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cmb2001 Flashcards

1
Q

what is gene expression

A

process by which information in genes is decoded into proteins
DNA transcribed to RNA which is translated into proteins

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2
Q

what is transcription

A

transfer of genetic information from dsDNA to ssRNA (mRNA)

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3
Q

what are promoters

A

cis acting DNA regulatory element through which transcription is initiated and controlled

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4
Q

eukaryotic promoters

A
  • core promoter elements - cpg islands
  • ten intitation occurs at lower rate and at several start sites
    associated with regions with a high frequency of CG sequences cpg islands
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5
Q

UAS and enhancer

A

activators binding sites

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6
Q

URS and silencer

A

repressor binding sites

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7
Q

CpG islands

A
  • in mammals most C residues followed by G are methylated
  • generally C residues in CpG islands escape methylation in mammals
  • methylation of CpG islands is associated with silencing (txn switched off)
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8
Q

tools for identifying promoter elements

A
  • sequence comparison
  • reporter analysis
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9
Q

what is sequence comparison

A
  • identification of the TATA box
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10
Q

what is reporter analysis

A
  • reporter genes encode proteins whose levels can easily be measure
  • e.g. GFP, luciferase, lacZ
  • amount of reporter protein provides a measure of gene expression
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11
Q

what can reporters be used to identify

A
  • where a gene is expressed
  • when it is expressed
  • what signal it responds to
  • what factors and sequences control its expression
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12
Q

general transcription factors

A
  • bacterial RNA polymerase requires sigma-factor to recognise promoter DNA
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13
Q

what does the job of sigma factor in eukaryotes

A
  • RNA pol specific
  • multi component factors
  • form a complex on TATA box
  • recruit RNA pol II to the promoter
  • direct initiation at start site
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14
Q

steps of transcription initiation by RNA pol II

A
  • helicase activity of TFIIH separates the template strand at the start site - requires ATP hydrolysis
  • as pol II begins transcribing it is extensively phosphorylated on the C terminal domain
  • TFIID TFIIA may stay behind
  • TFIIB, TFIIE, TFIIH are release
  • TFIIF moves down the template with pol II
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15
Q

what is the Carboxyl terminal domain

A
  • a series of repeats located at the C terminal end of the largest pol II
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16
Q

function of TFIID

A
  • binds to the TATA box
    recruit TFIIB
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17
Q

function of TFIIA

A
  • stabilises TFIID binding
  • anti repression function
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18
Q

function of TFIIB

A
  • recruits pol II - TFIIF
  • important for start site selection
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19
Q

function of TFIIF

A
  • stimulates elongation
  • destabilises non-specific RNA pol II - DNA interactions
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20
Q

function of TFIIE

A
  • recruits TFIIH and modulates its activity
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21
Q

function of TFIIH

A
  • promoter melting and clearance - enzyme XPB
  • CTD kinase activity
  • DNA repair and coupling
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22
Q

2 parts of TFIIH

A
  • core and CAK
  • CAK contains a kinase that phosphorylates the CTD of RNAP II
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23
Q

TFIID

A
  • TATA binding protein + TBP associated factos
  • TBP is the central subunit of TFIID
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24
Q

TBP vs TFIID

A
  • TBP can direct the assembly go the PIC on a TATA-containing promoter
    TBP alone cannot direct PIC assembly on a TATA-less promoter
  • TBP cannot support activated transcription
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25
Q

TAFs

A
  • promote the interaction of TFIID with the basal promoter
  • interact with activators
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26
Q

core promoter

A
  • consists of the region around the transcription start site
  • associated with elements such as the TATA box and the initiator element
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27
Q

enhancer

A
  • DNA regions close or far from the start site
  • binding sites for activator protein
  • often composed of multiple UAS elements
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28
Q

silencer

A
  • DNA regions close or far from the start site
  • binding sites for repressors
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29
Q

general transcription machinery

A
  • a set of factors that recruit RNA pol II to the promoter and direct initiation at the start site
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30
Q

pre-initiation complex

A
  • assembly of the basal machinery at the core promoter
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31
Q

activator

A
  • a factor that binds the gene specific regulatory sequences and stimulates transcription initiation
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32
Q

basal transcription activated txn

A
  • the level of transcription from a core promoter
  • increased levels of transcription mediated by an activator protein
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33
Q

classes of enhancer elements

A
  • common sequence elements e.g. GC box, octamer, CAAT box
    bind activations that are relatively abundant in the cell and constitutively active
  • response elements e.g. SRE HSE. bind factors whose activity is controlled in response to specific stimuli
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34
Q

combinatorial control of transcription

A
  • the type and combination of elements dictates when and at what level a gene is transcribed
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35
Q

how do activators contact the basal transcription machine

A
  • tracking
  • looping
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36
Q

types of activation domains

A
  • acidic patch - clusters of negative charged residues e.g. vp16
  • glutamine rich e.g. sp1
    proline rich e.g. jun
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37
Q

analysis of activators (in vitro)

A
  • DNA foot printing
  • electrophoretic mobility shift assay
  • transcription assay
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38
Q

what is an electrophoretic mobility shift

A
  • activator + radiolabelled probe DNA –> run on non-denaturing acrylamide gel
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39
Q

what are transcription assays

A
  • RNA pol II + GTPs + DNA template + radiolabelled rNTPs
  • requires the activator to both have a functional DNA binding domain and a function activation domain
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40
Q

analysis of activators (in vivo)

A
  • reporter assays
  • chromatin immunoprecripitation
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41
Q

how does chromatin immunoprecipitation work

A
  • cross link bound proteins to DNA
  • isolare chromatin and shear DNA
  • precipitate chromatin with protein-specific antibody
    reverse cross-link and digest protein
  • analyse DNA using - PCR or sequencing
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42
Q

how do activators work

A
  • promoter binding of an additional activators
    stimulate complex assembly
    release stalled RNA polymerase
  • modulation of chromatin
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43
Q

how to activate stalled RNA pol II

A
  • heat shock genes such as hsp70
  • heat shock activates HSF transcription factor which interacts with RNA pol II and release it from the pause after 50nts
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44
Q

which transcription factors are involved in recruitment

A
  • TFIID
  • TFIIB
  • mediator
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45
Q

what is the function of mediators

A
  • provides a bridge between activators and RNA pol II
  • activator interactions aid recruitment of RNA pol II and therefore enhance PIC formation
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46
Q

general role of activators

A
  • can promoter the binding of additional activators
  • can increase the rate of PIC formation
  • may stimulate post recruitment steps
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47
Q

function of chromatin

A
  • to compact DNA
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48
Q

what is the composition of chromatin

A
  • composed primarily of histones
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49
Q

what are the two types of histones

A
  • core histones
  • linker histones
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50
Q

what are nucleosomes

A
  • DNA wrapped twice around an octamer of histone proteins
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51
Q

what is an octamer

A
  • central H3-H4 tetramer and 2 flanking H2A-H2B dimers
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52
Q

function of linker histones

A
  • H1 bind to the DNA between nucleosomes
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53
Q

3 pieces of evidence that chromatin inhibits transcription

A
  • in vitro reconstitution experiments
  • in vivo nucleosome positioning experiments
  • genetic studies of saccharomyces cerevisae
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54
Q

how does the in vitro reconstitution experiment prove that chromatin inhibit transcription

A
  • RNA pol II + transcription + chromatin template –> no transcription
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55
Q

what are the roles of nucleosomes in the nucleus

A
  • compaction of DNA
  • forms a template for DNA transcription
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56
Q

3 main mechanisms for modulating the structure of chromatin

A
  • histone variants
  • post transcriptional modification of histones
  • ATP dependent chromatin remodelling
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57
Q

histone variants

A
  • all except H4
  • histone variants confers novel structural and functional properties of the nucleosome which affect the chromatin dynamics
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58
Q

post translational modification of histones

A
  • acetylation
  • methylation
  • ubiquitylation
  • phosphorylation
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59
Q

consequences of histone modification

A
  • directly alter chromatin folding/structure
  • control the recruitment of non-histone proteins to chromatin
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60
Q

how are histone acetyl transferases recruited

A
  • activators recruit HATs to specific promoter
  • some HATs are part of the general transcription machinery
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61
Q

how does acetylation mediate transcriptional activation

A
  • direct influence on chromatin structure
  • directs the recruitment of bromodomain proteins
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62
Q

why are bromodomains important

A
  • specific acetylates lysine residues are recognised by proteins with bromodomains
  • bromodomain proteins often promoter transcription
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63
Q

2 examples of bromodomains

A
  • BDF1 - binds acetylated H4 and recruits TFIID
  • TAFII250 - TFIID subunit, also binds acetylated H4
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64
Q

how does histone methylation occur

A
  • histone methylation can occur on lysine
  • methylation does not affect charge so minor influence on chromatin structure
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65
Q

importance of lysine methylation

A
  • specific methylated lysines are recognised by specific proteins
  • methyl-lysine residues can function either as activating or repressing marks
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66
Q

enzyme involved in ATP dependent chromatin remodelling

A
  • SNF2-related ATPase
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67
Q

steps of ATP dependent chromatin remodelling

A
  • sliding
  • unwarpping
  • eviction
  • spacing
  • histone variant exchange
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68
Q

how does SWI/SNF remodel chromatin

A
  • catalytic subunit is snf2
  • hydrolyses ATP in the presence of DNA or nucleosome
  • uses energy from ATP hydrolysis to track along DNA and induce torsion
  • this results in disruption of histone DNA interactions and movement of the nucleosome
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69
Q

roles of SWI/SNF complexes

A
  • cell cycle control via interaction with rb and cyclin E
  • in development, deletion in mice results in embryonic lethality
  • tumour suppressor pathways; mutations are associated with a variety if tumour types
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70
Q

role of SWI/SNF in cancer

A
  • the tumour suppressor activity of the SWI/SNF complexes most likely due to roles in facilitating transcription factor function
71
Q

chromatin modifying factors in repression of transcription

A
  • histone deacetylases
  • ATP dependent remodellers
  • histone methylases
72
Q

HDAC co-repressor complexes

A

recruited to promoters by interaction with site-specific DNA binding proteins
e.g. SIN3 co-repressor complexes

73
Q

what is the role of ATP dependent remodelling complexes

A

mediate transcriptional repression

74
Q

two types of chromatin

A
  • euchromatin - gene rich, potential to be transcribed
  • heterochromatin - gene poor, repetitive regions, transcriptional silencing
75
Q

3 features of heterochromatin

A
  • hypoacetylation
  • specific histone H3 methylation
  • association of specific silencing factors
76
Q

function of HP1 and heterochromatin

A
  • binding of HP1 is thought to compact nucleosomal arrays
  • act as platform form the recruitment of further activities that prevent recruitment
77
Q

heterochromatin - X-chromosome inactivation

A
  • females have 2 X chromosomes one of which is inactivation
  • this equalises the number of X linked genes expressed in males and females
  • the inactivated X-chromosome is seen in the nucleus as a condensed structure that is assembled into a specific form of heterochromatin
78
Q

what is a Barr body

A
  • inactivated X chromosome in the nucleus as a condensed structure
  • formation of the Barr body is controlled by non-coding RNAs Xist and Tsix
79
Q

role of NF-kappa B

A
  • allows the cell to respond to external challenges
  • regulates expression of target genes to help programmed the response, to allow cells to survive or recover
80
Q

what is the NF-kappa B family

A
  • the real homology domains encodes the DNA binding and dimerisation functions of NF-KB
  • p50 and p52 are proteolytically processed from their precursor proteins p105 and p100
  • p100 and p105 contain ankyrin repeats in their c-terminal that allow them to function as IKB-like inhibitors
  • TA1/TA2, TAD, SD1, SD2 - non conserved transcriptional activation domains
  • LZ - leucine zipper like domain
81
Q

describe the ubiquitin - proteasome pathway

A
  • E3 ubiquitin ligase facilitates the attachment of ubiquitin chains to a target protein
  • this pathway is protein degradation
  • Ub is conjugated to protein that are destined for degradation by an ATP-dependent process
  • 5 ub molecules attach to the protein substrate
  • ub is removed and the protein is linearised and injected into the central core
  • the in proteasome the protein is digested to peptides
  • peptides to amino acids by peptidases
  • used in antigen presentation
82
Q

why is NF-KB induced

A
  • inflammatory cytokines
  • bacterial products
  • viral proteins and infection
  • DNA damage
  • cell stress
83
Q

what does NF-KB regulate

A
  • the immune and inflammatory response
  • stress response
  • cell survival and cell death
  • cell adhesion
  • proliferation
84
Q

activation of NF-KB

A
  • when the cell is stimulated, IKB is phosphorylated by ubiquitination
  • NF-KB is released
  • NF-KB translocated to the nucleus
85
Q

enzymes involved in canonical pathway

A
  • TNF
  • IL-1
  • LPS
86
Q

enzymes involved in non-canonical pathway

A
  • LPS
  • CD40
  • Lymphotoxin receptors LMP1
87
Q

responses to infection regulation by NF-KB

A
  • inflammation
  • proliferation
  • survival
  • tumour promotion and metastasis
  • angiogenesis
  • cell death and anti-proliferative effects
88
Q

how are beta interferons produced

A
  • viral infections
  • spacing and orientation sites allows for appropriate protein-protein contacts
89
Q

what is the role of activators

A
  • transcription factors binding at the beta interferon enhancer all interact to form the enhanceosome complex
90
Q

role of the enhanceosome complex

A
  • forms an interaction interface to allow the high affinity recruitment of transcriptional cofactors such as p300 and cap
91
Q

what are coactivators interactions weak

A
  • favour the formations of coactivator complexes only at the promoters and enhancers
92
Q

what activators are present at the beta IFN enhancer

93
Q

what levels of regulation of NF-KB give transcriptional specificity

A
  • stimulus e.g. TNF or IL1
  • phosphorylation and degradation of IKB alpha, beta or E
  • translocation of NF-KB to the nucleus modification of NF-KB subunits
  • DNA binding and gaining access to the promoter/enhancer
  • transactivation
  • specific transcriptional response
94
Q

what is transactivation

A

interaction with the basal transcription complex and co activators

95
Q

purpose of ubiquitination

A
  • when proteins undergo ubiquitination they can be targeted for degradation by the proteasome
96
Q

summarised function of NF-KB

A
  • NF-KB complexes are held in an inactive form
  • bound to an inhibitory protein
  • once phosphorylated by the IKK complex it becomes ubiquinated and degraded
  • NF-KB is free to translocate to the nucleus
97
Q

what is the first step of the transcription response

A
  • shows how the degradation of different IKB proteins can lead to activation of different NF-KB dimers
98
Q

what happens to NF-KB in the nucleus

A
  • subject to regulation
  • by post translational modifications
  • or by interactions with nuclear transcriptional regulators
99
Q

role of beta interferon

A

role in antiviral response, inhibited by cars cov2

100
Q

what is the enhanceosome

A
  • creates a landing and for transcriptional regulators such as p300/cbp
  • can lead to beta interferon gene transcription
101
Q

what is hypoxia

A
  • the lowering of the oxygen. concentration compared to normal levels cells are exposed to
102
Q

what is the medical relevance of hypoxia

A
  • high altitude diseases
  • cancer
  • rheumatoid
  • ageing
  • neurodegenerative diseases
  • schizophrenia
  • gastrointestinal disease
  • chronic kidney disease
  • diabetes
  • stroke/ischemia
103
Q

consequences hypoxia

A
  • translational block
  • transcriptional program
  • chromatin structure changes
  • microRNA signature
  • DNA replication block
104
Q

what are the cellular responses to hypoxia

A
  • restoration of oxygen homeostasis
  • cell survival
  • cell death
105
Q

what are the different hypoxia inducible factors

A
  • HIF 1alpha - ubiquitously expressed in all tissues
  • HIF 2alpha - expression restricted in certain tissues
  • HIF 3alpha - lacks c-terminus. functions as a dominant negative inhibitor for HIF-1alpha and HIF-2alpha
106
Q

how is HIF-1alpha regulated in normoxia

A
  • under normoxia, proline hydroxylases and FIH inhibiting HIF enzymes use oxygen to hydroxylate key residues within the HIF-1alpha subunit
  • hydroxylation of the ODDs signals for the VHL binding and ubiquitination
  • leads to proteasomal degradation
107
Q

how is HIF-1alpha regulated in hypoxia

A
  • PHDs and FIH are inhibited
  • HIF 1alpha is stabilised
  • HIF 1alpha dimerises with HIF 1beta
  • activate target gene transcription through recruitment of co-activators
108
Q

what are the targets of HIF

A
  • oxygen supply
  • transcription
  • cellular metabolism
  • cell death
  • HIF control
  • cell growth
109
Q

structure of p53 tumour suppressor

A
  • trans - transactivation domain
  • p - proline rich domains
  • NLS - nuclear localisation sequence
  • tet - tetramerization domain
110
Q

what is the regulation pathway of p53 pathway

A
  • p53 is inactivated by its negative regulators mdm2
  • when DNA is damaged, p53 and mdm2 complex dissociates
  • p53 then induces cell cycle arrest
111
Q

role of mdm2

A
  • is an e3 ubiquitin ligase
  • major role is ubiquitination of p53, leading to degradation
  • when DNA is damage it becomes phosphorylated
  • over expression inactivates p53, preventing apoptosis
112
Q

role of ARF

A
  • p14arf is a tumour suppressed
  • induced by oncogene
  • disrupts interaction between the p53 and mdm2
  • inhibits ubiquitin ligase
  • increased levels of transcriptionally active p53
113
Q

what is LFS syndrome

A
  • hereditary conditions
  • cancer risk passed from generation to generation
  • mutation in TP53 gene
114
Q

how is eukaryotic gene expression regulated

A
  • transcription control
  • RNA processing control
  • translational control
  • protein activity control
115
Q

what is the primary transcript process

A
  • events coupled to transcription via the RNA pol ii CTD which acts as a landing pad
  • capping
    splicing
  • poly adenylation
  • editing
116
Q

what are the steps of the 5’ m7G cap synthesis

A
  • RNA initially contains triphosphate at 5’ end
  • capping
  • methylation alters chemical behaviour of bases
117
Q

functions of the m7G cap

A
  • protects mRNA from degradation by 5’-3’ nucleases
  • facilitate splicing
  • facilitates export from the nucleus
  • functions mediated through protein binding
118
Q

essential proteins for translation

A
  • CBP80/CBP20 in nucleus - processing/export
  • eIF4 complex in cytoplasm - translation
119
Q

function of 5’ cap

A
  • capping linked to transcription
  • the cap is a protein binding element
120
Q

what are the conserved sequences in introns

A
  • 5’ splice site
  • 3’ splice site
  • branch site
121
Q

what are the step of splicing

A
  • 2 trans-esterification reaction
  • cut at 5’ splice site
  • creation of bond between the 5’ end of intron and branch site
  • cut at 3’ splice site to release intron lariat
  • ligation of two exons
122
Q

what is the spliceosome

A
  • enzymatic complex that catalyses the removal of introns
  • requires ATP
123
Q

proteins included in the spliceosome

A
  • RNA binding proteins
  • ATPases
  • GTPases
124
Q

function of anti-sm

A
  • anti-sm antibodies react against the sm proteins
  • present in lupus
125
Q

how is the alternative splicing mechanism regulated

A
  • activators - binds to intronic and exonic splicing enhancers (ISE & ESE)
  • repressors - binds to intronic and exonic splicing silencers (ISS & ESS)
126
Q

diseases caused by mutations in splicing

A
  • spinal muscular atrophy - common in genetic cause of infant mortality
  • retinitis pigmentosa - reduced visual capabilities and blindness
  • myotonic dystrophy - a muscle wasting disease
127
Q

what is polyadenylation

A
  • addition of poly A tail to end of mRNA
  • endonuclease cleavage
  • splicing of AAUAAA
  • G/U rich tract just downstream of polyA site
128
Q

what are the proteins required for polyadenylation

A
  • cleavage and polyadenylation specificity factor - binds AAUAAA
  • cleavage of stimulatory factor - binds G/U
129
Q

function of polyA tail

A
  • enhances export of RNA
  • stabilises 3’ end of mRNA
  • enhances translation of mRNA
130
Q

what are the two classes of RNA editing

A
  • insertion/deletion
  • modification
131
Q

what is the significance of RNA editing in medicine and development

A
  • disease - atherosclerosis
  • brain function
  • development
  • parasites
132
Q

what are the effects of mRNA editing

A
  • creation of start/stop codons by U insertions
  • creation of start/stop codons by C to U changes
  • creation new open reading frames by nucleotide insertions
  • changes in encoded amino acids and splice site choice by base conversion
  • removal of stop codons by base conversions
133
Q

how is RNA edited by deamination

A
  • deaminated adenosine is inosine
  • inosine is similar to guanosine
134
Q

what enzyme is involved in cytidine deamination

A
  • pre mRNA editing carried out by the APOBEC-1 enzyme
135
Q

importance of A to I editing

A
  • decrease in ca2+ permeability of channels containing the R version
  • editing carried out by ADAR2
136
Q

importance of nuclear pore

A
  • RNA is an acid (hydrophobic) so need help getting out of the nucleus
137
Q

importance of localised mRNA

A
  • localised protein synthesis -
  • generate cell polarity
  • prevents expression in the wrong place
  • promotes efficiency of subsequent protein targeting
  • local control of translation
138
Q

importance of localisation of dendritic mRNA

A
  • synaptic proteins produced at the synapse, which gives us synaptic plasticity and spine morphogenesis
139
Q

how does localisation effects diffusion

A
  • mRNAs are local entrapped by anchor proteins in the cytoplasm
  • local entrapment
    -anchor proteins at the site where you want them
140
Q

which transport mechanisms are used to localised mRNAs

A
  • active transport
  • passive transport
141
Q

what are the step in catalysing aminoacy-tRNA syntheses

A
  • amino activation
    amino acid and ATP bind catalytic site, nucleophilic attach by alpha carboxylic acid, oxygen yielding aminoaceyl-adenylate
  • hydroxyl group of adenine 76 to tRNA attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP
142
Q

steps of translation

A
  • peptide bond formation is catalysed by the ribosome
  • tRNAs deliver the amino acids
  • tRNA are present in the P and A sites - the expanding polypeptide chain is attached to the p-site tRNA
143
Q

how is eukaryotic translation initiated

A
  • small subunit binds the CAP, moves to the first AUG, encoding the initiating methionine of the protein
  • most frequently found in the Kozak consensus sequence
144
Q

what are the 5 factors in translation initiation

A
  • eIF1A - met-tRNA binding to 40s
  • eIF3 - 80s dissociation, binds many other eIFs
  • eIF1- AUG recognition
  • eIF2 - GTPase, met-tRNA binding, binds eIF5
  • eIF5 - stimulates eIF2 GTPase, GAP for eIF2
145
Q

what is 43s association

A
  • interaction - eIF3 with eIF4G
  • RNA unwinding - eIF4F unwinds cap-proximal sequence
146
Q

what is scanning

A
  • 5’ proximal AUG used
  • mutation of natural AUG leads to use of next one
  • mutation of initiator tRNA leads to use of next one
  • requires eIF4F
146
Q

what are the final step of translation initiation

A
  • eIF2 needs to be recycled to generate ternary complex for further initiation event
  • eIF5B - GTPase that promoted sub-unit joining
147
Q

what are the final steps of eukaryotic translation

A
  • translocation required to move the tRNAs and mRNA through the ribosome
  • when reach termination codon, translation stop and the ribosome dissociates
148
Q

what are the key regulation points of translation

A
  • formation of eIF4F
  • 43S binding
  • function of eIF2B/ternary complex formation
148
Q

what are the key points of eIF2B

A
  • present at much lower levels than eIF2
  • its activity governs levels of active eIF2-GTP
  • down regulated in responses to stresses
  • regulation through phosphorylation of eIF2, competitive inhibitors
149
Q

what are eIF2 kinases

A
  • PKR - activated by double stranded RNA
  • PERK - a mediator of the unfolded protein response
  • GCN2 - a regulator of translation in response to amino acid availability
  • HRI - linking global availability to protein synthesis
150
Q

what is PKR

A
  • antiviral defence strategy
  • increased when cells are exposed to interferons
  • when PKR binds dsRNA it dimerises and is activated
151
Q

how is iron metabolism regulated

A
  • regulated by the expression of fe-storage/transport proteins
152
Q

where are iron response elements found

A
  • found in the 5’ or 3’ UTRs of iron regulated mRNAs
  • bound by iron regulatory proteins, IRP1 IRP2
153
Q

function of IRP1

A
  • binding can block or activate translation
  • binding also affects mRNA stability
154
Q

where is there RNA degradation

A
  • damaged mRNA
  • incorrectly transcribed/ processed mRNA
  • control gene expression
156
Q

what is phase 1 of mRNA degradation

A
  • decapping enzymes
  • endonucleases
  • edadenylases
  • initiate breakdown of the RNA
156
Q

why is casein mRNA important

A
  • increases half life in response to prolactin
  • polyA tail length increases
  • 3’ UTR of RNA binds proteins which aid stabilisation
157
Q

phase 2 of mRNA degradation

A
  • the exosome - the main 3’ to 5’ exonuclease in the cell - involved in RNA turnover and processing
  • XRN1 - functions after decapping of the mRNA
158
Q

what causes deadenylation-dependent decay

A
  • mechanisms whereby all mRNAs gradually lose their polyA tails
159
Q

what causes deadenylation-independent decay

A
  • auto regulation - rps288 binds its own message
  • edc3 is an activator of decapping enzymes
  • nucleases targeting specific substrate
  • PMR1 cleaves albumin mRNA
160
Q

what is nonsense mediated decay

A
  • where mistakes in the RNA are detected, RNA is targeted for degradation
161
Q

what do premature stop codons result from

A

errors in:
- transcription
- splicing
- editing
- polyadenylation
- mutations

164
Q

what is the nonsense mediated decay mechanisms

A
  • EJCs are removed from the mRNA by the ribosome
  • once the ribosomes reach the PTC, and EJC remains downstream, specific factors interact with the RNA degradation machinery
  • process is known as surveillance
165
Q

miRNA vs siRNA

A
  • siRNA - complimentary to target RNA, viral defence mechanism, leads to degradation of the target RNA
  • miRNA - not complimentary, regulatory mechanism, leads to block in translation
166
Q

importance of 3’ UTR changing length

A
  • during embryonic development 3’ UTR get longer
  • mRNA proliferating cells get longer
  • longer 3’ UTR has more possibilities of binding sites for miRNAs
167
Q

what are the clinical uses of siRNAs

A
  • fitsuiran - lowers antithrombin, reduces bleeding in haemophiliacs
  • STP705 - knocks down both TGF-beta1 and COX-2 gene expression
168
Q

what is miRNA used for

A

translational regulation

169
Q

what are siRNAs used for

A

target mRNAs for cleavage
important tools for manipulating gene expression

CM2001 (13 decks)

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