CMB2001 Lecture 1-10 Flashcards
1
Q
What are the elements present in eukaryotic promoters?
A
- TATA box
- Initiator (Inr)
- Downstream core promoter element (DPE)
- TFIIB recognition element (BRE)
2
Q
How can reporter genes be used to analyse promoter sequences?
A
amount of reporter protein provides a measure of gene expression
3
Q
How many major RNA polymerases are present in eukaryotic cells?
A
- RNA pol I
- RNA pol II
- RNA pol III
4
Q
What gene classes do the RNA polymerases transcribe?
A
RNA pol I = rRNA
RNA pol II = mRNA
RNA pol III = tRNA
5
Q
What is gene expression?
A
a process by which information in genes (DNA) is decoded into protein.
6
Q
What are promoters?
A
cis acting DNA regulatory element through which transcription is initiated and controlled
7
Q
What effect do UAS and enhancer have on binding sites?
A
function as activator binding sites
8
Q
What effect do URS and silencer have on binding sites?
A
function as repressor binding sites
9
Q
What are cpG islands?
A
DNA methylation regions in promoters known to regulate gene expression through transcriptional silencing of the corresponding gene.
10
Q
What factor allows RNA polymerase to bind and recognize the promoter?
A
Sigma factor
11
Q
What are the roles/characteristics of sigma factors in eukaryotes?
A
- are RNA specific
- multi-component factors
- form a complex TATA box
- recruit RNA pol II to the promoter
- Direct initiation at start site
12
Q
What are the characteristics of TFIIH?
A
- composed of 9-10 subunits
- can be divided into 2 parts = CORE + CAK
- CAK module contains one part of the kinases the phosphorylates CTD of RNAP II
13
Q
What are the characteristics of TFIID?
A
- is the central RNA pol II transcription factor
- TFIID = TATA box protein + TBP associated factors
14
Q
What is the properties/role of TBP?
A
TBP can direct the assembly of the PIC on a TATA-containing promoter but TBP can not direct PIC alone.
15
Q
What do TAFs do?
A
- promote the interaction of TFFID with the basal promoter
- interact with activators
16
Q
What is the role of ‘common’ sequence elements and where are they located?
A
- located close to the core promoter (promoter proximal)
- bind activators that are relatively abundant in the cell and constantly active
17
Q
What is the function of response elements?
A
bind factors whose activity is controlled/induced in response to specific stimuli
18
Q
What is an enhancer?
A
an activated binding site that operates irrespectively of its location
19
Q
What does it mean if eukaryotic activators are modular?
A
composed of separable independent functioning parts = in eukaryotic activators, the DNA binding domain and activation domains are in different separable functional domains
20
Q
What are the characteristics of eukaryotic activation domains?
A
- acidic patch (cluster of -ve charged residues)
- glutamine rich (high gln content)
- proline rich
21
Q
What are the in vitro approaches for analysis of activators?
A
- DNA footprinting
- Electrophoretic Mobility Shift Assay (gel shift)
- Transcription assays
22
Q
What does a transcription assay require?
A
requires activator to have a function activator domain and a DNA binding domain
23
Q
What are the in vivo approaches for analysis of activators?
A
- Reporter assays
- Chromatin Immunoprecipitation (ChIP)
24
Q
What components of the PIC complex do activators interact with to promote assembly?
A
- TFIID (via TAFs)
- TFIIB
- Mediator
- Modulation of Chromatin
25
What is the role of heat shock with activators?
Heat shock activates HSF transcription factor which interacts with RNA pol II and releases it from the pause
26
What happens in the absence of heat shock in regards to activators?
RNA pol II pauses after ~50nts
27
What is a mediator?
a 22 polypeptide complex that connect activators and transcription factors. Can exist on its own or associated with RNA pol II (through C-terminal domain)
28
What is the function of a mediator?
- provides a bridge between activator proteins and RNA pol II
- mediator activator interactions aid recruitment of RNA pol II and enhance PIC formation
29
How is eukaryotic DNA packaged?
assemble DNA in chromatin
30
What is the composition of Chromatin?
- Composed primarily of Histones
- 2 types of Histones.. Core histones and Linker histones
31
What are the characteristics of core histones?
- highly conserved and present in all eukaryotes
- main part of forming the histone
32
What is the characteristic of linker histones?
- H1 histone which bind to the DNA between nucleosome (keep DNA wrapped around the nucleosome)
33
How do core histones bind DNA?
the core histones form repeating units called nucleosomes.
34
How are nucleosomes organised?
1. DNA passes directly from one nucleosome to the next
2. linker histones (histone H1) bind to the DNA between nucleosomes
3. 30nm fibre is formed
35
What evidence proves that chromatin inhibits transcription?
1. In vitro reconstruction experiments
2. In vivo nucleosome positioning experiments = shows that nucleosomes are disrupted or lost during transcriptional activities
3. Genetic studies in Saccharomyces Cerevisiae
36
What are the 3 major mechanisms for modulating chromatin structure?
1. Histone variants
2. Post-transcriptional modification of Histones
3. ATp dependent chromatin remodelling
37
How do we get histone variants?
histone variants are encoded genes that differ from the highly conserved major types.
38
What do histone variants do?
Histone variants confers novel structural and functional properties of the nucleosome which affect the chromatin dynamics.
39
What histones have variants?
all conventional histones except for H4
40
What are the post-translational modification of histones?
- majority of modifications happen in the tail of histones (N-tails)
- Histone modification plays a key role in controlling gene expression
41
What are the 4 possible post translational modifcations?
- acetylation
- methylation
- ubiquitylation
- phosphorlyation
42
What is the fundamental repeating subunit of chromatin called?
nucleosomes
43
What is the composition of nucleosomes?
147bp of DNA wrapped twice around an octomer of histone proteins
44
What are the enzymes that control the acetylation and methylation status of chromatin?
- acetylation = mediated by HATs, reversible by HDAcs
- methylation = occurs on lysine
45
How are HATs recruited?
activator protein interacts with the subunit of the HAT proteins (TRA1) which then brings a histone a sample of transferase.
46
How does acetylation mediate transcriptional activation?
1. Direct influence on chromatin structure
2. Directs the recruitment of BROMODOMAIN proteins
47
What do bromodomain proteins do?
bromodomain proteins promote transcription by recognising specific acetylated lysine residues
48
How do histone acetylation and methylation influence chromatin structure and transcription?
Acetylation = key component of transcriptional activation
Methylation = context dependent = can activate or repress transcription
49
What does the histone code suggest?
suggests that there are enzymes that write the code, erase the code and read the code.
50
What are examples of enzymes that "write" the code?
- Histone acetyl transferases
- Histone methyl transferases
- Kinases
51
What are examples of enzymes that "erase" the code?
- Histone deacetylases
- Demethylases
- Phosphateses
52
What are examples of enzymes that "read" the code?
- Bromodomain proteins
- Chromodomain proteins
- PWWP proteins
- PhD
53
What are the roles of ATPase?
- slide nucleosomes
- unwrap DNA from nucleosomes
- evict nucleosomes
- space nucleosomes
- histone variant exchange
54
How does SWI/SNF remodel chromatin?
It hydrolyses 1000ATP molecules per minute in the presence of DNA or nucleosomes.
- uses ATP energy to introduce torsion and pushes the DNA in to the nucleosome
55
How do ATP dependent and HAT complexed co-operate?
- HATs and ATP-dependent remodellers are recruited to the same promoters
56
What are the major ATP-dependent chromatin remodelling complexes?
- SWI2/SNF2
- ISWI
- CHD/Mi2
- Ino80
57
How are ATP-dependent remodelling complexes recruited to DNA?
- NURD uses energy from ATP hydrolysis to move nucleotides along DNA by spacing them in a tight orderly fashion
58
What impact do histone deacetylases generally have on transcription?
it is a co-repressor
59
What it a co-repressor?
a molecule that represses the expression of genes
60
What regions of chromosome are commonly associates with heterochromatin?
gene poor and repetitive regions.
61
What is heterochromatin?
chromosome material of different density from normal, in which the activity of the genes is modified or suppressed.
62
What are the two basic types of chromatin?
Euchromatin = light staining
Heterochromatin = dark staining
63
What are the biochemical features of heterochromatin?
1. hypoacetylation
2. specific histone H3 methylation (lys9 & lys27)
3. association of specific silencing factors
64
What is the role of a chromodomain and give an example?
chromodomains recognise and bind to methylated lysine residue.
- HP1 is a chromodomain for H3 lyse9me2/3
65
What does HP1 form?
forms nuclesomal arrays
66
how does HP1 form nucleosomal arrays?
HP1 interacts with itself by bringing adjacent nucleosomes close together = has surfaces that attract binding which prevent the recruitment of RNA pol II.
67
What is the role of NF-kappaB, P53 and HIF pathway?
they all allow the cell/organism to respond to environmental threats.
68
What is NF-kappaB?
the nuclear factor of the kappa immunoglobulin light chain in B cells.
69
What is the role of NF-kappaB?
Transcription factors that regulate inflammation, DNA damage, cell death, cell adhesion and proliferation.
70
How does NF-kappaB bind to DNA?
forms a dimer to allow it to bind to DNA
71
What is NF-kappaB induced by?
- inflammatory cytokines
- bacterial products
- viral proteins & infection
- DNA damage
- Cell stress
72
What does NF-kappaB regulate?
- immune and inflammatory response
- stress response
- cell survival and cell death
- cell adhesion
73
What are the characteristics for the IkB family?
- have ancored repeat domains that form a stacked alpha helical structure
- are inhibitory proteins
- bind the NF-kappB complex in the cytoplasm and render it inactive
74
What are the two NF-kappaB pathways and what is the difference between the two?
1. Classical pathway = IKKb is the primary subunit that activates the pathway
2. Non-colonial pathway = mediated by IKKa involves the phosphorylation of P100 = allows the activation of the complex of kappa B containing P50
75
What happens when NF-kappaB is out of control?
causes the NF-kappaB activator to be turned on all the time = causes inflammatory diseases
76
What does a dimer do in regards to NF-kappaB?
Dimers give different levels of regulation = dimer will bind NFkB site with a specific orientation.
77
How is the function of NF-kappaB regulated?
NF-kappaB reaches the cytoplasm, it can be modified by a range of other proteins. Can determine whether we get activation or repression of gene expression
78
What is hypoxia?
lowering of the O2 concentrations compared to the normal levels that cells are exposed to.
79
What physiological processes is hypoxia involved in?
- embryo development
- high altitude living
- intense muscle exercise
80
How do cells react to low oxygen?
response is to shut down as much protein translation as possible so that the cell can conserve energy.
81
What is the difference between HIF-1alpha, HIF2alpha and HIF-3alpha?
HIF-1alpha = ubiquitously expressed in all tissue
HIF2alpha = expression restricted in all tissue
HIF-3alpha = expression restricted to certain issues and lacks C-terminus transactivation domain
82
What is the function of HIF-3alpha expresison?
functions as a dominant negative inhibitor for HIF-1alpha and HIF-2alpha.
83
What is the structure of HIFs?
- modular structure with different domains
- HIFs have a helix loop domain at the end terminus which mediates DNA binding
84
What happens if HIF-1alpha and HIF-2alpha have the C-terminal transactivation domain?
allows them to activate transcription = have a nuclear localisation sequence + passive domain
85
What is the structure and function of ODD in regards to HIFs?
contain two proline residues that can become hydroxylated = allows them to respond in low oxygen levels.
86
How is HIF-1alpha regulated in levels of high oxygen?
PHD proteins that mediate hydroxylation of HIF-1alpha at proline residues at ODD domain
87
What are the roles of hydroxylated prolines in the regulation of HIF-1alpha?
hydroxylated prolines allow the recruitment of VHL that binds HIF-1alpha = stimulates the ubiquination of HIF-1alpha
88
What is the regulation of HIF under normal conditions?
under normal conditions, PHD enzymes hydroxylate HIF-1alpha in its ODD domain.
FIF alos hydroxylates the aspara gene domain in the c-terminal = acts to inhibit transcription.
89
What is the regulation of p53 activity?
- Mdm2 becomes inactivated and p53 becomes activated
- activation of p53 = primary response is to produce cells cycle arrest, allows the DNA repair program to repair the DNA before the cell cycle has restarted.
90
What is the role of p53 if DNA damage is too severe?
p43 can flip its activity in response to damaged DNA and induce apoptosis to remove the damaged cell.
91
What is the role of Mdm2?
- Mdm2 is an E3 ubiquitin ligase = promotes the ubiquitination of p53 leading to its degradation by the proteasome.
92
What is the role of ARF?
- activation of tumour suppressor protein p14
- induced its expression by out of control proliferation.
- p14 binds to Mdm2 to block its ability to interact with p53 = stabilisation of p53
93
Why do we get cancer?
- all cancer cells find ways to inactivate p53
- can occur through the mutation of ARF, ATM, or p53
- can occur through viral infection affect E6
- can occur through amplification of MDm2
94
What is the difference between transcription and translation of eukaryotes and prokaryotes?
eukaryotes = transcription and translation are separate
prokaryotes = transcription and translation are linked/coupled
95
What is the structure/characteristics of eukaryotic mRNA?
- have a start codon and an end codon with an open reading frame that can be translated
- m7G cap structure at 5' prime end for protection
- poly A tail of 250 A's
- cap structure and pol A tail are added after transcription
96
What is the structure and synthesis of the 5' m7G cap?
- present on all RNA pol II RNAs
- Ribose is added to 5' to 5' end
- methylation alters chemical behaviour of base
97
What is the function 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
98
What is the function of spilicing?
is to produce a continuous reading frame without introns
99
What is the two-step splicing of introns?
2 trans-esterification reactions:
Step 1 = cut at 5' splice site, creation of bond between 5' end of intron and branch site.
Step 2 = cut at 3' splice site to release intron lariat ligation of two exons
100
What is the splicesome?
enzymatic complex that catalyses the removal of introns. It requires ATP
101
What are the proteins included in the splicesome?
- RNA-binding proteins
- ATPases
- GTPases
102
What are snRNPs?
RNA protein complex that are essential for splicing. Form a ring by staking and RNA passes through the ring on the structure.
103
What are the 3 diseases mutations causing defects in splicing?
1. Spinal muscular atrophy = most common genetic cause of infant mortality
2. Retinitis pigmentosa = reduced visual capabilities and blindness
3. Mytonic dystrophy = a muscle wasting disease
104
What is dystrophin found to do?
a gene linked to Duchenne muscular dystrophy
105
What is polyadenylation?
the addition of conserved AAUAAA 10-35 nucleotides upstream of the poly(A) site
106
What are the proteins required for polyadenylation?
- CPSF (cleavage and polyadenylation specificity factor = binds AAUAAA
- CstF = cleavage stimulatory factor = binds G/U
- Cleavage factors I and II
- PolyA polymerase
107
What is the functional significance of the polyA tail?
- enhances export of RNA
- stabilizes the 3' end of the mRNA
- enhances translation of mRNA
108
What is RNA editing?
nucleotide alterations which result in different or additional nucleotides in the mature RNA.
109
In what major classes of RNA does ENA editing occur?
-mRNA
- tRNA
- ribosomal RNA (rRNA)
110
What are the effects of mRNA editing?
- can create start and stop codons by inserting a U.
- can change encoded amino acids
- can change splice sites
111
How does N6-methylation alter the mRNA?
- changes protein binding
- affects the ability of reader proteins to bind
- affects the stability and degradation of the mRNA
112
What happens when RNA editing is done by deamination?
Inosine is recognized as guanosine = critical channel position becomes occupied by R = encoded in the mRNA by CIG
113
What is the effect of A to I editing in the Q/R site of glutamate receptors?
- editing yields a decrease in Ca2+ permeability of channels containing the 'R' version.
114
What changes the property of the ribosome?
- methyl group from the 2'-O-methylation changes the property of the ribosome
115
What are the different pathways mediated by the different classes of RNA?
tRNA = uses exprT
mRNA = uses exp5
116
Why is mRNA localized?
- for localized protein synthesis
- to generate cell polarity
- prevents expression of genes in the wrong place
- promotes efficiency of subsequent protein targeting = produce where the protein is needed
- local control of translation
117
What is diffusion based localization?
local entrapment = protein binds to anchored proteins at site = mRNA diffuse through the cell but they are slowly enriched at the site that they are needed
118
What does eukaryotic translation depend on and why?
- depends of 5' CAP
- CAP moves to the first AUD and encodes the initiating methionine of the protein.
119
How is mRNA circularised?
polyA tail is bound by PolyA binding protein, the protein links round and bind eIF4G in the eIF4F complex and cicuses the mRNA
120
Why is mRNA circularised?
helps the subunit come back to the CAP and sped up translation
121
What are the proteins needed for mRNA circularisation?
eIF4E, eIF4G and PAB
122
What is eIF2B and what is its function?
- eIF2B is a subunit of eIF2
- eIF2B level governs levels of active eIF2-GTP and thus overall initiation rate
123
When is eIF2B activated and when is it inactivated?
- eIF2B down-regulated in responses to stresses such as viral infections and amino acid deprivation
- eIF2B down-regulated in diabetic muscle
- eIF2B activated by insulin
124
How is translation initiation regulated?
- activation of eIF2B by phosphorylation = causes essential amino acid production to stop under stress
125
What is the regulation of iron metabolism?
- levels of iron in the cell regulate the expression or iron storage/transport of protein
126
How do iron levels affect mRNA translation and stability?
- low iron = repress storage proteins to release more iron
- dependent on IRP1/2 protein binding which binds on the 5' region of UTR so translation is repressed.
- TfR1 production is increased produced through mRNA stabilization
127
What happens if we have too much iron?
- activate the production of storage and used proteins = translation activation through iron binding to IRP1 and IRp2 degradation
128
Why do we have RNA degradation?
- to remove damaged mRNA
- to remove incorrectly transcribed/processed mRNA
- control gene expression
129
Why is mRNA circular during translation?
- to monitor mRNA integrity
- brings ribosomes ending translation close to the AUG to "recycle" onto the 5' end
- protects mRNA from turnover
130
What is the role of the exosome?
degrades the RNA from 3' end to the 5' end = involved in RNA turnover and processing
- multiple nuclease activities = RRP6 & RRP44
131
What is the role of XRN1?
5' to 3' exonuclease = cannot degrade with a CAP
- involved in RNA turnover and processing
- involved in transcription termination
- functions after decapping of the mRNA
132
What is deadenylation-dependnt decay?
mRNA gets older = polyA tail shortens by enzymes.
when polyA tail becomes too short mRNA can go to exosome to be degraded
or
mRNA goes through 5' to 3' decay and gets decappedd and degraded from the 5' end.
133
What is the role of nonsense mediated decay?
prevents translation of mutant mRNAs by recognizing premature stop codon and mistakes in the RNA.
133
What is the role of nonsense mediated decay?
prevents translation of mutant mRNAs by recognizing premature stop codon and mistakes in the RNA.
133
What is the role of nonsense mediated decay?
prevents translation of mutant mRNAs by recognizing premature stop codon and mistakes in the RNA.
134
How do cells measure if there is a STOP codon randomly introduced?
work out the distance between stop codon and the splice sites, if the distance is more than 55 nucleotides between = potential nonsense decay target
135
What is the difference between miRNA and siRNA?
siRNA:
- 21-23 nucleotide RNA
- Perfect complimentary to target RNA
- though to be mainly viral defence mechanism
- leads to the degredation of the target RNA
miRNA:
- 21-23 nucleotide RNAs
- imperfect complimentary to target RNA
- key gene regulatory mechanism in the cell
- leads to block in translation
136
Name the 6 sigma factors in eukaryotes.
1. TFIIA
2. TFIIB
3. TFIID
4. TFIIE
5. TFIIF
6. TFIIH
