Control Of Eukaryotic Gene Expression Flashcards
(107 cards)
1
Q
What is gene expression?
A
Process by which information in genes (DNA) is decoded into protein.]
Multistep process - Transcription and Translation
2
Q
What is transcription?
A
Transfer of genetic information from dsDNA to ssRNA (mRNA)
3
Q
What is required for transcription?
A
- 1 strand is used as a template to build up single stranded copy of template using RNA polymerase, along with ribonucleotide triphosphates (ATP, UTP, CTP, GTP).
- RNA copy created that is complementary to template strand.
4
Q
What do promoter regions do?
A
- Contains the signals that tell RNA polymerase to bind and start transcribing.
- Promoter dictates when a gene is switched on and how efficiently.
5
Q
Describe transcription in prokaryotes
A
- RNA polymerase + sigma factor makes contacts with sequences upstream of transcribed region.
- Promoter has sequence elements located ~35 & 10bp upstream from transcription start site.
- RNA polymerase makes initial contact with promoter sequence to form closed complex, DNA still base-paired.
- RNA polymerase prises DNA open over transcription start site, gives access to template strand.
- Template strand used to make RNA copy.
- After ~10 nucleotides have been formed, sigma factor is released.
6
Q
What is a promoter?
A
cis acting DNA regulatory element through which transcription is initiated and controlled.
7
Q
Details of bacterial promoter region
A
- Transcription start-site (+1)
- ~10 base-pairs upstream is -10 sequence (hexamer).
- Spacer between 16-18bp, and then -35 sequence (hexamer) around -35bp.
- These are consensus sequences (strongest bacterial promoters).
- All signals located in small region upstream of transcription start-site.
8
Q
What and where are the core promoter elements?
A
TATA box (-30bp)
Initiator (located over start-site)
DPE (+30bp)
BRE (next to TATA box)
9
Q
What are eukaryotic promoters divided into?
A
Core (basal) region and regulatory region.
10
Q
What are CpG islands?
A
- C-G rich sequences, feature of just mammalian promoters
- Approx. 60-70% of protein coding genes lack obvious TATA and initiator element.
- Transcription initiation occurs at lower rate and at several start sites.
- Associated with regions (~100-1000bp) with a high frequency of CpG islands.
- In mammals, most C residues followed by a G are methylated (to 5-methyl C)
- However generally C residues in CpG islands escape methylation (hypomethylated)
- Important for promoter function
- Methylation of CpG islands is associated with silencing (transcription switched off)
11
Q
What is the UAS/Enhancer?
A
Upstream Activator Sequences, activator binding sites – increase amount of transcription initiation.
12
Q
What is the URS/Silencer?
A
Upstream Repressor Sequences – binding site for proteins that repress/inhibit transcription initiation.
13
Q
What might you find in the regulatory region?
A
UAS/Enhancer
URS/Silencer
Can be proximal or many kb away.
Combination of these elements dictates at what level a gene will be switched on and which signals it will respond to.
14
Q
2 tools for identifying promoter elements.
A
- Sequence comparison:
- Used to identify TATA box
- Take sequences of regions that were known to be just upstream of transcription start site and try and identify a common sequence.
- No functional information about what the sequence does. - Reporter analysis:
- Reporter genes encode proteins whose levels can be easily measured, enzymatically or down a microscope (GFP, luciferase, lacZ)
- Amount of reporter protein provides a measure of gene expression.
- Used to identify when a gene is expressed, where it is expressed/which tissue, which signals it responds to, what factors and sequences control its expression.
15
Q
Name 3 major eukaryotic RNA polymerases, what they transcribe and where.
A
- RNA pol 1
* Transcribes rRNA genes.
* Nucleolus - RNA pol 2 - most important
* Transcribes mRNA, snRNAs, miRNAs.
* Nucleus - RNA pol 3
* Transcribes tRNA, 5S RNAs, U6 RNA, 72 RNA
* Nucleus
16
Q
Compare bacterial RNA polymerase and yeast RNA polymerase 2.
A
Bacterial RNA Pol:
- B subunit, B prime subunit, 2 a subunits, w subunit
- Similar crab claw structure
Yeast RNA Pol 2:
- 12 subunits
- More complicated
- Some direct counterparts
- Similar crab claw structure
17
Q
What do sigma factors do?
A
Recognise promoter regions in bacteria
18
Q
Which is the most important sigma factor?
A
Sigma 70 - housekeeping
19
Q
What does the job of sigma factor in eukaryotes?
A
General transcription factors:
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
All required to bring RNA pol to promoter and allow transcription initiation.
Multi-component factors (complexes), except B.
20
Q
How doe general transcription factors work?
A
Form a complex on TATA box, recruit RNA pol 2 to the promoter & direct initiation at start-site.
21
Q
Summarise PIC assembly
A
- Initiation – TFIID interacts with TATA box.
- Once TFIID is assembled, TFIIA can be assembled.
- … and TFIIB. Important because this is the signal that allows the recruitment of the polymerase.
- RNA polymerase joins with TFIIF.
- Assembly of TFIIE and TFIIH.
-Equivalent of closed complex in bacteria.
22
Q
Describe transcription initiation by RNA pol 2
A
- TFIIH separates the template strand at the start-site (Open Complex)- Requires ATP hydrolysis.
- As Pol II begins transcribing (promoter clearance) it is extensively 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.
- TFIID and TFIIA may stay behind, remaining bound.
- TFIIB, TFIIE and TFIIH are released.
- TFIIF moves down the template with RNA Pol II.
23
Q
Function and number of subunits of TFIID
A
13
TBP + TAF
Binds to the TATA box
Recruits TFIIB
Trilobular structure - TBP in the middle.
Saddle-like structure
24
Q
Function and number of subunits of TFIIA
A
3
Stabilises TFIID binding
Anti repression function
25
Function and number of subunits of TFIIB
1
Recruits RNA pol 2 - TFIIF
Important for start site selection
26
Function and number of subunits of TFIIF
2
Stimulates elongation
Destabilises non-specific RNA pol 2 - DNA interactions
27
Function and number of subunits of TFIIE
2
Recruits TFIIH and modulates TFIIH activity
28
Function, structure and number of subunits of TFIIH
9
Divided into:
- Core + CAK
- The CAK module contains one of the kinases that phosphorylates the CTD of RNAP 2
- Contains an ATPase called XPB (or Ssl2) that plays a key role in promoter melting.
Promoter melting and clearance
CTD kinase activity
DNA repair coupling
29
TBP vs TFIID
- TBP can direct the assembly of the PIC on a TATA-containing promoter (in vitro) (TAFs not required)
- TBP alone can NOT direct PIC assembly on a TATA-less promoter (most promoters).
- TBP can NOT support “activated” transcription.
30
Function of TAF?
- Promote interaction of TFIID with the basal promoter
- Interact with activators.
31
What are the different types of UAS/Enhancer elements?
1. Common sequence elements
- Often located close to the core promoter
- Bind activators that are relatively abundant in the cell and constantly active.
2. Response elements
- Bind factors whose activity is controlled in response to specific stimuli
e.g. SRE - binds SRF, induces growth factors
HSE - binds heat shock factor, induces heat shock
32
How are activators bound to enhancer elements brought into contact with the basal transcription machinery?
Possible models:
- Tracking
- Looping
33
What type of domains are found in activator proteins? What do they do?
DNA binding domain: binds to a DNA sequence specific to the activator
Activation domains: interact with other proteins to increase transcription.
- Generally thought to be unstructured
- Often characterised according to their amino acid composition
34
Eukaryotic Activators are often modular. What is meant by this?
'Each of a set of independent units that can be used to construct a more complex structure'
35
Explain the basis of Reporter Assays, Electrophoretic Mobility Shift Assays and in vitro Transcription Assays?
Reporter Assays:
- In vivo
-
Electrophoretic Mobility Shift Assays:
- In vitro
- Activator and radiolabelled probe DNA run on non-denaturing acrylamide gel = measure the ability of an activator to bind to a specific sequence.
Transcription Assays:
- In vitro
- RNA pol 2 + GTFs + DNA template + radiolabelled rNTPs = requires activator to have functional domains
36
What is a mediator and why is it important?
An additional complex required for activation.
Important as many activators cannot activate transcription in minimal in vitro transcription systems.
Provides a bridge between activators and RNA Pol 2, aid recruitment of RNA Pol 2, enhance PIC formation.
37
What are the mechanisms by which activators stimulate transcription?
1. Activators promote binding of additional activators
2. Increase rate of PIC formation by increasing TFIID binding, TFIIB binding and RNA pol 2 recruitment.
3. May stimulate post recruitment steps, promoting the release of stalled RNA pol 2.
4. Modulation of chromatin to allow complex formation.
38
What are the major activator targets?
1. TFIID (via TAFs)
2. TFIIB
3. Mediator
39
Describe 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 PCR and sequencing
40
What is the fundamental repeating subunit of chromatin called?
Nucleosome
41
Composition of nucleosome
~147 bp of DNA wrapped twice around an octamer of histone proteins.
Octamer = central H3-H4 tetramer + 2 flanking H2A-H2B dimers
42
What is the difference between core and linker histones?
Core histones are highly conserved.
43
What histone can direct the formation of the 30 nm fibre in vitro?
In vitro linker histones
44
What evidence indicates that chromatin blocks access of the transcription machinery?
1. In vitro reconstitution experiments
2. In vivo nucleosome positioning experiments
3. Genetic studies in S. cerevisiae.
Shown that nucleosomes are disrupted/lost during transcriptional activation
45
What are histone variants?
- Encoded by genes that differ from the highly conserved major types
- Variants are expressed at very low levels compared to their conventional counterparts.
- All the conventional histones, except H4*, have variants.
- Confer novel structural and functional properties of the nucleosome which affects the chromatin dynamics.
46
In which region are histones subjected to extensive post-translational modification?
N-terminal tails
47
What are the enzymes that control the acetylation and methylation status of chromatin?
Acetylation - HATs and HDACs
Methylation - HKMTs and lysine demethylases.
48
How do histone acetylation and methylation influence chromatin structure and transcription?
Acetylation - relaxes the chromatin, allows transcription
Methylation - minor influence on chromatin structure. Either increase or decrease transcription depending on the amino acid.
49
Composition of chromatin
Composed of histones
Controlled by: histone variants, PTM of histones, ATP dependent chromatin remodelling
50
Composition of histones
N-terminal tail - highly basic, rich in Lys and Arg
Globular domain - a-helices and loops
51
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. In vitro linker histones result in the formation of a thicker 30nm fibre
52
What are the names and the characteristics of the major ATP-
dependent chromatin remodelling complexes?
SWI/SNF - all have a SNF2-related ATPase.
Helicase and NTP driven nucleic acid translocase superfamily 2.
53
How do the major ATP-
dependent chromatin remodelling complexes remodel chromatin?
- The first complex to be isolated was the SWI/SNF complex from yeast
- The catalytic subunit of SWI/SNF is called Snf2 (or Swi2)
- It hydrolyses 1000 ATP molecules per minute in the presence of DNA or nucleosomes.
- Snf2 is 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
54
How can ATP-dependent remodelling complexes be recruited to DNA?
SWI/SNF and the GCN5 HAT regulate the same genes in yeast.
These HATs and ATP-dependent remodellers are commonly recruited to the same promoters
Bromodomains in Snf2 help tether it to acetylated nucleosomes
HATs & ATP-dependent remodellers function co-operatively
55
What impact do histone deacetylases generally have on transcription?
Repression of transcription
56
What is a co-repressor?
A molecule that binds to a repressor and makes it bind to the operator tightly, decreasing transcription.
57
What is heterochromatin and its key characteristics?
A type of chromatin. Gene poor, repetitive regions, transcriptional silencing.
Biochemical features: hypoacetylation, specific histone H3 methylation, association of specific silencing factors.
58
Which regions of chromosomes are commonly associated with heterochromatin?
Usually occurs around the centromere.
59
What are the key steps that lead to the formation of H3 K9me-dependent heterochromatin?
60
What type of protein motif is involved in the binding of methylated histone H3K9
histones?
61
How are silencing assays used to study heterochromatin?
62
How does X-chromosome inactivation occur?
In females, there are 2 X chromosomes, so one has to be inactivated.
This equalises the number of X-linked genes expressed in males and females.
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
63
What do the NF-kB, p53 and HIF pathways do?
Allow the cell to respond to environmental threats
64
How does NF-kB allow the cell to respond to external threats?
By regulating the expression of a wide range of target genes, it helps programme the response to these, either allowing the cell/organism to survive and recover or sometimes inducing death.
65
Which enzymes are involved with NF-kB?
p50, p52, p100, p105
66
What is E3 Ubiquitin ligase?
A protein that facilitates the attachment of ubiquitin chains to a target protein
67
What is NF-kB induced by?
Inflammatory cytokines
Bacterial products
Viral proteins & infection
DNA-damage
Cell Stress
68
What does NF-kB regulate?
The immune and inflammatory responses
Stress responses
Cell survival and cell death
Cell adhesion
Proliferation
69
What are the 3 core subunits of the IkB kinase (IKK) complex?
2 catalytic subunits IKKa and IKKB, and regulatory subunit NF-kB essential modifier (NEMO/IKKy)
70
What are the inhibitors of NF-kB?
IκBα, IκBβ, IκBε and Bcl-3.
Contain ankyrin repeat motifs in their C termini.
71
Which two pathways activate NF-kB?
Canonical and non-canonical pathway.
Canonical - mediated by IKKB components. Activates p50
Non-canonical - mediated by IKKa. Activates p52
72
How is transcriptional specificity gained?
1. Phosphorylation and degradation of the IkBs
2. Translocation of NF-kB to the nucleus modification of NF-kB subunits.
3. DNA binding and gaining access to the promoter/enhancer
4. Transactivation
73
What is hypoxia?
A lowering of the O2 concentrations compared to normal levels that cells are used to
NOT the absence of oxygen
74
Cellular responses to hypoxia and how they are brought about
Restoration of oxygen homeostasis
Cell survival
Cell death
Brought about by:
Transcriptional program
Chromatin structure changes
Translational blocks
75
What is a HIF?
Hypoxia Inducible Factor
A heterodimeric TF - HIFa and HIF1B
76
3 subunits of HIF-a
HIF-1a - expressed in all tissues
HIF-2a - expression restricted
HIF-3a - expression restricted and lacks C terminus transactivation domain
77
What does HIF control?
Oxygen supply - most central
Transcription
Cellular metabolism and growth
Cell death
78
What is the role of Mdm2?
- E3 ubiquitin ligase
- Promotes the ubiquitination of p53, leading to its degradation by the proteosome.
- Keeps p53 levels low
79
What does p53 do?
Tumour suppressor that promotes cellular and genetic stability.
80
The role of ARF
- Tumour suppressor whose expression is induced by oncogenes as a result of increased cellular proliferation.
- Results in increased levels of p53.
81
What is Li-Fraumeni syndrome?
- Hereditary genetic condition
- Cancer risk can be passed from generation to generation in a family
- Mainly caused by a mutation in p53
82
When are cap and poly A tail added to the polypeptide?
Post-transcriptionally - not encoded in the genome.
Protect the outer layer from degradation
83
What is capping?
2 step event:
1. GpppN structure
2. Methylation - alters chemical behaviour of base
84
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
- Functions mediated through protein binding
85
How is splicing carried out?
Two trans-esterification reactions
1. Cut at 5' splice site, creation of bond between 5' end of intron and branch site
2. Cut at 3' splice site to release intron lariat, ligation of two exons
86
What are snRNPs?
- Small nuclear ribonucleoprotein particles
- Stable RNA-protein complexes in the nucleus
- Catalyse splicing
- RNAs base-pair with conserved sequences in the intron
86
What is the spliceosome?
- Enzymatic complex that catalyses the removal of introns
- Requires ATP
- Proteins include: RNA-binding proteins, ATPases, GTPases
- Contains several snRNPs
87
Different types of alternative splicing
Exon skipping
Intron retention
Mutually exclusive exons
Alternative 5' splice site
Alternative 3' splice site
88
3 diseases caused by defects in splicing
Spinal muscular atrophy
Retinitis pigmentosa
Myotonic dystrophy
89
Functional significance of the poly A tail
Approx 250nts long - unusual
Enhances export of RNA
Stabilises the 3' end of the mRNA
Enhances translation of mRNA
Protein-binding element
90
What is RNA editing?
Nucleotide alterations which result in different or additional nucleotides in the mature RNA
91
Where does RNA editing occur?
mRNA, tRNA, rRNA
92
Two classes of editing
Insertion/deletion
Modification
93
What happens in marked nucleotide base modification?
Methyl group is added
93
What happens in altered identity base modification?
I is read as G (equivalent of an A to G change), pairs with C.
94
Why localise mRNA?
- Prevents expression in the wrong place
- Promotes efficiency of subsequent protein targeting
- Local control of translation
95
Types of localisation
Diffusion based - mRNAs freely diffuse in the cytoplasm and are locally entrapped by anchor proteins
Active transport based - mRNA recognised by specific trans-acting factors in the nucleus. Cytoplasmic factors ensure transport along a polarised cytoskeleton.
95
Composition of a ribosome
1/3 protein
2/3 RNA
96
2 step reaction of aminoacyl-tRNA synthetases
1. Amino acid activation – amino acid and ATP bind catalytic site, nucleophilic attack by a-carboxylic acid oxygen yielding aminoacyl-adenylate (aa-AMP)
2. Hydroxyl group of adenine 76 of tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP
97
What is needed for 43S association?
1. Interactions – eIF3 w eIF4G
2. RNA unwinding: Most 5’ UTRs have at least some structure. eIF4F (4A) unwinds cap-proximal sequence.
98
What does elF4A complex do?
Binds cap - also interacts with PAB to circularise the mRNA
99
Describe what small subunit does in eukaryotic translation
Small subunit – as part of the initiation complex, is recruited to the cap and then scans along until it finds the start AUG
Then large subunit joins
Translocation required to move the tRNAs and mRNA through the ribosome
When reach termination codon, translation stops and the ribosome dissociates
100
What is PKR?
- Antiviral defence strategy
- Expression is typically low, increases when cells are exposed to interferons - produced and released by cells infected by viruses
- When PKR binds dsRNA it dimerises and is activated
101
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
102
Important points of regulation of translation
eIF2 phosphorylation regulates translation initiation
Many different stresses lead to eIF2 phosphorylation
Iron levels regulate the production of iron binding proteins
This leads to a switch between iron import and storage
Binding of iron-responsive protein 1(IRP1) to the mRNA can block or activate translation
IRP1-binding can also affect mRNA stability
103
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