CMB2001 Control of Eukaryotic Gene Expression Flashcards
1
Q
What is Gene expression?
A
Process by which information in genes (DNA) is decoded into protein.
2
Q
What is transcription?
A
Transfer of genetic information from dsDNA to ssRNA (mRNA)
3
Q
Describe the process of initiation of transcription in prokaryotes
A
-σ70 combines interacts with RNA polymerase, working together to recognise promoter regions and initiate transcription, forming an open complex.
4
Q
After how many bases does σ70 detach?
A
~10 bases
5
Q
What are promoters?
A
Cis acting DNA regulatory element through which transcription is initiated and controlled.
6
Q
What are the regions that make up Eukaryotic promoters?
A
-Regulatory region
-Core (basal) region
7
Q
Which region of eukaryotic promoters are further upstream?
A
Regulatory region
8
Q
Name some core (basal) promoter elements
A
-TATA box
-Initiator (Inr)
-MTE (Motif ten element)
-DPE (Downstream core promoter element)
-BRE (TFIIB Recognition element)
-CpG islands
9
Q
Describe what occurs when CpG islands are methylated?
A
They are silenced (ie transcription is switched off)
10
Q
What class of eukaryote contains CpG islands?
A
Mammalia
11
Q
Do Eukaryotic promoters have all elements?
A
NO
12
Q
What is the UAS found in promoter regions?
A
Upstream activating sequence, an activator binding site
13
Q
What is the URS found in promoter regions?
A
Upstream repressing sequence, A repressor binding site
14
Q
What are the two tools to identify promoter elements?
A
-Sequence comparison
-Reporter analysis
15
Q
What does sequence comparison of promoters consist of?
A
-Lining up sequences with known consensus sequences
-Helps identify key characteristics eg TATA boxes
16
Q
What is the limitation with sequence comparison of promoters?
A
Does not tell us function and mechanism
17
Q
What does reporter analysis of promoters consist of?
A
-Attach a regulatory sequence to a reporter gene
-Using regulatory sequence in question to express a “reporter” gene which is easy to measure in terms of quantity of expression
18
Q
Give examples of reporter genes
A
-GFP
-Luciferase
-LacZ (β-galactosidase)
19
Q
Why is the quantity of reporter protein significant?
A
Provides a measure of gene expression
20
Q
What can reporter analysis be used to identify?
A
-When a gene is expressed
-Where it is expressed
-What signals it responds to
-What factors and sequences control its expression
21
Q
Name some major eukaryotic RNA polymerases
A
-RNA pol I
-RNA pol II
-RNA pol III
22
Q
What is the target gene of RNA pol I?
A
rRNA
23
Q
Where is RNA pol I located?
A
In the nucleolus
24
Q
What is the target gene of RNA pol II?
A
-mRNA
-snRNAs
-miRNAs
25
Where is RNA pol II located?
In the nucleus
26
What are the target genes of RNA pol III?
-tRNA
-5S RNAs
-U6 RNA
-7S RNA
27
How many subunits make up Eukaryotic RNA Pol II?
12 Subunits
28
Where is RNA pol III located?
In the nucleus
29
What are General Transcription Factors (GTFs)?
A set of factors that recruit RNA pol II to the promoter and direct initiation at the start-site
30
Name some Eukaryotic General transcription factors?
-TFIIA
-TFIIB
-TFIID
-TFIIE
-TFIIF
-TFIIH
31
Where do General Transcription Factors form a complex on?
TATA box
32
Describe the order of addition in the formation of the Pre Initiation Complex (PIC)
-IID binds
-IIA binds
-IIB binds
-RNA pol II and IIF binds
-IIH and IIE binds
33
What does the job of σ70 in eukaryotes?
General Transcription Factors (GTFs)
34
Describe Transcription initiation by RNA pol II
-Helicase activity of TFIIH separates template strand at start site
-As Pol II begins transcribingg it is extensively phosporylated on C-terminal domain
35
Describe the C terminal domain of RNA pol II
Series of repeats located at the C-terminal end of the largest subunit (β' homologous)
36
What occurs to the General transcription factors as transcription is initiated?
-TFIID and TFIIA may stay behind
-TFIIB, TFIIE and TFIIH are released
-TFIIF move down the template with Pol II
37
Describe the function of TFIID and the number of subunits
-Binds to the TATA box
-Recruits TFIIB
13 subunits
38
Describe the function of TFIIA and the number of subunits
-Stabilises TFIID binding
-Anti repression function
3 subunits
39
Describe the function of TFIIB and the number of subunits
-Recruits RNA pol II-TFIIF complex
-Important for start site selection
1 subunit
40
Describe the function of TFIIF and the number of subunits
-Assists TFIIB in recruiting RNA pol II
-Stimulates RNA Pol II elongation
2 subunits
41
Describe the function of TFIIE and the number of subunits
-Helps recruit TFIIH and modulates TFIIH activity
2 subunits
42
Describe the function of TFIIH and the number of subunits
-Promotes melting and clearance
-CTD Kinase activity
-DNA repair coupling
9 subunits
43
Name the two parts of TFIIH GFT
Core and CAK
44
What is the function of the CAK module of TFIIH?
Contains one of the kinases that phosphorylates the CTD of RNAP II
45
What enzyme does TFIIH contain that melts promoters?
An ATPase called XPB or Ssl2
46
How does Ssl2 or XPB helices form open complexes?
-Uses energy from ATP hydrolysis to push DNA into the cleft where RNA polymerisation is catalysed
-Creating torsional stress that contributes to open complex formation
47
What does TFIID consist of?
TATA binding protein (TBP) and TBP associated factors (TAFs)
48
Describe the properties of TATA binding protein (TBP)?
-CAN direct assembly of the PIC on a TATA containing promoter
-CANNOT direct PIC on a TATA-less promoter
-CANNOT support "activated" transcription
49
What do TBP associated factors (TAFs) do?
-Promote the interaction of TFIID with basal promoter elements
-TAFs interact with activators to promote transcription initiation
50
What is an enhancer?
-DNA regions close or far from the start-site
-Binding sites for activator proteins
51
What are enhancers often composed of?
Multiple UAS elements
52
What are Silencers?
-DNA regions close or far from the start-site
-Binding sites for repressor proteins
53
What is the basal transcription machinery?
Another name for GTFs and RNA pol II
54
What is the Pre initiation complex?
Assembly of the basal machinery at the core promoter
55
What is an activator?
A factor that binds to gene specific regulatory sequences (ie UAS & Enhancer elements) and stimulates
transcription initiation.
56
How is transcription controlled?
By changing the efficiency of the Pre Initiation Complex
57
What are the classes of UAS/enhancer elements?
-Common sequence elements
-Response elements
58
Describe Common sequence enhancer elements
-Often located close to the core promoter
-Bind activators that are relatively abundant in the cell and constantly active
59
Describe Response enhancer elements
-Bind factors whose activity is controlled in response to specific stimuli
60
Give an example of a response enhancer element
-SRE binds to serum response factor, and is induced by Growth factors
-HSE binds to Heat shock factor, and is induced by Heat shock
61
What is combinatorial control of transcription?
The type and combination of elements dictates when and at what level a gene is transcribed.
62
How close do enhancers have to be to ORF to work?
Enhancers work irrespective of location or orientation
63
How can enhancers work at any location or orientation?
Activators associated with Enhancer elements are brought into contact with GTF/RNA Pol II sat the core looping out intervening DNA
64
How cab eukaryotic activators work when separated?
-Eukaryotic activators are modular
-Working even when separated
-They often are separated
65
What are the domains found in Eukaryotic activators?
-Activation domains
-DNA binding domain
-Flexible protein domain often joining these
66
How may we manipulate Eukaryotic activators?
-We may combine modules from different activators
-eg the activation domain of one activator with the DNA binding domain of a different activator
67
Give some examples of DNA binding domains
-Leucine zipper
-Zinc finger
-Homeodomain
-Helix loop helix
68
Name some characteristics of activation domains
-Often characterised according to amino acid composition
-Lack of sequence conservation
-Generally thought to be unstructured
-Contain multiple short segments that work together in an additive fashion
-Interact with other proteins in the transcriptional machinery
69
Give some in vitro approaches to analysing activators
-DNA foot printing
-Electrophoretic mobility shift assays (gel shift)
-Transcription assays
70
Give some in vivo approaches to analysing activators
-Reporter assays
-Chromatin immunoprecipitation
71
Describe electrophoretic mobility shift assays (gel shift) as a method to analyse activators
-Measures ability of a protein to bind to a certain DNA sequence
-Combine activator with radiolabelled probe DNA, then run this on non denaturing acrylamide gel.
72
Describe transcription assays as a method to analyse activators
-Mix RNA Pol II, GTFs, DNA template and radiolabelled rNTPs.
-Measuring RNA transcript
73
What do transcription assays to analyse activators require?
-Requires the activator to both have a functional DNA binding domain and a functional activation domain
74
Describe Reporter assays as a method to analyse activators
-Producing a plasmid with a gene encoding protein X
-Producing a plasmid with a X binding site and reporter gene
-Measuring level of reporter gene expressed
75
Give the method for chromatin immunoprecipitation
-Cross link bound proteins to DNA
-Isolate chromatin
-Precipitate chromatin with protein-specific antibody
-Reverse cross link and digest protein
-Analyse the DNA using PCR or sequencing
76
How do activators work?
-Promote binding of an additional activator
-Stimulate complex assembly, with activators interacting with TFIIB which aid TFIID binding to the TATA box
-Release Stalled RNA polymerase
-Modulate chromatin
77
Describe the composition of activation mediators
-Approximately 22 polypeptides
-Can exist on its own or associated with RNA pol II (through the C terminal domain)
-Composed of Head, Middle and Tail domains
78
What are the function of activator mediators?
-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 enhance PIC formation
79
Where may RNA polymerases stall, and how is this solved?
RNA pol II can stall at or near to the promoter, which active activator proteins releasing stalled RNA pol II
80
What is the basic function of chromatin?
To compact DNA
81
What is the composition of Chromatin?
Primarily of small basic proteins called histones
82
What are the two basic types of histones?
Core and Linker histones
83
What are core histones split into?
-Globular domain made up of ⍺ helices and loops
-N terminal tail which are highly basic, rich in Lys and Arg
84
What are the repeating units of core histones?
Nucleosomes
85
Describe the composition of a nucleosome
147bp of DNA wrapped twice around an octamer of histone proteins
86
Describe the composition of the histone octamer formed in a nucleosome?
Central H3-H4 tetramer + 2 flanking H2A-H2B dimers
87
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
88
What evidence is there that chromatin inhibits transcription?
-In vitro reconstitution experiments
-In vivo nucleosome positioning experiments
-Genetic studies in budding yeast
89
Describe in vitro reconstitution experiments that demonstrate that chromatin inhibits transcription
-RNA pol II + transcription factors + naked DNA template = Transcription
-RNA pol II + transcription factors + chromatin template = No transcription
90
Describe in vivo nucleosome positioning experiments that demonstrate that chromatin inhibits transcription
Numerous experiments have shown that nucleosomes are disrupted or lost during transcriptional activation
91
Describe the genetic studies in budding yeast that demonstrate that chromatin inhibits transcription
Nucleosome depletion is linked to the expression of many inducible genes
92
What mechanisms may cells use the modulate the dynamic structure of Chromatin?
-Histone variants
-Post translational modification of histone
-ATP dependent chromatin remodelling
93
Describe how histone variants may modulate chromatin structure
-Histone variants differ from highly conserved major types
-Expressed at very low levels
-All conventional (except H4) have variants
-Histone variants creates new structural and functional properties of the nucleosome which affect chromatin dynamics.
94
Give some examples of post-translational modification of histones
-Acetylation
-Methylation
-Ubiquitylation
-Phosphorylation
95
How may post translational modification of histones modulate chromatin?
-Histone modification state has been proposed to involve a code that sets its transcriptional state BY
-Directly altering chromatin folding/structure
-Could control the recruitment of non histone proteins to chromatin
96
What enzymes are involved in the acetylation of histone lysines?
Histone acetyl transferases (HATs)
97
What enzymes are involved in the deacetylation of histone lysines?
Histone decatylases (HDACs)
98
How may chromatin be acetylated?
Through Histone Lysine acetylation
99
Is acetylation a key component in transcriptional activation or silencing?
ACTIVATION
100
What are the two types of multisubunit complexes of Histone Acetyl transferases (HATs)?
GNAT family and MYST family
101
How are HATs recruited?
-Activators recruit HATs to specific promoters
-Many HAT complexes contain a specific subunit that interacts with activators
-Also some HATs are part of the general transcription machinery
102
How does acetylation mediate transcriptional activation?
-Direct influence on chromatin structure, as it causes the conversion of charged N termini to uncharged termini, causing histones to be exposed
-Directs the recruitment of BROMODOMAIN proteins, which often promote transcription
103
Where can histone methylation occur?
On Lysine and Arginine residues on histones
104
What enzymes are responsible for Histone methylation?
Histone Lysine Methyl transferases (HKMTs)
105
What enzymes are responsible for Histone demethylation?
Lysine Demethylases
106
How do HKMTs differ from HATs?
They may mono, di or tri methylate a lysine
107
Does methylation have a large effect on chromatin structure?
It does not affect charge so probably has only minor if any influence on chromatin structure
108
Are methyl-lysine residues on histones activators or repressors?
Depending on context they may do either, eg
-H3 Lys9 represses
-h3 Lys4 activates
109
Give some examples of environmental challenges that a cell may respond to?
-DNA damage
-Infection
-Hypoxia
-Physical stress
110
Give some cellular responses that may occur in response to environmental changes
-Gene expression
-Programmed death
-Repair
-Immune response
111
Give some pathways that allow the cell to respond to environmental changes
-NFκB
-p53
-HIF
112
What does NFκB stand for?
Nuclear Factor of the kappa Immunoglobulin light chain in B cells
113
What does the NFκB pathway allow for?
-Cells to respond to external challenges or threats
-By regulating expression of certain genes
114
What are the two subunits that make up the Mammalian NFκB family?
-RelA
-p50
-RelB
-c Rel
-p105
-p100
-p52
115
What region of DNA encodes DNA binding in NFκB?
Rel Homology domain
116
What region of DNA encodes the dimerisation functions of NFκB?
Rel Homology domain
117
How are p50 and p52 produced (NFκB)?
They are proteolytically processed from their precursor proteins p105 and p100
118
What do ankyrin repeats in p100 and p105 allow for?
To function as IκB like inhibitors
119
What is E3 Ubiquitin ligase responsible for?
Attachment of ubiquitin chains to a target protein
120
What is the Ubiquitin proteasome pathway responsible for?
Protein degradation
121
Describe what occurs after a chain of 5 ubiquitin molecules are attached to a protein substrate?
The 26S proteasome recognises this, and the protein is digested to peptides.
122
What induces NFκB?
-Inflammatory cytokines
-Bacterial products
-Viral proteins and infection
-DNA damage
-Cell stress
123
What does NFκB regulate?
-Immune and inflammatory responses
-Stress responses
-Cell survival and cell death
-Cell adhesion
-Proliferation
124
What makes up the NFκB complex?
-p50 + RelA dimer (interact with DNA)
-IκB (inhibitor protein)
125
Describe the process whereby NFκB is activated?
-Ligand is exposed to cell
-IKK kinase is activated, phosphorylating IκB kinase complex
-This complex phosphorylates IκB, which is then ubiquinated
-IκB is proteolytically degraded
-NFκB translocates to nucleus
126
What are the three core subunits of the IκB Kinase (IKK) complex?
IKKγ (regulatory), IKK⍺ (catalytic), and IKKβ (catalytic)
127
Name some inhibitors of NFκB
-IκB⍺
-IκBβ
-IκBε
-Bcl 3
128
What is the action of IκB?
It inhibits NFκB
129
What is the same in both the canonical and non canonical pathway in the activation of NFκB?
Ubiquitination, proteasomal degradation and nuclear localisation of NFκB complex
130
Give some inducers of the canonical pathway to activate NFκB
-TNF
-IL1
-LPS
131
Give some inducers of the non-canonical pathway to activate NFκB
-LPS
-CD40
-LMP1
132
Give some diseases associated with improper NFκB functioning
-Cancer
-Arthritis
-AIDS
-Asthma
-Diabetes
-Artherosclerosis
133
How many activators/modulators of NFκB are there?
>200
134
How does diversity in genes benefit the NFκB system?
A multigene family composed of dimers allows the formation of many different types of NFκB dimers with different properties.
135
How do different subunits of NFκB allow for different properties and functions?
Different subunits have subtly different DNA binding specificity and so can target different genes and position themselves on DNA.
136
What may conjugate NFκB after it has been translocated to the nucleus (to regulate)?
-Kinases
-Acetylases
-Phosphatases
137
What may regulate NFκB after it has been translocated to the nucleus?
-Corepressors
-Coactivators
-Heterologous transcription factors
138
How can we regulate NFκB's activity in the nucleus?
-Transcriptional repression
-Transcriptional activation
-Promoter targeting and selectivity
139
How may NFκB gain access to NFκB-dependent genes hidden by their chromatin state?
NFκB can recruit chromatin remodellers or rely on other proteins to change the state
140
What levels of regulation combine to give transcriptional specificity when NFκB is stimulated?
-Phosphorylation and degradation of IκB⍺, IκBβ or IκBε
-Translocation of NFκB to the nucleus and modification of subunits
-DNA binding and gaining access to promoter/enhancer (chromatin remodelling and cooperative DNA binding with other transcription factors_
-Transactivation, the interaction with the basal transcription complex and coactivators
141
How may COVID19 affect NFκB regulated genes, affecting symptoms and prognosis?
-May affect levels of expression in IFNβ cells
-INCREASING INFLAMMATION
-Decreasing relative anti-viral properties
142
What is the function of beta interferon (IFNβ)?
If unregulated, and IFNβ are multimerised, they can act as a viral inducible promoter and respond to other inducers.
143
How may NFκB act in a coactivator interaction surface?
It participates in a "combination lock", with only the p50/RelA dimer working at the IFNβ enhancer.
144
Where do coactivator complexes favourably form?
At the promoters and enhancers where an appropriate interface is required.
145
How may NFκB directly induce transcription after chromatin remodelling?
By recruiting the BTM - Basal transcription machinery
146
How may the alternative (non-canonical) pathway differ from the classical (canonical) pathway with NFκB?
Phosphorylation of the p100 subunit leads to proteolytic processing to p52
147
How can activation of gene transcription with NFκB be likened to a combination lock.
Each number of the combination lock is a different TF, or specifically modified TF or chromatin remodelling factor. It is only when all the numbers of the combination are entered correctly (ie all the TFs etc are recruited to the gene promoter/enhancer) that the lock is opened (that is transcription occurs).
148
How may parallel signalling pathways regulate the transcriptional specificity found in NFκB?
Parallel signalling pathways (e.g. kinases) also active in the cell can regulate each of these steps. For example, by phosphorylating NFκB subunits or activating other transcription factors that work co-operatively or antagonistically with NFκB
149
What environmental challenge activates the p53 pathway?
DNA damage
150
What environmental challenge activates the HIF pathway?
Hypoxia
151
What is Hypoxia
Hypoxia can be defined as a lowering of the O2 = concentrations compared to the normal levels cells are exposed to
152
What may the hypoxia response be used for in embryonic development?
Development in
-Placenta
-Heart
-Bone
-Vasculature
153
What may the hypoxia response be utilised to adapt to?
-High altitude living
-Intense muscle exercise
154
What diseases may hypoxia be related to?
-High altitude diseases
-Stroke/Ischemia
-Neurodegenerative diseases
-Ageing
-Cancer
-Rheumatoid arthritis
-Schizophrenia
155
How do cells respond to hypoxia?
-Restoration of oxygen homeostasis
-Cell survival
-Cell death
156
Through what pathways may cells respond to Hypoxia?
-Transcriptional program (HIF)
-Translational block
-Chromatin structure changes
-DNA replication block
-microRNA signature
157
What is HIF?
-Hypoxia Inducible Factor
-Heterodimeric transcription factor (HIF-⍺ and HIF-1β)
158
What subtypes is there for the HIF-⍺ subunit?
-HIF-1⍺ Which is ubiquitously expressed in all tissues
-HIF-2⍺ Which is restricted to certain tissues
-HIF-3⍺ Which is restricted to certain tissues and functions as a dominant negative inhibitor for 1 and 2.
159
What is the ODD region found in the HIF subtypes genome?
The oxygen dependent degradation domain
160
What regulates the function of HIF?
Proline hydroxylases (PHD)
161
What do Proline hydroxylases require?
Oxygen, meaning in an anoxic state they will not inhibit HIF
162
What do Proline Hydroxylases do in the presence of oxygen?
-PHDs hydroxylate prolines on HIF1⍺
-Allowing binding sites for VHL
-Which binds to HIF1⍺ and leads to ubiquitation
163
How is HIF1⍺ regulated on multiple levels?
-HIF1⍺ mRNA evades block on translation
-Lack of oxygen inactivates the PHD proteins
164
What happens to HIF1⍺ in hypoxia?
In hypoxia, PHDs and FIH are inhibited, and HIF-1α is stabilized and able to dimerize with HIF-1β and activate target gene transcription through recruitment of co- activators.
165
What pathways does HIF control?
-Oxygen supply
-Transcription
-Cellular metabolism
-Cell death
-HIF control
-Cell growth
166
What may occur to hypoxic tumours?
-Activation of HIF stimulates angiogenesis, bringing nutrients and oxygen to the tumour
-It also leads to increased evasion and metastasis
167
What environmental challenge activates the p53 pathway?
DNA damage
168
Give some domains found in p53
-Transactivation domain
-Proline rich domain
-Nuclear localisation sequence
-Tetramerisation domain
169
How may NFκB, p53 and HIF interact?
-Many functions overlap
-Depending on context they may either function cooperatively or antagonistically
170
What inactivates p53?
Its regulator, Mdm2
171
What change happens to p53 if activated?
Dissociation with its negative regulator, Mdm2
172
What occurs when p53 is separated from Mdm2?
p53 will either
-induce a cell cycle to allow repair and survival before restarting the cycle
-Apoptosise the cell, eliminating damaged cells
173
What is the role of p14 ARF?
-Tumour suppressor with expression induced by oncogenes as a result of increased cellular proliferation
-Disrupts the interaction between the p53 tumour suppressor and Mdm2
174
How does p14 ARF disrupt the interaction between p53 and Mdm2?
ARF binds Mdm2 and inhibits its ubiquitin ligase activity
175
How does Mdm2 function?
-It is an E3 ubiquitin ligase
-Promotes ubiquitation of p53
-leading to degradation by the proteasome
176
How does DNA damage lead to p53 and Mdm2 separating?
p53 is phosphorylated at serine 15 by the ATM or ATR kinases, and Mdm2 is phosphorylated, disrupting their interaction
177
What do cancer cells do in reaction to p53?
They will activate/inhibit the p53 either by
-Mutating ARF
-Mutating ATM
-Mutating p53
-Amplifying Mdm2
178
How do prokaryotic and eukaryotic transcription and translation differentiate?
-Prokaryotic transcription and translation is coupled
-Eukaryotic transcription and translation are separate
179
At what levels can eukaryotic gene expression be regulated?
-Transcription control
-RNA processing control
-Translational control
-Protein activity control
180
Name some features added to eukaryotic mRNA post transcription
-m7G cap
-PolyA tail
181
Are m7G caps and PolyA tails encoded in the genome?
NO
182
What modifications to pre mRNA occur after transcription
-Capping
-Splicing (alternative splicing)
-Polyadenylation
-Editing
183
Describe the steps involved in synthesis of the m7G cap
-GpppN structure is formed
-Methylation occurs, altering chemical behaviour
184
What are the functions of the m7G cap?
-Protects mRNA from degradation by 5'-3' nucleases
-Facilitates splicing
-Facilitates export from the nucleus
-Critical for translation of most mRNAs
185
What mediates the functions of the m7G cap?
-CBP80/CBP20 in nucleus
-elF4 complex is cytoplasm
186
What conserved sequences are found in exon and intron boundaries?
-5' splice site
-3' splice site
-Branch site
187
What do the conserved sequences in introns do?
Recruit the splicing machinery required to remove the intron and join the exons
188
Describe the steps in the splicing of introns
1 - Cut at 5' splice site
- Creating a bond between 5' end of intron and branch site
2 - Cut at 3' splice site to release intron lariat
- Ligation of two exons
189
Through what kind of reaction are introns spliced?
Transesterification
190
What is the spliceosome?
-Enzymatic complex that catalyses the removal of introns
-Requiring ATP
-Large complex containing over 200 proteins
191
Give some examples of proteins in the spliceosome
-RNA binding proteins
-ATPases
-GTPases
-snRNPs
192
What are snRNPs?
Small nuclear ribonucleo-protein particles
193
What do snRNPs do?
catalyse splicing
194
Do snRNPs code for a protein enzyme?
-No they do not, they are non coding DNA.
-They form a stable RNA-protein complexes in the nucleus
195
What is alternative splicing?
The process where exons from the same gene are joined in different combinations, leading to different mRNA transcripts
196
Give some examples of types of alternative splicing
-Exon skipping
-Intron retention
-Mutually exclusive exons
-Alternative 5' or 3' site
197
What do activators bind to to regulate alternative splicing?
Intronic and Extrinsic splicing enhancers
198
What do repressors bind to to regulate alternative splicing?
Intronic and Extrinsic splicing silencers
199
What is polyadenylation?
The addition of a polyA tail at the 3' end of mRNA
200
What is polyadenylation initiated by?
The polyadenylation signal, a sequence of AAUAAA 10-35 nucleotides upstream of the polyA site.
201
What proteins are required for polyadenylation?
-Cleavage and Polyadenylation specificity factor (CPSF)
-Cleavage stimulatory factor (CstF)
-Poly(A) polymerase
202
What occurs after the polyadenylation signal is bound to?
-Endonuclease cleavage
-Addition of As by PolyA polymerase
203
What is upstream of the polyadenylation signal?
U-rich upstream element (USE)
204
What is downstream of the polyadenylation signal?
G/U or U rich tract just downstream of polyA site
205
What is the significance of the polyA tail?
-All mRNAs have 3' polyA tail
-Enhances export of RNA
-Enhances translation
-Stabilises the 3' end of the mRNA
206
What is RNA editing?
Nucleotide alterations which result in different or additional nucleotides in the mature RNA
207
What are the three major classes of RNA, of which RNA editing occurs?
mRNA, tRNA and ribosomal RNA
208
What are the two classes of RNA editing?
-Insertion/deletion
-Modification
209
Give some examples of types of base modification in RNA editing
-Marked nucleotide
-Altered identity
210
What is marking a nucleotide in base modification in RNA editing?
-Addition of methyl group, changing the type of proteins that may bind
211
What is altering identity in base modification in RNA editing?
Changing groups on a base (eg NH2 for O)
212
What is RNA editing by deamination?
-Through enzymatic deamination
-Inosine is recognised as guanine, therefore this is equivalent to an A to G change.
213
Are all classes of RNA exported from the nucleus by the same pathway?
NO! They each have different pathways
214
How can mRNA be changed in terms of location?
RNA can be distributed in higher concentrations to certain areas of the cell
215
Why localise mRNA?
-Localise protein synthesis
-Generate cell polarity
-Prevent expression in the wrong place
-Local control of translation
-Promotes efficiency of subsequent protein targeting
216
Describe diffusion based RNA localisation
mRNAs freely diffuse in the cytoplasm and are locally entrapped by anchor proteins
217
Describe Active transport based RNA localisation
-mRNA recognised by specific trans acting factors in the nucleus
-Cytoplasmic factors ensure transport along a polarised cytoskeleton
218
How much of a ribosome is protein and RNA?
1/3 Protein, 2/3 RNA
219
What does R represent in tRNA?
Purine (G or A)
220
What does Y represent in tRNA?
Pyrimidine
221
What does ᴪ represent in tRNA?
Pseudouridine
222
Describe tRNA charging
1 - Amino acid activation. Amino acid and ATP bind catalytic site, nucleophilic attack by carboxylic acid oxygen yielding aminoacyl-adenylate
2 - Hydroxyl group of adenine 76 of tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl tRNA and AMP
223
Where are tRNAs present in ribosomes?
Polypeptide chain site (P) and Aminoacylated tRNA binding site (A)
224
What is the role of Factor G (eEF2 in eukaryotes)?
Hydrolyses GTP, catalysing mRNA movement across the ribosome
225
What is the role of the 5' cap on mRNA?
-Eukaryotic translation initiation depends upon this
-Small subunit binds to cap, scans the first AUG, encoding the initiating methionine of the protein
-Ternary complex binds, forming the 43s pre initiation complex
226
What is the purpose of circularising mRNA when translation is initiated?
-Moniters integrity of mRNA
-Brings ribosomes ending translation close
-Several other key translation factors
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At what points is translation regulated?
-Formation of EIF4F
-43S binding
-Function of EIF2B
-Ternary complex formation
228
What is required for 43S association with mRNA?
-eIf3 interacting with eIF4G
-RNA unwinding most 5' UTRs have at least some structure
229
What is the function of EIF2B?
-Its activity governs level of active EIF2-GTP and this overall initiation rate
-EIF2B activity is down regulated in response to stresses
-Impairs generation of ternary complex, reducing translation initiation of mRNA
230
How is EIF2B regulated?
Through EIF2 phosphorylation, which competitively inhibits eIF2B
231
What are the three subunits of eIF2?
eIF2⍺ - Phosphorylated on Ser51 by PKR, PERK, GCN2 HRI
eIF2β - binds eIF2B, eIF5
eIF2γ - GTPase, Met-tRNA
232
Name some eIF2 kinases
-PKR
-PERK
-GCN2
-HRI
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What is eIF2 kinase PKR activated by?
double stranded RNA (Viral infection), inhibiting translation initiation
234
What is eIF2 kinase PERK?
A mediator of the unfolded protein response (endoplasmic reticulum stress), inhibiting translation initiation
235
What is eIF2 kinase GCN2?
A regulator of translation in response to amino acid availability, inhibiting translation initiation
236
What is the function of eIF2 kinase HRI?
Linking global availability to protein synthesis
237
Describe eIF2 kinase PKR
-PKR expression increases when cells are exposed to interferons
-Which are produced and released by cells infected by viruses
-When PKR binds dsRNA it dimerises and is activated
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What is the function of UTRs in mRNA?
-Untranslated regions
-Influence stability in mRNA
239
What are Iron response elements (IREs)?
-Hairpin loops with a conserved loop sequence and a bulge within the stem
-Found in the 5' or 3' UTRs of iron regulated mRNAs
-Bound by Iron Regulatory proteins (IRP1 and IRP2)
240
What are iron response elements bound by?
Iron regulatory proteins (IRP1 and IRP2)
241
What metal can be linked to regulation of gene expression?
Iron
242
Why degrade RNA?
-To remove damaged mRNA
-To remove incorrectly transcribed mRNA
-To control gene expression
243
Give an example of how a hormone can influence mRNA degradation
-Casein mRNA increases on stimulation by prolactin (70 fold)
-But transcription only increases 2 fold
-As half life increases dramatically in response to prolactin as PolyA tail length increases
244
Describe eukaryotic mRNAs during translation
-Circular
-This monitors mRNA integrity (will not be circular if it has lost cap or polyA)
-Brings ribosomes ending translation close to the AUG
245
What does most decaying mRNA decay from?
Deadenylation-dependent decay
246
As the polyA decays, what degrades the mRNA?
The exosome
247
As the M7G cap is degraded, what degrades the mRNA?
XRN1
248
Give some examples of mRNA decapping enzymes
-DCP1
-DCP2
249
Give some examples of mRNA deadenylase enzymes
Ccr/Not Complex
250
Give some examples of mRNA endonuclease enzymes
-Argonaute
-Swt1
-Smg6
251
Describe the mRNA exosome?
-3' to 5' exonuclease
-Multisubunit complex
-Involved in RNA turnover and processing
-RRP6 and RRP44 nuclease
-Rest of the subunits function in RNA binding and unwinding
252
Describe XRN1
-5' to 3' exonuclease
-Involved in RNA turnover and processing
-Also involved in transcription termination
253
What stimulates deadenylation dependent decay?
By certain sequences, strands, etc
254
Give some examples of routes of stimulation of deanylation-dependent decay
-AU rich element or ARE
-Nonsense codon
-miRNA
-C for major coding deterinant
-miRNA recognition site
255
Describe nonsense mediated decay of deadenylation-dependent decay.
-Mistakes in RNA are detected, and RNA is targeted for degradation
-As premature stop codons occur
-Exon junction complexes are not removed from downstream, which interact with RNA degradation machinery
256
Give examples of how premature stop codons (PTCs) can be introduced
-Transcription
-Splicing
-Editing
-Polyadenylation
-Mutations
257
What is siRNA?
Small inhibitory RNA
258
What is RNAi?
RNA interference
259
What is miRNA?
Micro RNA
260
What is RISC?
RNA induced silencing complex
261
Describe siRNAs
-21-23 nucleotide RNAs
-Perfect complimentary to target RNA
-Thought to be mainly viral defence mechanism
-Leads to the degradation of the target RNA
261
Describe the transition in the formation of open complex from closed complex during transcription initiation in prokaryotes
CLOSED:
-RNA polymerase holoenzyme binds to the promoter region, which contains -35 TTGACA and -10 TATAAT box upstream of transcription start site, of which the σ factor binds to. DNA is double stranded.
OPEN:
-σ factor facilitates melting of the DNA helix around the -10 region, exposing the template strand. RNA polymerase stabilises the melted DNA by forming a transcription bubble.
262
Describe miRNAs
-21-23 nucleotide RNAs
-Imperfect complimentary to target RNA
-Key gene regulatory mechanism in the cell
-Leads to block in translation
262
Where are UAS or URS regions found?
In the regulatory region of eukaryotic promoters
263
Describe how siRNA works
-RISC recognises the siRNA duplex and loads it
-RISC removes one of the siRNA strands "passenger:
-The remaining "guide" strand binds to the RISC and guides it to the target mRNA
-siRNA-RISC complex cleaves the target mRNA, causing degradation
263
What are the subunits that make up bacterial RNA polymerase?
-β
-β'
-⍺ x 2
-𝜔
264
Describe how miRNA works
-miRNA controls gene expression
-By binding to the mRNA in the cytoplasm
-This marks the mRNA for degradation by Argonaute protein
264
Describe the pre initiation complex
CONSISTS OF
-RNA Polymerase
-General transcription factors including
-TFIID
-TFIIA
-TFIIB
-TFIIF
-TFIIH
265
Describe different examples of 3' UTR length changes?
-During embryonic development 3' UTRs frequently get LONGER
-mRNAs in proliferating cells tend to have shorter 3' UTRs
265
Give the functions of the Pre Initiation Complex
-Promoter recognition
-DNA unwinding
-RNA polymerase II positioning
-Transition to the open complex
-C terminal domain phosphorylation
266
What drives differing 3' UTR length changes?
Alternate polyadenylation sites
266
Name some activation domains
-Acidic patch (clusters of -ve charged residues)
-Glutamine rich (high glutamine content)
-Proline rich
267
What is the benefit of longer 3' UTRs?
More possibility of binding sites for miRNAs
267
Describe the composition of activator mediators
-Consists of ~22 polypeptides
-Can exist on its own or associated with RNA pol II
-Composed of 3 domains: Head, Middle and Tail
268
Describe the structure of core histones
-N terminal tail (highly basic)
-Globular domain (⍺-helices and loops)
269
Give the key superfamily involved in ATP-dependent chromatin remodelling
SWI2/Snf2 ATPase superfamily
270
Describe ATP dependent chromatin remodelling
-Utilises energy from ATP hydrolysis to change interactions between DNA and histones
-Either relaxes chromatin (forming euchromatin) or condenses (forming heterochromatin)
271
Give some mechanisms of chromatin remodelling
-Nucleosome sliding (repositioned along DNA, exposing certain DNA regions)
-Ejection (removing entire histone octamers)
-Histone variant exchange (swapping histones)
-DNA unwrapping (making specific sequences more accessible)
272
Describe the SWI/SNF class of ATP-dependent chromatin remodelling complex
-Uses energy from ATP hydrolysis to track along DNA and induce torsion
-Promotes nucleosome sliding and ejection, facilitating activation
273
What may mutations in the SWI/SNF class of ATP-dependent chromatin remodelling complex result in?
-Mutations in the genes encoding subunits of the SWI/SNF complexes are collectively present in nearly 25% of cancers
-As it also acts as a tumour suppressor
274
What factors may be involved in manipulating recruitment of chromatin in order to repress transcription?
-Histone Deacetylases
-ATP dependent remodellers
-Histone methylases
275
Give the 4 major groups of histone deactylases
-Classical HDACs (zinc dependent)
-Class III Sir2 family (NAD dependent)
276
What are the two types of chromatin, and describe them
-Euchromatin (gene rich, with potential to be transcribed)
-Heterochromatin (gene poor, with repetitive regions, associated with transcriptional silencing), examples include centromeres and telomeres
277
Give some biochemical features of heterochromatin
-Hypoacetylation
-Specific histone H3 methylation
-Association of specific silencing factors
278
What do chromodomain proteins often bind to?
Methylated lysine residues on heterochromatin
279
Give a key chromodomain protein, and its function
-Heterochromatin Protein 1
-Forms and maintains heterochromatin
-Compacts nucleosomal arrays
280
What are barr bodies
-Condensed, inactivated structures formed by one of the X chromosomes in females assembling into a specific form of heterochromatin
-Formation is controlled by non coding RNAs Xist and Tsix
281
How may covid-19 lead to hypoxia?
-Acute respiratory distress syndrome (ARDS) caused by filling alveoli with inflammatory liquid
-Damage to pulmonary vasculature
-Impaired oxygen diffusion
-Hyperactivation of the immune system
-Haemoglobin and oxygen transport dysfunction
282
Give examples of negative feedback in transcription factor pathways
-p53 induces its inhibitor Mdm2, which causes its proteolytic degradation
-HIF1⍺ induces the PHD proteins, which causes its proteolytic degradation
-NFκB induces its inhibitor IκB⍺ that removes it from the nucleus and retains it in the cytoplasm
283
How do cancer cells and p53 interact?
Almost all cancer cells find ways to inactivate p53, or regulators of p53
284
Describe Li-Fraumeni syndrome
-LFS is a hereditary genetic condition
-Caused by mutation in gene coding for p53
285
Describe how p53 and NFκB may cross talk
-May suppress each other (eg by upregulating IκB⍺, or inducing MDM2)
-Both may bind to promoters of target genes involved in apoptosis, cell cycle regulation and inflammation
-Share modulators (CBP, ATM/ATR kinases, ROS)
286
What is dystrophin linked to?
Duchenne muscular dystrophy
287
Give some diseases associated with mutations causing defects in mRNA splicing
-Spinal muscular atrophy
-Retinitis pigmentosa
-Myotonic Dystrophy
288
What is RNA editing involved in in medicine and development?
-Disease (eg atherosclerosis)
-Brain function
-Development
-Parasites
289
Give some effects of mRNA editing
-Creation of start codons (by inserting U)
-Creation of new open reading frames by nucleotide insertion
-Changes in encoded amino acids and splice site choice
-Creation/removal of stop codons
290
What is cytidine deamination of apoB protein?
Pre-mRNA editing where a cytidine is deaminated to uridine by APOBEC1 enzyme, forming two different forms of the apoB protein
291
Name which type of ribosome is prokaryotic and eukaryotic
Prokaryotic = 70S
Eukaryotic = 80S
291
Describe the steps of translational elongation
-Binding of Aminoacyl tRNA (escorted by elongation factor) to the A site
-GTP hydrolysis and release of elongation factor (EF-Tu or eEF1A)
-Peptide bond formation between aminoacyl in A site and P site
-Ribosome translocates, with deacylated tRNA in P site moving to the E site and releasing. Facilitated by EF-G or eEF2
292
Describe the steps in the scanning process of translation
-Formation of preinitiation complex assembles, with EIF4F facilitating the binding to the cap
-This forms a 48S complex which scans for the start codon
-Once bound and stabilised, the large ribosomal subunit joins, forming the initiation complex
