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Biomed year 2 sem 1 > CMB2001- gene expression > Flashcards

CMB2001- gene expression Flashcards

1
Q

CpG islands

A

areas with high freq of CG sequences
- associated with lower rate of Txn initiation

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2
Q

CpG island methylation =

A

silencing

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3
Q

UAS + enhancer =

A

activator binding sites

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4
Q

URS+ silencer =

A

repressor binding sites

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5
Q

Tools for identifying promotor elements

A

Sequence comparison
- identifying TATA box
Reporter analysis
- measure levels of proteins encoded by reporter genes

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6
Q

RNA pol I target and location

A

rRNA
Nucleolus

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7
Q

RNA pol II target and location

A

mRNA, snRNAs, miRNAs
nucleus

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8
Q

RNA pol III target and location

A

tRNA, 5S/ U6/7S RNAs
nucleus

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9
Q

GTF =

A

General transcription factor

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10
Q

PIC =

A

Pre initiation complex

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11
Q

sigma factor recognising promotor DNA (bacterial RNApol)

A
  1. RNA pol specific
  2. multicomponent factors
  3. form a complex on TATA box
  4. recruit RNA pol II to the promoter
  5. Direct initiation at start-site
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12
Q

CTD =

A

C-terminal domain
- series of repeats located at the C-terminal end of the largest pol II subunit

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13
Q

Transcription initiation by RNA pol II

A

helicase activity of TFIIH separates template strand at start site (requires ATP)
-> open complex
-> pol II is phosphorylated on CTD as pol begins transcribing

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14
Q

properties of TFIID

A

binds tata box (core promoter)
recruits TFIID

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15
Q

TFIID structure

A

central RNA pol II transcription factor
TBP (TATA Binding Protein) + TAFs (TBP associated factors = TFIID

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16
Q

TFIIH properties

A

promoter melting and clearance
CTD kinase activity
DNA repair coupling

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17
Q

TFIIH stucture

A

2 subunits: CORE + CAK
CAK contains kinase -> phosphorylates CTD of RNApol2

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18
Q

TBP properties

A
  • can directly assemble PIC on TATA containing promoter
  • cannot act alone without TATA
    cannot support activated transcription
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19
Q

TAF properties

A

promote interaction of TFIID with basal promoter
interaction with activators

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20
Q

GC box: sequence and factor

A

GGGCGG
Sp1

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21
Q

Octamer: sequence and factor

A

ATTTGCAT
Oct-1

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22
Q

CAAT box: sequence and factor

A

GGCCAATCT
NFY

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23
Q

common sequence elements

A

promoter proximal
constantly (constitutively) active

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24
Q

SRE

A

Response element
binds: serum response factor (SRF)
inducers: growth factors

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25
Q

HSE

A

Response element
binds: heat shock factor
inducer: heat shock

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26
Q

activation domains

A

lack of sequence conservation/structural information
multiple short segments that work in an additive way
interact with other proteins in the transcriptional machinery (e.g. TAFs)

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27
Q

characterising activation domains

A

by amino acid composition:
acidic patch (VP16)
glutamine rich (SP1)
proline rich (Jun)

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28
Q

in vitro analysis of activators

A

DNA footprinting, Electrophoretic Mobility Shift Assays (gel shift), Transcription assay

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29
Q

in vivo analysis of activators

A

reporter assays, chromatin immunoprecipitation

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30
Q

chromatin immunoprecipitation

A
  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 with PCR and ChiP-Seq
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31
Q

how activators work

A
  1. promote binding of an additional activator
  2. stimulate complex assembly (recruitment)
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32
Q

discovery of mediators

A
  • many activators cannot activate in vitro transcription
  • suggests that another factor is needed
    -> disovered mediators
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33
Q

composition of mediators

A
  • large complex (~22 polypeptides)
  • can exist alone or associated with RNA pol II
  • three domains: head, middle, tail
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34
Q

function of mediators

A
  • many interact with specific mediator subunits
  • provides bridge between activators and RNA pol II
  • mediator-activator interactions aid recruitment of RNA Pol II -> enhance PIC formation
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35
Q

how activators control transciption

A
  1. promote binding of additional activator
  2. stimulate complex assembly
  3. release stalled RNA pol II
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36
Q

chromatin

A

protein complex that packs DNA

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37
Q

Chromatin is primarily composed of:

A

histones

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38
Q

two basic histone types

A

core histones and linker histones

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39
Q

4 highly conserved core types of histones

A

H2A, H2B, H3, H4

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40
Q

N-terminal tail of core histones

A

highly basic
rich in Lys and Arg

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41
Q

globular domain of core histones

A

alpha helices and loops

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42
Q

Repeating unit of chromatin

A

Nucleosomes

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43
Q

composition of histone octamer

A

central H3-H4 tetramer + 2 flanking H2A-H2B dimers

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44
Q

organisation of nucleosomes

A
  • DNA passes directly from one nucleosome to next -> 10nm fibre
  • linker histones (e.g. H1) bind to DNA between nucleosomes
  • 30nm fibre is formed in vivo
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45
Q

In vitro experimental evidence that chromatin inhibits transcription

A

Experimentally:
RNA Pol II + transcription factors + naked DNA template -> transcription
RNA Pol II + transcription factors + chromatin template -> NO transciption

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46
Q

Chromatin inhibits…

A

transcription

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47
Q

Histone variants control chromatin structure

A

Histone Variants
- encode by genes that differ from highly conserved major types
- expressed at lower levels than conventional counterparts
- all but H4 have variants
- variants have novel structural and functional properties -> affects chromatin dynamics

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48
Q

post transcriptional modification of histones

A
  • could directly alter chromatin folding/structure
  • could control recruitment of non-histone proteins to chromatin
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49
Q

Enzymes for histone acetylation

A

acetylation mediated by HATs (Histone Acetyle Transferases)
acetylation readily reversed by HDACs (Histone Deacetylases)

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50
Q

Histone acetylation and transcriptional activation

A

High levels of acetylation = high levels of transcription
- direct influence on chromatin structure
- directs recruitment of bromodomain proteins

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51
Q

Histone methylation enzymes

A

methylation: Histone lysine methyl transferases
demethylation: lysine demethylation
- can add up to 3 methyl groups
- can’t be readily reversed by hydrolysis

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52
Q

histone methylation

A

doesn’t affect charge -> prob only has a minor effect on chromatin structure

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53
Q

the histone code

A

code telling the transcriptional state of DNA

54
Q

ATP-dependent chromatin remodelling

A
  • cells have multiple remodelling complexes
  • ## all have SNF2-related ATPase
55
Q

bromodomains bind…

A

acetylated lysines

56
Q

what can ATPase do to chromatin remodelling

A
  • sliding
  • unwrapping
  • eviction
  • spacing
  • histone variant exchange
57
Q

catalytic subunit of SWI/SNF

A

Snf2 or Swi2

58
Q

Snf2

A

related structurally to DNA helicase
- molecular motor: uses ATP to track alon DNA -> induce torsion -> disruption of histone-DNA interactions
- pushes DNA from a place where it isn’t accessible on the nucleosome to a place where it is

59
Q

ATP-dependent and HAT complexes co-operate

A
  • commonly are recruited to the same promoters
  • bromodomains in Snf2 help hold it onto acetylated nucleosomes
60
Q

Classical HDACs

A

class 1, 2, 4 enzymes
Zinc Dependent

61
Q

Class 3 HDACs

A

Sir2 family
NAD co-factor required

62
Q

NuRD complex

A
  • belongs to Mi2/CHD family
  • spaces out nucleosomes v regularly + tightly together -> stops transcription
63
Q

biochemical features of heterochromatin

A
  • hypoacetylation
  • specific H3 methylation
  • association of specific silencing factors
  • if acetylated, cannot be methylated (HP1 recognises methylation)
64
Q

HP1

A

chromodomain protein
- often recognises and binds to methylated lysine residues

65
Q

NF-kB =

A

nuclear factor of the kappa immunoglobin light chain in B cells

66
Q

NF-kB transcription factor pathway

A
  • allows response to external threats
  • regulating gene expression helps program responses to these threats -> call can survive + recover or induce death
67
Q

precursor of p50

A

p105

68
Q

precursor of p52

A

p100

69
Q

RHD

A

Rel homology domain
- encodes DNA binding and dimerization of NFkB

70
Q

E3 ubiquitin ligase

A

protein that facilitates the attachment of ubiquitin chains to a target protein

71
Q

NF-kB induced by

A
  • inflammatory cytokines
  • bacterial products
  • viral proteins and infection
  • DNA damage
  • cell stress
72
Q

NF-kB regulates

A
  • stress response
  • cell survival/cell death
  • cell adhesion
  • proliferation
73
Q

IkB

A

NF-kB inhibitor
IkB is phosphorylated upon activation

74
Q

IKK

A

phosphorylates IkB -> IkB is ubiquitinated and degraded -> NF-kB can translocate to the nucleus

75
Q

abberant activation of NF-kB pathway ->

A

many human dieases and inflammation

76
Q

conditions associated with hypoxia

A

high altitude disease
stroke/ischaemia
diabetes
chronic kidney diesaese
GI diseases
schizophernia
neurodegenerative dieseases
ageing
RA
cancer

77
Q

cell’s reaction to low O2

A
  • restoration of oxygen homeostasis
  • cell survival
  • cell death
78
Q

HIF =

A

Hypoxia Inducible Factor

79
Q

pathways controlled by HIF

A
  • oxygen supply
  • transcription
  • cellular metabolism
  • cell growth
  • HIF control
  • cell death
80
Q

p53

A

tumour suppressor and transcription factor

81
Q

p53 structure

A

typical domain structure: distinct binding and multimerisation domains

82
Q

negative regulator of p53 activity

A

mdm2

83
Q

DNA damage or stress ->

A

Mdm2 dissociation -> p53 activation -> cell cycle arrest -> allow for repair or apoptosis

84
Q

mechanism of mdm2 dissociation

A

DNA damage -> p53 phosphorylated at ser15 by ATM/ATR kinases
mdm2 is also phosphorylated -> disrupts interaction between p53 and Mdm2

85
Q

P14ARF=

A

tumour suppressor, induced by oncogenes when cell proliferation is increased

86
Q

role of ARF

A

disrupts interaction between p53 and mdm2
can inhibit mdm2 -> increased levels of active p53

87
Q

pre-mRNA

A

precursor to mRNA

88
Q

primary transcript processing

A

events coupled to transcription:
- capping
- splicing
- polyadenylation
- editing
via RNA pol 2 CTD

89
Q

cap and polyA tail

A

added post-transcriptionally
- not encoded in the genome

90
Q

m7G cap functions

A
  • protects mRNA from degredation by 5’->3’ nucleases
  • facilitates splicing
  • facilitates export from nucleus
  • critical for translation of most mRNAs
  • functions mediated through protein binding
    CBP80 in nucleus for processing/export
    eIF4 complex in cytoplasm for translating
91
Q

conserved sequences in introns

A

5’ and 3’ splice sites, branch sites
- sequences define the limits of exon and intron
- sequences recruit the splicing machinery needed to remove intron/join exons

92
Q

2 steps of intron splicing

A

2 trans-esterefication reactions
1. cut at 5’ splice site, create bond between 5’ end of intron and branch site
2. cut at 3’ splice sites -> release intron -> ligation of two exons

93
Q

spliceosome

A
  • enzymatic complex for intron removal
  • requires ATP
94
Q

snRNPs

A

= small nuclear ribonucleo-protein particles
- non coding RNA
- splicing is catalysed by snRNAs

95
Q

alternative splicing

A

expands the proteome - number of proteins is greater than the number of genes in the genome

96
Q

splicing is a ….. catalysed event

A

RNA

97
Q

polyadenylation

A

Nascent RNA -> endonuclease cleavage -> addition of As by polyA polymerase -> polyadenylated mRNA

98
Q

functional significance of the polyA tail

A

enhances RNA export
stabilised 3’ end of mRNA
enhances mRNA translation

99
Q

proteins required for polyadenylation bind sequences

A

CPSF: cleavage and polyadenylation specificity factor
CstF: cleavage stimulatory factor
polyA polymerase

100
Q

RNA editing

A

nucleotide alterations -> different/additional nucleotides in the mature RNA
- changes coding sequence/proterties of mRNA

101
Q

two classes of RNA editing

A

insertion/deletion
modification

102
Q

effects of mRNA editing

A

-creation of start (AUG) codons
-new open reading frames
-creation of stop codons

103
Q

N6-methyladenosine

A
  • most prevalent internal euk mRNA modifyer
  • regulated by writers/readers/erasers
104
Q

APOBEC-1 enzyme

A

edits mRNA-> cytidine deamination
- linked to cholesterol control, cancer dev, inhibits viral replication

105
Q

A to I editing in the Q/R site of glutamate receptors

A
  • L-glutamate = maj excitory NT
  • editing -> decrease in Ca2+ permeability of channels containing R version
  • editing carried out by ADAR2
106
Q

purpose of mRNA localisation

A
  • generate cell polarity
  • prevents expression in wrong place
  • promotes efficience of protein targeting
  • local control of translation
107
Q

prokaryote ribosome

A

70S

108
Q

eukaryote ribosome

A

80S

109
Q

ribosome catalyses…

A

peptide bond formation

110
Q

kozac sequence

A

ACCACCAUGG
- sequence before and after start codon

111
Q

eIF2B

A

regulator of translation
- subunit of eIF2, governs eIF2-GTP levels
- down regulated in response to stress

112
Q

eIF2 phosphorylation (stress)

A

essential amino acids/ER stress/ heavy metals ->eIF2-P -> eIF2B -> protein synthesis

113
Q

where is Fe found in the cell

A

heme and iron-sulfur clusters

114
Q

cannonical IRE

A

TfR1B

115
Q

IREs with additional 5’ or 3’ unpaired nucleotides

A

L-ferritin
DMT1
HIF-2a

116
Q

IRE =

A

Iron Response Element

117
Q

iron levels regulate…

A

production of iron binding proteins -> switch between iron import and storage

118
Q

IRP! binding to mRNA ->

A

blocks or activates translation
- also affects mRNA stability

119
Q

Reasons for degrading RNA

A
  • damaged RNA
  • badly processed mRNA
  • to control gene expression: rapidly alters expression by just turning it off
120
Q

casein mRNA

A

mRNA increases 70-fold when stimulated by prolactin (transcription only increases 2-fold)
- half-life increases dramtically in response to prolactin

121
Q

phase 1 of mRNA degradation

A

remove cap and polyA tail with decapping enzymes and deadenylases
break up the strand so exonucleases can work

122
Q

phase 2 of mRNA degradation

A

open ends -> very vulnerable to exonucleases
exonucleases break mRNA down from either end

123
Q

decapping enzymes

A

DCP1, DCP2

124
Q

endonucleases

A

argonaute, Swt1, Smg6

125
Q

deadenylases

A

Ccr/Not complex

126
Q

Exosome

A

main 3’ to 5’ exonuclease
multiple nuclease activities: RRP6, RRP44
- all other subunits function in RNA binding and unwinding

127
Q

XRN1

A

5’ to 3’ exonuclease
- also involved in transcription termination
- can’t work unless mRNA cap is removed

128
Q

NMD

A

= nonsense mediated decay
mistakes in RNA detected -> RNA targeted for degradation
- premature stop codons -> errors

129
Q

RNAi

A

RNA interference

130
Q

siRNA

A

small inhibitory RNA
- viral defense mechanism
- leads to degradation of the target RNA

131
Q

miRNA

A

micro RNA
- key gene regulatory mechanism
- leads to block in translation

132
Q

RISC

A

RNA induced silencing complex

Biomed year 2 sem 1 (7 decks)

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