Eukaryotic Transcription And Control Of Gene Expression Flashcards
1
Q
What is gene expression
A
Process by which information from a gene is used in the synthesis of a functional product
2
Q
What processes does protein-coding gene functional products need
A
Transcription, mRNA processing, translation and post-translational modification
3
Q
What processes does non-coding gene functional RNA need
A
Ribosomal RNA (rRNA): translation Transfer RNA (tRNA): translation Small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA): RNA processing MicroRNA (miRNA) and long non-coding RNA (lnRNA): regulation of gene expression
4
Q
What are the products of gene expression responsible for
A
Structure (cytoskeleton, membranes, cell wall)
Biochemical reactions (catabolism and anabolism)
Cellular and intercellular communication
Gene expression
Other things cells do
5
Q
When can gene expression be controlled and how
A
Transcription- which genes are copied into mRNA measures by transcriptome analysis
Co-transcription/ mRNA processing- alternative splicing giving different forms of a protein
Post-transcription- mRNA stability and translation efficiency determines how much protein is made from each mRNA
Post-translational- covalent modifications, localization and degradation alter protein function or abundance
Inc-RNA- multiple stages
6
Q
Why is controlling gene expression important
A
Development- all cells in multicellular organisms have the same DNA but look and function differently because different cell types make different genes. Can be altered when expression isnt properly controlled.
Cancer= loss of gene expression
Environment- gene expression changes in response to the environment and allows organisms to adapt to different enviro conditions
Biotech/ medicine- altering gene expression can be used to alter characteristics
7
Q
Where can transcription occur in eukaryotic cells
A
Nucleus
Nucleolus
Mitochondria and chloroplasts
8
Q
Why does transcription occur in mitochondria and chloroplasts
A
From endosymbiosis… mitochondria and chloroplasts came from engulfing bacteria
9
Q
Why is the nucleolus dark
A
From the amount of transcription of rRNA occurring
10
Q
What does RNA polymerase I produce and where is it located
A
Most rRNA in the nucleolus
11
Q
What does RNA polymerase II produce and where is it located
A
mRNA, snoRNA, some snRNA, miRNA and lncRNA in the nucleoplasm
12
Q
What does RNA polymerase III produce and where is it located
A
tRNA, 5s rRNA, some snRNA and other small nRNAs in nucleoplasm
13
Q
What does RNA polymerase IV and V produce and where is it located (plants only)
A
Regulatory ncRNA in nucleoplasm
14
Q
What are the RNA polymerases in mitochondria and chloroplasts and what RNAs do they make
A
Nuclear encoded polymerase (NEP) in mitochondria and chloroplasts make rRNA, tRNA and mRNA
Plastid encoded polymerase in chloroplasts make rRNA, tRNA and mRNA
15
Q
What are the substrates required for RNA transription
A
Nucleotriphosphates (NTPs) ATP, GTP, CTP and UTP
16
Q
Which DNA strand is complementary to the RNA transcript
A
Template strand
17
Q
What end are new nucleotides added to
A
3’ end of RNA transcript
18
Q
What are nucleotides joined together by
A
Phosphodiester bonds
19
Q
Structure of RNA polymerase II
A
Multiple subunits (Rpb#)
Catalytic subunits Rpb1 and Rpb2 related to prokaryotic RNA pol catalytic subunits
Additional subunits required for stability and transcription through chromatin in eukaryotes
20
Q
How does RNA polymerase II work
A
DNA enters cleft and is unwound
Clamp is closed when active
Wall bends the template into active site
NTPs enter the pore
Template is exposed to NTPs in the catalytic site. When correct NTP binds a new phosphodiester bond forms
Bridge changes conformation and pushes paired nucleotide from active site
DNA/RNA hybrid helix formed which is 8-9bp long from active site= stability
Rudder separates DNA and RNA and RNA transcript leaves the exit
21
Q
How does proof reading from RNA polymerase II occur
A
A mismatch is detected due to tension in DNA/RNA hybrid helix= less stable
Pol II moves backwards until mismatch is in the pore
Nuclease activity cleaves/ cuts out mismatch and the mismatch and following RNA degrades
Transcription resumes
22
Q
What is the RNA polymerase II C-terminal domain (CTD)
A
C-terminal domain of Rpb1 subunit. Not structured and not catalytic. Heptapeptide repeat (multiple copies of same seven amino acid sequence)
23
Q
What is the CTD heptapeptide sequence
A
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
24
Q
What is the function/ what happens to the polymerase II CTD
A
Reversibly phosphorylated during transcription
Ser 2 and Ser 5- kinase adds phosphate group, phosphatases remove phosphate group
Different phosphorylation patterns alter interactions with other proteins as CTD interacts with proteins. Addition of phosphates= protein interaction. Removal= no protein interaction
25
What does RNA polymerase II need help with
To recognise and bind DNA, especially when it is packaged into chromatin
26
What are the important parts of the protein-coding gene for RNA polymerase
Core/ proximal promoter- where RNA poll II is recruited to. Close to TSS (transcription start site)
Cis-regulatory element (CRE)- short DNA sequences involved in regulation of transcription (cis= same molecule). Can be distal to core promoter
27
What are the two types of CREs
Enhancers- increase transcription
| Silencers- reduce transcription
28
Features of nucleosomes
DNA coils around histones to form nucleosomes. 2x H2A/H2B dimers and 2x H3/H4 dimers
N terminal tails stick out
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What are ‘writers’ and ‘readers’
Writers- add/ remove marks on histone tails
| Readers- recognise marks on histone tails
30
What is acetylation (histone tail modification)
Acetylation (ac)- acetyl group added
Writers: added, histone acetyltransferases (HATs), removed histone deacetylases (HDACs)
Modifies lysine residues
Functions: removes positive charge from lysine and recruits specific proteins/ readers
31
What is methylation (histone tail modification)
Methylation (me)- methyl groups added- one two or three
Writers: added histone methyltransferases (HMTases), removed histone demethylases
Modifies lysine and arginine residues
Functions: recruit specific proteins/ readers- different patterns and amounts recruit different readers CHARGE REMAINS ON LYSINE
32
What are chromatin remodellers
ATP dependent
Disrupt interacts between DNA and histones and make changes to the chromatin
Work in conjunction with histone chaperones
Number of complexes in eukarytotes
33
What different modifications can chromatin remodellers do
Nucleosome assembly
Nucleosome sliding
Nucleosome eviction (removes a whole nucleosome)
Unwrapping
Dimer replacement
Dimer eviction (removes some of the histones eg H1 and H2)
34
Stages of transcription and other things that happen
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Pol II recruitment
Initiation and early elongation
Productive elongation
Termination
Other things: CTD changes phosphorylation states, RNA pol II receives help and pre-mRNA processing
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35
How do activators work to recruit RNA pol II
Are transcription factors- recognise and bind a CRE based on DNA sequence to initiate/ increase transcription. Multiple activators normally bind to the promoter of each gene
36
What are the two activator domains
DNA binding domain- binds cis regulatory element, hold the activator domain in the vicinity of the promoter
Activation domain- protein to protein interactions, other activators and co-activators
37
Repressors and RNA pol II recruitment
Repressors also bind a CRE (silencer) but reduce transcription
Repressor is similar but has a repressor domain that recruits co-repressors instead of an activation domain
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What are co-activators and their functions
Often protein complexes
Protein-protein interactions: range of activators (many genes) and general transcription factors/ RNA pol II
Chromatin modifications
39
How is the core protein made accessible to RNA pol II
Co-activators acetylate histone H3 and H4 and may make other histone tail modifications eg mathylation
Nucleosome remodellers recruited to acetylated H3/H4 and make interactions with activators/ coactivators
Nucleosome remodellers slide or evict nucleosomes (fully for partially by removing H2A and H2B dimers
40
How is the preinitiation complex formed
Consists of general transcription factors (GFTs) names TFII letter
Recruits RNA pol II to TSS
Formation occurs in different orders- TFIID normally recruited first and binds to DNA sequences in core promoter eg TATA box
TFIIA and B bind early- stabilises TFIID and prepares for RNA pol II
TFIIF binds to RNA pol II and brings to complex
TFIIE helps recruit TFIIH
TFIIF, RNA pol II, TFIIE and TFIIH may be recruited together as a holoenzyme
41
How does initiation occur
PIC formation and RNA polymerase is recruited
Initiation: TFIIH helicase activity opens transcript bubble and GFTs help feed template strand into RNA pol II
Abortive initiation: RNA pol II unstable, multiple short transcripts produced (<12bp) and transcription bubble is distorted
Promoter clearance occurs
42
How does promoter clearance work and why
To become stable RNA pol II
Adopt active conformation: clamp closure ~3nt. DNA/RNA hybrid helix forms with TFIIB going into RNA pol II channel to help stabilisation ~10nt
Breaks contact with GTFs: TFIIH kinase phosphorylates Ser5 of CTD which alters interactions with GFTs (TFIIF). DNA/RNA hybrid helix formation pushes TFIIB from exit channel
Promoter clearance complete when transcription bubble collapses and RNA pol II is stable and moves away
43
What does pre-mRNA processing involve
Addition of 5’cap
Addition of poly-A tail
Splicing of introns
44
Why does pre-mRNA need to be processed
Protection- RNA unstable and exonuclease with degrade exposed 5’ or 3’ end
Introns- many protein-coding genes contain introns that need to be removed/ spliced from mature mRNA
45
What happens in the addition of 5’ m7G cap (3 parts)
1. Uncapped mRNA will be degraded from 5’ triphosphate from first NTP
2. Capping enzyme recruited by Ser5P of CTD, triphosphatase activity cleaves 5’ phosphate, guanylyltransferase activity uses GMP to add an inverted G, = stable but poor translation efficiency
3. RNA methyltransferase (RNMT) recruited by Ser5P of CTD, methylates N7 of guanine base (m7)= stable and high translational efficiency
Occurs early in transcription (pre-mRNA virtual)
46
How does early elongation move to productive elongation
P-TEFb (positive transcription elongation factor) complex kinase subunit phosphorylates Ser2 of CTD just after capping, mRNA <100nt
47
How does productive elongation work
PTEFb phosphorylation recruits elongation factors that help RNA pol II in many ways and pre-mRNA processing (splicing)
48
How do elongation factors (EFs) help RNA pol II in productive elongation
Reach full processivity/ stability
Transcribe DNA in chromatin- EFs have similar roles to co-activators at the core promoter, HAT activity H3/H4 acetylation which loosens and recruits readers, nucleosome remodellers evict (full or partial) nucleosomes in front of RNA pol II and replace after transcription
49
What does pre-mRNA splicing involve
Involves two transesterification reactions (breaking of one bond coupled to making of another) with phosphodiester bonds
Done by the spliceosome
50
How does the transesterification reactions work
1- 2’OH of A nucleotide near 3’ splice site attacks 5’ splice site and breaks phosphodiester bond between last nt exon1 and first nt intron making phosphodiester bond between start (5’ end) of intron and 2’ carbon of A. Forms a branch within intron= intron lariat
2- OH at 5’ splice site attacks 3’ splice site breaking phosphodiester bond between last nt in intron and first nt in exon 2. Makes phosphodiester bond between last nt exon 1 and first nt exon 2. Lariat degraded
51
Features of the spliceosome
Requires ATP to catalyse splicing
Complex of splicing factors (U#) some recruited by RNA pol II with Ser2 on CTD
Splicing factors consist of snRNAs and proteins (small nuclear ribonucleoproteins snRNPs or SNERPs, RNA molecules form complex shapes and catalytic site of spliceosome largely formed by RNA molecules- base pairing to make stem loop structures)
Ribozyme- ribonucleic acid enzymes
52
How do splicing factors bind recognition sequences in mRNA
Some splicing factors associate with Ser2 P on CTD of RNA pol II which recruits U1, BBP and U2 associated factors (U2A) which recognise consensus sequences by base pairing in pre-mRNA as it exits RNA pol II
Sites recognised: 5’ splice site- GU, branch point- A, 3’ splice site- polypyrimidine tract/ AG
53
How does the spliceosome catalyse intron splicing (how does it work after splicing factors bind recognition sequences in mRNA)
Rearrangement: U2 is at the branch point and the A complex forms
U4/U6 which are intact through bp and U5 are recruited, U1/U2 pair and B complex forms bringing U1 (5’ splice site) and U2 (3’ splice site) close
Transeserification 1 occurs where U4 and U6 separate
U1 and U4 dissociate forming the C complex
Transesterification 2 occurs, lariat is degraded, snRNPs are recycled and exon jumping complex (EJC) is recruited
54
What does the exon jumping complex do
Marks sites where splicing is complete leading to signals eg for nuclear export
55
What are the changes in the CTD during productive elongation
Ser5 P levels decrease (removed by a phosphatase) and Ser2 P levels increase (being added by P-TEFb)
56
How does addition of the polyA tail and termination work
Low Ser5 and high Ser2 of CTD recruits CPSF and CstF
CPSF and CstF transfer to mRNA when cleavage signal exits pol II (not base pairing)
Additional cleavage factors are recruited and mRNA is cleaved between where CPSF and CstF are bound leaving 2 pieces of RNA
57
What happens to the mRNA upstream of the cut site (CPSF side) during addition of polyA tail and termination
Poly-A-polymerase is recruited by CPSF which adds multiple As to 3’ end of mRNA without a template (not DNA dependent polymerase)
PolyA binding proteins bind, 3’ end of transcript is protected from exonuclease and can be recognised by ribosome
58
What happens to the mRNA downstream of the cut site (CstF side) during addition of polyA tail and termination
5’ end of cleaved mRNA no longer protected by 7mG-cap
Exonuclease is recruited by CTD and degrades mRNA that is associated with pol II
DNA/RNA hybrid helix is disturbed by exonuclease and reduces stability
RNA pol II terminates transcription and dissociates from DNA
CTD is phosphorylated so it can be recycled and associated with TFIIF again
59
What are activators
Bind to CREs and can recruit co-activators as first step in recruiting RNA Pol II. They are regulated in response to developmental or environmental signals
60
How can activators being the key for gene expression control be seen
Observing impact when an activator is ‘active’ in the wrong place or at the wrong time (ectopic expression eg fly with extra thorax and wings or fly with legs as antennae) or missing (not active eg mutant leaf lacking hairs) at the right time or place- often due to mutation
61
What does activator ‘activity’ mean
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DNA binding domain (DBD) activity/ role is its ability to bind DNA and recognise/ bind a CRE (cis-regulatory element)
Activation domain (AD) activity/ role is its ability to recruit co-activators
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62
In what ways can activator activity be controlled
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Presence/ absence
Conformation
Complex formation
Inhibitors
Localisation
Normally multiple forms of regulation on a single activator
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63
How does presence/ absence control activator activity
Presence of activator causes gene expression
Absence of activator causes degradation
Activator is present or absent in response to an active activator causing transcription of that particular activator
64
How does conformation control activator activity
The shape/ structure of the activator at DBD, AD or NLS. Wrong conformation= hidden and inactive. Right conformation= exposed and active.
Can occur through ligand binding or post-translational modifications (such as signal-> kinase-> phosphorylation)
65
How does complex formation control activator activity
Heterodimers or larger complexes. Limited number of activators can support a much larger number of transcriptional patterns
One activator might be in different complexes leading to different responses
Each activator activity is controlled
66
How does inhibitors control activator activity
Controlled by presence/ absence of an inhibitor or by post-translational modification
Inhibitor stops activator from working. Can be PTM-ied eg by signal-> kinase-> phosphorylation
67
How does localisation in cell control activator activity
Needs to be in the nucleus- controlled by nuclear localisation signal (NLS)- aa sequence which is recognised by nuclear import machinery. NLS conformation of the activator or PTM matters
Can occur through stimulation of nuclear entry via PTM or release from the membrane- protein translated into transmembrane domain (ER or plasma membrane). Signal can cause protease to cleave out of the transmembrane domain and go to the nucleus when required
68
How is transcription of a gene controlled/ what things do this
Multiple signals
Multiple activators (with each activity regulated in response to signals)
Multiple activator interactions (complexes)
Multiple CREs
69
How can activators be studied- 2 ways
RT-qPCR- measure the expression level of a known gene (can design primers)
RNA-seq- measure the expression level of all genes in a sample
70
What are the different chromatin states
Heterochromatin- tightly packaged, low transcription, repetitive sequences (transposons and telomeres) with few protein coding genes. Closed so activator cannot access CREs and not transcribed
Euchromatin- not tightly packaged, high transcription, rich in protein-coding genes. Open so activator can access CREs and transcribed
71
How is chromatin state of euchromatin dynamic
Changes between open and closed within
Provides a method to control transcription (open or closed chromatin determines if an activator can bind)
Modifying chromatin can change packaging and transcription
72
What is the histone code and what is its effect on transcription
Specific modifications evoke certain chromatin-based functions
Act in combination to generate biological outcome (control)
Writers: proteins that add or remove specific modifications- act in response to a signal
Readers: recognise specific histone modifications- can act to alter chromatin structure. Acetylated histone tails recruit readers associated with open chromatin. Methylated histones can recruit readers associated with open or closed chromatin depending on histone, residue and the number of residue groups
73
What is epigenetics
Any potential stable and heritable change in gene expression that occurs without a change in DNA sequence
= above or on top of genetics
Describes phenomena in which genetically identical cells or organisms express their genomes differently causing phenotypic differences
Can be a result of histone tail modifications
74
Example used to describe epigenetics
Identical twin sister mice (same DNA), difference= methylation on a single gene (Agouti gene)
Obese and yellow mouse- unmethylated and expressed all the time
Brown mouse- methylated and gene not expressed
75
What is chromatin immuniprecipitation (ChIP)
Used to detect interactions between proteins and DNA within cells-
Used to find where a protein is bound to DNA and relies upon having an antibody that specifically recognises the protein of interest
Used to find DNA location of; transcriptional machinery (RNA pol II, co-activator, elongation factors), activators, chromatin modifying enzymes and histones with specific modifications
76
What is the process of chromatin immunoprecipitation
Formaldehyde treatment forms covalent bonds between DNA and proteins and between close (interacting) proteins- cross links, done while still in the cell
Then take the proteins/ DNA from the cell and break into fragments (sonication)
Immunoprecipitation- incubate DNA fragments with antibodies that bind the protein of interest- binds to target protein and any DNA. Other proteins are then washed away
Isolate DNA and analyse
Antibodies specific to things such as proteins, protein complexes, histone modifications
77
What is alternative splicing and the different options
Combining different exons for a gene to produce different mRNAs and proteins- most human and plant genes undergo this process (~99% of human genes… have ~25,000 protein coding genes and 100,000 proteins), less common in yeast
Introns spliced out
Exon skipping (most common form in plants and animals)
Intron inclusion (more common in plants)
78
Example showing alternative splicing in humans
Tropomyosin
Part of most actin filaments in animals
Two types; muscle- interacts with actin, myosin and sacamere (muscle contraction) and non-muscle- control and regulate cytoskeleton
Humans have 4 genes but 40 different protein isoforms
79
What can alternative-splicing influence
mRNA- stability and translation efficiency (especially in UTRs)
Protein- localisation, interactions with other proteins, regulation (post-translational modification)
80
Example of alternative splicing impacting protein localisation
ESRP1 gene- 2 splice variants
1= nuclear, seen with cherry (red fluorescent protein)
2= cytoplasmic, seen with cherry, differs to nuclei pattern
81
What type of control of gene expression is alternative splicing
Co-transcriptional control
82
How can alternative splicing be controlled
Determine if the first splicing factors (SFs: U1, BBP and U2AFs) bind to the consensus sequences
Influenced by other factors (proteins and ribonucleoproteins) that bind to sequences in the RNA and help or inhibit SF binding
Factors can be regulated (eg phosphorylation)
3’ splice site of intron 1 does not recruit SFs, U1 at 5’ splice site of intron 1 interacts with BBP and U2AFs at 3’ splice site of intron 2
83
What information in mRNA transcripts can influence gene expression and how
5’cap and poly-A tail
UTRs
Influence mRNA half life/ stability and translation (ribosome association)
Determines number of times translated and amount of protein made
84
How does the 5’ cap and poly-A tail influence mRNA stability
Deadenylase removes As one at a time from 3’ end and short poly-A tail promotes mRNA degradation (A~25)
5’ and 3’ ends held together in circle by proteins; 5’ end has eukaryotic translation initiation factor (eIF) and 3’ end has poly-A-binding proteins
CPE fight for protected- recruits proteins to add As to 5’ end of poly-A tail
Competition (balance) between eIF/poly-A binding proteins and deadenylase occurs. Signals alter the competition… can regulate CPE and deadenylase
85
What is half life of mRNA and another way to view it
Half the time mRNA is in the cytoplasm… time taken for deadenylation to win and mRNA to be degraded due to exposed 5’ and 3’ ends
86
What happens when 5’ and 3’ ends of mRNA are exposed
5’- exposed to decapping complex and exonuclease= degraded
3’- exposed to exonuclease and recognised by exosome complex (can recognise mRNA once linear as normally blocked by poly-A binding proteins) and further degraded mRNA
87
Features of the exosome complex
Multiprotein complex
Forms cylinder-like structure
Nucleases inside: endonucleases and exonucleases
Range of complexes with different functions: nuclear and cytoplasm, degradation and processing
88
What are RNA structures and how can they post-transcriptionally control gene expression
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RNA structures eg stem-loop
Cis elements/ sequences
uAUG on 5’UTR
uORF on 5’UTR
IRES
miRNAs
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89
How can RNA structures eg stem-loop do post-transcriptional control of gene expression
Can be 3’ or 5’ UTR, eg stem-loop
Can alter translation efficiency by blocking ribosome from binding
Can recruit regulatory proteins (enhance or inhibit translation)
90
How can cis elements/sequences do post-transcriptional control of gene expression
Can recruit regulatory proteins to enhance or inhibit translation
91
How can uAUG do post-transcriptional control of gene expression
Upstream translation initiation site
Two different AUGs that can be used to start translation
Regulated
Give different N-terminal amino sequences- adds extra aas if done to early= altered proteins and altered localisation
92
How can uORF do post-transcriptional control of gene expression
Upstream open reading frame
Start and stop codon
Can be recognised by the ribosome and translation
Regulated
Generally inhibits the translation of the downstream ORF= ribosome stalling
93
What is RNA interference (RNAi)
Process where RNA molecules inhibit gene expression
Triggered by dsRNA from multiple sources
dsRNA processed to produce an siRNA (small interfering RNA)
One strand incorporated into RNA-induced silencing complex (RISC)
Targets mRNA to control gene expression
Found in most eukaryotes- evolved as viral defense and co-opted into regulate gene expression
94
How does siRNA associate with Argonaute protein
5’ and 3’ ends are held so that it isnt degraded= stable complex and ends protected so bases are exposed and available for base-pairing to target RNA
95
Mechanism of RNAi
dsRNA in cytosol cut into small fragments (21-23bp) by dicer= RNase
Strands separate, one degraded, other incorporated into RISC and interacts with siRNA
RISC activity occurs: cleavage of target RNA, translational repression and destruction of target RNA, formation of heterochromatin/ closing of chromatin to stop transcription
96
What are dsRNA sources
Stem-loop
Sense and antisense transcripts
MicroRNA
97
Features of microRNAs
Type of siRNA which triggers RNAi
Endogenous- functions in the cell/organism where it is transcribed
Post-transcriptional control of gene expression in response to signals
Found in most eukaryotes- over 1000 miRNAs in humans, regulate ~30% of genes- one miRNA can have multiple target mRNAs
98
Production of miRNA
miRNA gene is in genomic DNA and undergoes transcription by RNA pol II (controlled like normal transcriptional control)
Produces primary miRNA with 5’ cap and poly-A tail
Microprocessor causes folding into dsRNA and also crops/ cuts off 5’cap and poly-A tail to form pre-miRNA
Means dsRNA is in the cytosol and acts as trigger RNA for RNAi
99
Function of miRNA
Control of gene expression using RNAi
100
How is RISC “reused”
Can dissociate and bind to new target= one RISC can target many mRNAs
101
How does the texel sheep example of miRNA work
In texel sheep, mutation in 3’UTR means two mRNAs can be recognised by miRNAs= reduced myostatin gene expression and increased muscle growth
102
Using miRNAs and RNAi as research tools
siRNAs can be used to degrade mRNA molecules of interest
Design siRNA that will be complementary to target gene eg antisense mRNAs (transcript in opposite direction) or artificial miRNAs- modify miRNA gene to contain target sequence in stem
Clone into plasmid for transformation into host cell- can be targeted to certain host cell type/ developmental stage by promoter
103
Using miRNAs and RNAi as research tools- studying activators
Eg plant with amiRNA targeting activator= activator level reduced and transcription of target genes reduced
Eg express mutated version of activator gene that is no longer regulated by a miRNA (change nucleotide sequence of miRNA binding site) and keep aa sequence the same= increased amount of protein
