFIIH
A
- core and CAK
- CAK contains a kinase that phosphorylates the CTD of RNAP II
23
Q
TFIID
A
- TATA binding protein + TBP associated factos
- TBP is the central subunit of TFIID
24
Q
TBP vs TFIID
A
- TBP can direct the assembly go the PIC on a TATA-containing promoter
TBP alone cannot direct PIC assembly on a TATA-less promoter - TBP cannot support activated transcription
25
TAFs
- promote the interaction of TFIID with the basal promoter
- interact with activators
26
core promoter
- consists of the region around the transcription start site
- associated with elements such as the TATA box and the initiator element
27
enhancer
- DNA regions close or far from the start site
- binding sites for activator protein
- often composed of multiple UAS elements
28
silencer
- DNA regions close or far from the start site
- binding sites for repressors
29
general transcription machinery
- a set of factors that recruit RNA pol II to the promoter and direct initiation at the start site
30
pre-initiation complex
- assembly of the basal machinery at the core promoter
31
activator
- a factor that binds the gene specific regulatory sequences and stimulates transcription initiation
32
basal transcription activated txn
- the level of transcription from a core promoter
- increased levels of transcription mediated by an activator protein
33
classes of enhancer elements
- common sequence elements e.g. GC box, octamer, CAAT box
bind activations that are relatively abundant in the cell and constitutively active
- response elements e.g. SRE HSE. bind factors whose activity is controlled in response to specific stimuli
34
combinatorial control of transcription
- the type and combination of elements dictates when and at what level a gene is transcribed
35
how do activators contact the basal transcription machine
- tracking
- looping
36
types of activation domains
- acidic patch - clusters of negative charged residues e.g. vp16
- glutamine rich e.g. sp1
proline rich e.g. jun
37
analysis of activators (in vitro)
- DNA foot printing
- electrophoretic mobility shift assay
- transcription assay
38
what is an electrophoretic mobility shift
- activator + radiolabelled probe DNA --> run on non-denaturing acrylamide gel
39
what are transcription assays
- RNA pol II + GTPs + DNA template + radiolabelled rNTPs
- requires the activator to both have a functional DNA binding domain and a function activation domain
40
analysis of activators (in vivo)
- reporter assays
- chromatin immunoprecripitation
41
how does chromatin immunoprecipitation work
- cross link bound proteins to DNA
- isolare chromatin and shear DNA
- precipitate chromatin with protein-specific antibody
reverse cross-link and digest protein
- analyse DNA using - PCR or sequencing
42
how do activators work
- promoter binding of an additional activators
stimulate complex assembly
release stalled RNA polymerase
- modulation of chromatin
43
how to activate stalled RNA pol II
- heat shock genes such as hsp70
- heat shock activates HSF transcription factor which interacts with RNA pol II and release it from the pause after 50nts
44
which transcription factors are involved in recruitment
- TFIID
- TFIIB
- mediator
45
what is the function of mediators
- provides a bridge between activators and RNA pol II
- activator interactions aid recruitment of RNA pol II and therefore enhance PIC formation
46
general role of activators
- can promoter the binding of additional activators
- can increase the rate of PIC formation
- may stimulate post recruitment steps
47
function of chromatin
- to compact DNA
48
what is the composition of chromatin
- composed primarily of histones
49
what are the two types of histones
- core histones
- linker histones
50
what are nucleosomes
- DNA wrapped twice around an octamer of histone proteins
51
what is an octamer
- central H3-H4 tetramer and 2 flanking H2A-H2B dimers
52
function of linker histones
- H1 bind to the DNA between nucleosomes
53
3 pieces of evidence that chromatin inhibits transcription
- in vitro reconstitution experiments
- in vivo nucleosome positioning experiments
- genetic studies of saccharomyces cerevisae
54
how does the in vitro reconstitution experiment prove that chromatin inhibit transcription
- RNA pol II + transcription + chromatin template --> no transcription
55
what are the roles of nucleosomes in the nucleus
- compaction of DNA
- forms a template for DNA transcription
56
3 main mechanisms for modulating the structure of chromatin
- histone variants
- post transcriptional modification of histones
- ATP dependent chromatin remodelling
57
histone variants
- all except H4
- histone variants confers novel structural and functional properties of the nucleosome which affect the chromatin dynamics
58
post translational modification of histones
- acetylation
- methylation
- ubiquitylation
- phosphorylation
59
consequences of histone modification
- directly alter chromatin folding/structure
- control the recruitment of non-histone proteins to chromatin
60
how are histone acetyl transferases recruited
- activators recruit HATs to specific promoter
- some HATs are part of the general transcription machinery
61
how does acetylation mediate transcriptional activation
- direct influence on chromatin structure
- directs the recruitment of bromodomain proteins
62
why are bromodomains important
- specific acetylates lysine residues are recognised by proteins with bromodomains
- bromodomain proteins often promoter transcription
63
2 examples of bromodomains
- BDF1 - binds acetylated H4 and recruits TFIID
- TAFII250 - TFIID subunit, also binds acetylated H4
64
how does histone methylation occur
- histone methylation can occur on lysine
- methylation does not affect charge so minor influence on chromatin structure
65
importance of lysine methylation
- specific methylated lysines are recognised by specific proteins
- methyl-lysine residues can function either as activating or repressing marks
66
enzyme involved in ATP dependent chromatin remodelling
- SNF2-related ATPase
67
steps of ATP dependent chromatin remodelling
- sliding
- unwarpping
- eviction
- spacing
- histone variant exchange
68
how does SWI/SNF remodel chromatin
- catalytic subunit is snf2
- hydrolyses ATP in the presence of DNA or nucleosome
- uses energy from ATP hydrolysis to track along DNA and induce torsion
- this results in disruption of histone DNA interactions and movement of the nucleosome
69
roles of SWI/SNF complexes
- cell cycle control via interaction with rb and cyclin E
- in development, deletion in mice results in embryonic lethality
- tumour suppressor pathways; mutations are associated with a variety if tumour types
70
role of SWI/SNF in cancer
- the tumour suppressor activity of the SWI/SNF complexes most likely due to roles in facilitating transcription factor function
71
chromatin modifying factors in repression of transcription
- histone deacetylases
- ATP dependent remodellers
- histone methylases
72
HDAC co-repressor complexes
recruited to promoters by interaction with site-specific DNA binding proteins
e.g. SIN3 co-repressor complexes
73
what is the role of ATP dependent remodelling complexes
mediate transcriptional repression
74
two types of chromatin
- euchromatin - gene rich, potential to be transcribed
- heterochromatin - gene poor, repetitive regions, transcriptional silencing
75
3 features of heterochromatin
- hypoacetylation
- specific histone H3 methylation
- association of specific silencing factors
76
function of HP1 and heterochromatin
- binding of HP1 is thought to compact nucleosomal arrays
- act as platform form the recruitment of further activities that prevent recruitment
77
heterochromatin - X-chromosome inactivation
- females have 2 X chromosomes one of which is inactivation
- this equalises the number of X linked genes expressed in males and females
- the inactivated X-chromosome is seen in the nucleus as a condensed structure that is assembled into a specific form of heterochromatin
78
what is a Barr body
- inactivated X chromosome in the nucleus as a condensed structure
- formation of the Barr body is controlled by non-coding RNAs Xist and Tsix
79
role of NF-kappa B
- allows the cell to respond to external challenges
- regulates expression of target genes to help programmed the response, to allow cells to survive or recover
80
what is the NF-kappa B family
- the real homology domains encodes the DNA binding and dimerisation functions of NF-KB
- p50 and p52 are proteolytically processed from their precursor proteins p105 and p100
- p100 and p105 contain ankyrin repeats in their c-terminal that allow them to function as IKB-like inhibitors
- TA1/TA2, TAD, SD1, SD2 - non conserved transcriptional activation domains
- LZ - leucine zipper like domain
81
describe the ubiquitin - proteasome pathway
- E3 ubiquitin ligase facilitates the attachment of ubiquitin chains to a target protein
- this pathway is protein degradation
- Ub is conjugated to protein that are destined for degradation by an ATP-dependent process
- 5 ub molecules attach to the protein substrate
- ub is removed and the protein is linearised and injected into the central core
- the in proteasome the protein is digested to peptides
- peptides to amino acids by peptidases
- used in antigen presentation
82
why is NF-KB induced
- inflammatory cytokines
- bacterial products
- viral proteins and infection
- DNA damage
- cell stress
83
what does NF-KB regulate
- the immune and inflammatory response
- stress response
- cell survival and cell death
- cell adhesion
- proliferation
84
activation of NF-KB
- when the cell is stimulated, IKB is phosphorylated by ubiquitination
- NF-KB is released
- NF-KB translocated to the nucleus
85
enzymes involved in canonical pathway
- TNF
- IL-1
- LPS
86
enzymes involved in non-canonical pathway
- LPS
- CD40
- Lymphotoxin receptors LMP1
87
responses to infection regulation by NF-KB
- inflammation
- proliferation
- survival
- tumour promotion and metastasis
- angiogenesis
- cell death and anti-proliferative effects
88
how are beta interferons produced
- viral infections
- spacing and orientation sites allows for appropriate protein-protein contacts
89
what is the role of activators
- transcription factors binding at the beta interferon enhancer all interact to form the enhanceosome complex
90
role of the enhanceosome complex
- forms an interaction interface to allow the high affinity recruitment of transcriptional cofactors such as p300 and cap
91
what are coactivators interactions weak
- favour the formations of coactivator complexes only at the promoters and enhancers
92
what activators are present at the beta IFN enhancer
- p50/relA
93
what levels of regulation of NF-KB give transcriptional specificity
- stimulus e.g. TNF or IL1
- phosphorylation and degradation of IKB alpha, beta or E
- translocation of NF-KB to the nucleus modification of NF-KB subunits
- DNA binding and gaining access to the promoter/enhancer
- transactivation
- specific transcriptional response
94
what is transactivation
interaction with the basal transcription complex and co activators
95
purpose of ubiquitination
- when proteins undergo ubiquitination they can be targeted for degradation by the proteasome
96
summarised function of NF-KB
- NF-KB complexes are held in an inactive form
- bound to an inhibitory protein
- once phosphorylated by the IKK complex it becomes ubiquinated and degraded
- NF-KB is free to translocate to the nucleus
97
what is the first step of the transcription response
- shows how the degradation of different IKB proteins can lead to activation of different NF-KB dimers
98
what happens to NF-KB in the nucleus
- subject to regulation
- by post translational modifications
- or by interactions with nuclear transcriptional regulators
99
role of beta interferon
role in antiviral response, inhibited by cars cov2
100
what is the enhanceosome
- creates a landing and for transcriptional regulators such as p300/cbp
- can lead to beta interferon gene transcription
101
what is hypoxia
- the lowering of the oxygen. concentration compared to normal levels cells are exposed to
102
what is the medical relevance of hypoxia
- high altitude diseases
- cancer
- rheumatoid
- ageing
- neurodegenerative diseases
- schizophrenia
- gastrointestinal disease
- chronic kidney disease
- diabetes
- stroke/ischemia
103
consequences hypoxia
- translational block
- transcriptional program
- chromatin structure changes
- microRNA signature
- DNA replication block
104
what are the cellular responses to hypoxia
- restoration of oxygen homeostasis
- cell survival
- cell death
105
what are the different hypoxia inducible factors
- HIF 1alpha - ubiquitously expressed in all tissues
- HIF 2alpha - expression restricted in certain tissues
- HIF 3alpha - lacks c-terminus. functions as a dominant negative inhibitor for HIF-1alpha and HIF-2alpha
106
how is HIF-1alpha regulated in normoxia
- under normoxia, proline hydroxylases and FIH inhibiting HIF enzymes use oxygen to hydroxylate key residues within the HIF-1alpha subunit
- hydroxylation of the ODDs signals for the VHL binding and ubiquitination
- leads to proteasomal degradation
107
how is HIF-1alpha regulated in hypoxia
- PHDs and FIH are inhibited
- HIF 1alpha is stabilised
- HIF 1alpha dimerises with HIF 1beta
- activate target gene transcription through recruitment of co-activators
108
what are the targets of HIF
- oxygen supply
- transcription
- cellular metabolism
- cell death
- HIF control
- cell growth
109
structure of p53 tumour suppressor
- trans - transactivation domain
- p - proline rich domains
- NLS - nuclear localisation sequence
- tet - tetramerization domain
110
what is the regulation pathway of p53 pathway
- p53 is inactivated by its negative regulators mdm2
- when DNA is damaged, p53 and mdm2 complex dissociates
- p53 then induces cell cycle arrest
111
role of mdm2
- is an e3 ubiquitin ligase
- major role is ubiquitination of p53, leading to degradation
- when DNA is damage it becomes phosphorylated
- over expression inactivates p53, preventing apoptosis
112
role of ARF
- p14arf is a tumour suppressed
- induced by oncogene
- disrupts interaction between the p53 and mdm2
- inhibits ubiquitin ligase
- increased levels of transcriptionally active p53
113
what is LFS syndrome
- hereditary conditions
- cancer risk passed from generation to generation
- mutation in TP53 gene
114
how is eukaryotic gene expression regulated
- transcription control
- RNA processing control
- translational control
- protein activity control
115
what is the primary transcript process
- events coupled to transcription via the RNA pol ii CTD which acts as a landing pad
- capping
splicing
- poly adenylation
- editing
116
what are the steps of the 5' m7G cap synthesis
- RNA initially contains triphosphate at 5' end
- capping
- methylation alters chemical behaviour of bases
117
functions of the m7G cap
- protects mRNA from degradation by 5'-3' nucleases
- facilitate splicing
- facilitates export from the nucleus
- functions mediated through protein binding
118
essential proteins for translation
- CBP80/CBP20 in nucleus - processing/export
- eIF4 complex in cytoplasm - translation
119
function of 5' cap
- capping linked to transcription
- the cap is a protein binding element
120
what are the conserved sequences in introns
- 5' splice site
- 3' splice site
- branch site
121
what are the step of splicing
- 2 trans-esterification reaction
- cut at 5' splice site
- creation of bond between the 5' end of intron and branch site
- cut at 3' splice site to release intron lariat
- ligation of two exons
122
what is the spliceosome
- enzymatic complex that catalyses the removal of introns
- requires ATP
123
proteins included in the spliceosome
- RNA binding proteins
- ATPases
- GTPases
124
function of anti-sm
- anti-sm antibodies react against the sm proteins
- present in lupus
125
how is the alternative splicing mechanism regulated
- activators - binds to intronic and exonic splicing enhancers (ISE & ESE)
- repressors - binds to intronic and exonic splicing silencers (ISS & ESS)
126
diseases caused by mutations in splicing
- spinal muscular atrophy - common in genetic cause of infant mortality
- retinitis pigmentosa - reduced visual capabilities and blindness
- myotonic dystrophy - a muscle wasting disease
127
what is polyadenylation
- addition of poly A tail to end of mRNA
- endonuclease cleavage
- splicing of AAUAAA
- G/U rich tract just downstream of polyA site
128
what are the proteins required for polyadenylation
- cleavage and polyadenylation specificity factor - binds AAUAAA
- cleavage of stimulatory factor - binds G/U
129
function of polyA tail
- enhances export of RNA
- stabilises 3' end of mRNA
- enhances translation of mRNA
130
what are the two classes of RNA editing
- insertion/deletion
- modification
131
what is the significance of RNA editing in medicine and development
- disease - atherosclerosis
- brain function
- development
- parasites
132
what are the effects of mRNA editing
- creation of start/stop codons by U insertions
- creation of start/stop codons by C to U changes
- creation new open reading frames by nucleotide insertions
- changes in encoded amino acids and splice site choice by base conversion
- removal of stop codons by base conversions
133
how is RNA edited by deamination
- deaminated adenosine is inosine
- inosine is similar to guanosine
134
what enzyme is involved in cytidine deamination
- pre mRNA editing carried out by the APOBEC-1 enzyme
135
importance of A to I editing
- decrease in ca2+ permeability of channels containing the R version
- editing carried out by ADAR2
136
importance of nuclear pore
- RNA is an acid (hydrophobic) so need help getting out of the nucleus
137
importance of localised mRNA
- localised protein synthesis -
- generate cell polarity
- prevents expression in the wrong place
- promotes efficiency of subsequent protein targeting
- local control of translation
138
importance of localisation of dendritic mRNA
- synaptic proteins produced at the synapse, which gives us synaptic plasticity and spine morphogenesis
139
how does localisation effects diffusion
- mRNAs are local entrapped by anchor proteins in the cytoplasm
- local entrapment
-anchor proteins at the site where you want them
140
which transport mechanisms are used to localised mRNAs
- active transport
- passive transport
141
what are the step in catalysing aminoacy-tRNA syntheses
- amino activation
amino acid and ATP bind catalytic site, nucleophilic attach by alpha carboxylic acid, oxygen yielding aminoaceyl-adenylate
- hydroxyl group of adenine 76 to tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP
142
steps of translation
- peptide bond formation is catalysed by the ribosome
- tRNAs deliver the amino acids
- tRNA are present in the P and A sites - the expanding polypeptide chain is attached to the p-site tRNA
143
how is eukaryotic translation initiated
- small subunit binds the CAP, moves to the first AUG, encoding the initiating methionine of the protein
- most frequently found in the Kozak consensus sequence
144
what are the 5 factors in translation initiation
- eIF1A - met-tRNA binding to 40s
- eIF3 - 80s dissociation, binds many other eIFs
- eIF1- AUG recognition
- eIF2 - GTPase, met-tRNA binding, binds eIF5
- eIF5 - stimulates eIF2 GTPase, GAP for eIF2
145
what is 43s association
- interaction - eIF3 with eIF4G
- RNA unwinding - eIF4F unwinds cap-proximal sequence
146
what is scanning
- 5' proximal AUG used
- mutation of natural AUG leads to use of next one
- mutation of initiator tRNA leads to use of next one
- requires eIF4F
146
what are the final step of translation initiation
- eIF2 needs to be recycled to generate ternary complex for further initiation event
- eIF5B - GTPase that promoted sub-unit joining
147
what are the final steps of eukaryotic translation
- translocation required to move the tRNAs and mRNA through the ribosome
- when reach termination codon, translation stop and the ribosome dissociates
148
what are the key regulation points of translation
- formation of eIF4F
- 43S binding
- function of eIF2B/ternary complex formation
148
what are the key points of eIF2B
- present at much lower levels than eIF2
- its activity governs levels of active eIF2-GTP
- down regulated in responses to stresses
- regulation through phosphorylation of eIF2, competitive inhibitors
149
what are eIF2 kinases
- PKR - activated by double stranded RNA
- PERK - a mediator of the unfolded protein response
- GCN2 - a regulator of translation in response to amino acid availability
- HRI - linking global availability to protein synthesis
150
what is PKR
- antiviral defence strategy
- increased when cells are exposed to interferons
- when PKR binds dsRNA it dimerises and is activated
151
how is iron metabolism regulated
- regulated by the expression of fe-storage/transport proteins
152
where are iron response elements found
- found in the 5' or 3' UTRs of iron regulated mRNAs
- bound by iron regulatory proteins, IRP1 IRP2
153
function of IRP1
- binding can block or activate translation
- binding also affects mRNA stability
154
where is there RNA degradation
- damaged mRNA
- incorrectly transcribed/ processed mRNA
- control gene expression
155
155
156
what is phase 1 of mRNA degradation
- decapping enzymes
- endonucleases
- edadenylases
- initiate breakdown of the RNA
156
why is casein mRNA important
- increases half life in response to prolactin
- polyA tail length increases
- 3' UTR of RNA binds proteins which aid stabilisation
157
phase 2 of mRNA degradation
- the exosome - the main 3' to 5' exonuclease in the cell - involved in RNA turnover and processing
- XRN1 - functions after decapping of the mRNA
158
what causes deadenylation-dependent decay
- mechanisms whereby all mRNAs gradually lose their polyA tails
159
what causes deadenylation-independent decay
- auto regulation - rps288 binds its own message
- edc3 is an activator of decapping enzymes
- nucleases targeting specific substrate
- PMR1 cleaves albumin mRNA
160
what is nonsense mediated decay
- where mistakes in the RNA are detected, RNA is targeted for degradation
161
what do premature stop codons result from
errors in:
- transcription
- splicing
- editing
- polyadenylation
- mutations
162
163
164
what is the nonsense mediated decay mechanisms
- EJCs are removed from the mRNA by the ribosome
- once the ribosomes reach the PTC, and EJC remains downstream, specific factors interact with the RNA degradation machinery
- process is known as surveillance
165
miRNA vs siRNA
- siRNA - complimentary to target RNA, viral defence mechanism, leads to degradation of the target RNA
- miRNA - not complimentary, regulatory mechanism, leads to block in translation
166
importance of 3' UTR changing length
- during embryonic development 3' UTR get longer
- mRNA proliferating cells get longer
- longer 3' UTR has more possibilities of binding sites for miRNAs
167
what are the clinical uses of siRNAs
- fitsuiran - lowers antithrombin, reduces bleeding in haemophiliacs
- STP705 - knocks down both TGF-beta1 and COX-2 gene expression
168
what is miRNA used for
translational regulation
169
what are siRNAs used for
target mRNAs for cleavage
important tools for manipulating gene expression
