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2) Promoter Clearance
Undergoes conformation change
o Strengthens binding
o Loses sigma or general transcription factors
Becomes phosphorylated
o TFIIH phosphorylates the CTD of the Rpb 1 subunit
o Pol II gets phosphorylated on the 5th serine of the CTD
3) Pausing
Pauses allowing capping of the RNA transcript
Further phosphorylation of the CTD results in continuing of elongation
o Then P-TEFb (transcription elongation factor) phosphorylates the Serine 2
residue on the CTD
Topoisomerases help to relieve supercoiling
o Terminators (for E.coli)
Intrinsic
Inverted repeats with small separation followed by UUUUU
The inverted repeats form a hairpin “stem loop” and weak dA -rU
Hairpin pulls the RNA transcript out of the active site stopping everything
Enzymatic terminators - ~60% of E.coli transcripts
Rho dependent termination sites : rich in C and free of secondary structures
Hexameric ATPase - transclocates along the RNA and displaces RNA from DNA
Regulation of Transcription in Prokaryotes
o Binding of sigma factors and general transcription factors to promoters only one way to control expression
o Operons - units of gene expression
Genes + promoter regions + operator regions (repressors / activator)
Repressor / Activators - proteins that decrease / increase transcription of genes
Inducers - bind to activators and repressors and ultimately stimulate gene exrepssion (example
allolactose inactavting the lac repressor, CAMP activating the CAP activator)
Corepressors - bind to repressor and ultimately inhibit gene expression (example: trp binding to trp
repressor)
Regulatory sequences
Typically upstream from the promoter
May overlap with the promoter (good for repressors - can bind and stop transcription
factors from binding)
May be far upstream - DNA looping with archirtectural DNA binding protein can be a
promoter
Eukaryotes contain elements 1000 of base pairs away called enhancers: these interact
with promoter regions through DNA looping
Eukaryotes are typically much more complex regulatory sequences and interactions
Often two components to the regulatory proteins: initial repressor or activator binds to the
DNA and then interactions with some co-activator or co-repressor which has no DNA
binding ability
o Mechanism examples in Bacteria
Trp operon
The trp operon is contains a cluster of genes that are responsible for the synthesis of the
amino acid tryptophan
The promoter region of the trp operon overlaps with a repressor regulatory sequence
The trp repressor when activated can bind to the repressor sequence
Tryptophan binds to allosteric site of the repressor resulting in a conformational change
Tryptophan is a corepressor
Additonal regulation via attenuation
o Region 1 2 3 4 trp genes
o Region 1 is rich in codons for tryptophan
o Takes advantage of tight transcription / translation coupling
o Ribosome pauses at 1 in low tryptophan environment allowing a hairpin loop
between complimentary sequences on 2/3 (antiterminator)
This prevents the formation of a Rho independent terminator loop
between region 3 and 4
Translation proceeds through trp genes
o With high tryptophan levels no pause leaving time for the Rho independent
loop terminator to form between ¾
o I’m confused about why the 2/3 stem loop doesn’t stop transcription but the
3/4 does?
Lac Operon
{ lac operon }
LacI | CAPO - promoter - LacO - LacZ lacY lacA
Repressor B-galactosidase , galactosidase permease, acetylase
CAP ‘catabolic activator protein’ helps polymerases bind to the promoter region
Camp binds to CAP
CAP either binds upstream of promoters and interacts with N-terminal domain of the
alpha subunit (class I promoter) or binds overlapping the promoter and interacts with N
terminal domain: both interaction stabilize RNA pol on the promoter and facilitate
isomerization of closed to open complex
2 different regulators:
o CAP
what: activator
where: CAP operator
Inducer: CAMP (hunger signal) via allosteric interaction
CAMP + CAP binds to CAP operator
Camp expressed in reponse to low glucose
promoted by low glucose
o Lac repressor expressed by LacI continuously
What: repressor, tetrameric
Where: LacO lac operator, +10 location, inverted repeat centered
around 10; binds to O1 and then also O2 / O3 and alternates
between (if one mutates still can bind to the other) (looping)
Repressor inactivated by high lactose
Inducer: allosteric inhibition by allolactose - if a lot of lactose,
repressor is inactive
IPTG - non metabolizable artificial inducer
Two component regulators: sensor kinases and response regulator
PhoR/PhoB
o PhoR = sensor, binds to phosphate ions in the periplasmic region, allosteric
inhibition; activated at low phosphate levels; transmembrane; acts as a kinase
for PhoB on the interior
o Phosphorylated PhoB now active
o The PhoB then binds to specific sequences in the promoters of PhoA/B/S/E
which encode various proteins like phosphate pumps
GlnA regulation
o GlnA genes: synthesis of glutamine from ammonia and glutamic acid
o NtrB phosphorylates NtrC in response to low glutamine levels
o NtrC binds to upstream regulatory activator sequences at -108 and -140
o Looping of the DNA brings it into contact with polymerase containing sigma
54 and enables it to form open complex
o ATPase activity of NtrC then stimulates the sigma 54 polymerase by opening
Mechanism in Eukaryotes
3 different things
o CpG island promoter (see above on houskeepin) - typically close to the core
promoters, divergent, only one direction result in transcription (no TATA box
in this case) - bound by SP1 proteins which specifically recognizes the GC
BOX
o TATA box
o Proximal promoter elements - nearby the core promoter (LacO, CAP operator,
trp operator in prokaryotes), so call ‘cis’ promoter proximal elements
o Enhancers - 1000s of bp away, use DNA looping and DNA architectural
proteins to contact/interact with polymerases (UAS in yeast, upstream
activating sequences)
Techniques
o DNA affinity chromatography for purification of binding proteins
1) Column containing all types of DNA, elute non DNA binding
proteins
2) Column with GC box DNA solid phase, binds only rare proteins
that bind to GC box like SP1
o DNA footprinting
Why doesn’t the presence of the protein on the particular strands of
interest slow it down relative to the other lanes
o EMSA Electrophoretic Mobility Shift Assay - In Vitro
Can detect multiple proteins ! ‘shift’ and ‘supershift’ -
the more bound proteins the greater the shift (unlike
DNA footprinting) - can bind a protein with antibody
for an addiotional shift
Fluorescently labeled DNA probe run in each lane
Lanes are also loaded with fractions from the column
chromatography: the fraction containing the protein of interest will
have dark spot shifted from other lanes because of increased steric
bulk of DNA from protein binding
How is this quantitative ?
Under the correct experimental conditions, the
interaction between the DNA (or RNA) and protein is
stabilized and the ratio of bound to unbound nucleic
acid on the gel reflects the fraction of free and bound
probe molecules as the binding reaction enters the gel.;
If the starting concentrations of protein and probe are
known, and if the stoichiometry of the complex is
known, the apparent affinity of the protein for the
nucleic acid sequence may be determined54334334ctd
o In vivo assay for transcription factors
Put in plasmid with gene responsible for making transcription
factor X
Also put in plasmid with some easily identifiable reporter gene
(GFP/luciferase/galactosidase) and possible X binding site
See if prescience of protein X quantitatively increases
concentration of Y reporter
o Identifying promoter proximal cis acting elements
If we have a suspected promoter proximal element next to a known
promoter make a 5’ deletion series of steadily shorter elements
Ligate into plasmids next to a reporter gene
Note which ones have high transcription activity
Transcription activity should disappear after a certain
point (the point at which proximal element is sliced off)
The length cut between a different strand will reveal differences in
activity
Computer assisted search for enhancers
Look for highly conserved nearby sequences in
multiple species
; sequences will be conserved for genes that are very
similar
Example: limb development SALLI enhancer
o DNA microarrays for studying global DNA expression in variable
environments
1) Purify mRNA via a DNA affinity column for Poly A tails
2) use a reverse transcriptase to get the complimentary DNA
sequences, dye with fluorescent markers, do this for multiple
populations with different colors
3) make a DNA microarray with many different short DNA
sequences representing different genes: mix it with the two
populations of cDNA allowing hybridization
4) based on color we can see which genes are expressed in
which environment
sequence specific transcription factors: DNA binding domains
o Helix turn Helix
o Zinc finger
2 histodine and 2 cysteine coordinate to zinc actom
beta sheet and alpha helix - use alpha helical side chain to
sample ; zinc atoms hold helix to beta sheet
Van der walls or hydrogen bonding interaction in the major
groove !
Typically in a tandem array ~ 3, sample only 2-3 bp each,
often need ~ 6 bp for a regulatory sequence recognition
o Helix loop helix - MAX family of proteins (may be homodimers or
heterodimers) ex. Myc for making IPs cells
Typically present in dimers
Often contain many positive amino acids to interact with
negative backbone
Also uses a helical structure for sampling on the N terminal
domain
C terminal domain binds to the other end
o Leucine Zippers - homo or heterodimers
Examples: Fos, Jun, yeast GCN4
Similar conservation of sequences between different species
DNA binding region + 6 amino acid spacer + leucine zipper
(every 7 amino acids)
Leucine’s from two monomers face each other and hold two
monomers together coiled-coil dimer
Basic amino acids in DNA binding regions interact with major
grooves
o Heterodimeric transcription factors
Common for leucine zippers and helix loop helix
More possible combinations with each other and repressors
Dealing with Chromatin
o Heterochromatin vs. Euchromatin
Hetrochromatin - highly condensed DNA - inactive ground
state -- antirepression
Euchromatin - decondensed into 30nm coil activation
Activated state
‘gradually thaw’
o Nucleosomes
Inihibit transcription factor binding
Inihbit formation of PIC complex and core promoter binding
around transcription start site
Polymerase can’t elongate
o Activate chromatin for transcription
Activators HATs histone acetyltransferases acetylate
lysine residues on histones recuit nucleosome remodeling
enzymes that contain bromodomains that binds to acetylated
lysines
3 mechanisms
direct covalent modification (HAT) by acetylation
removes + charge, stops tail binding
ATP dependent nucleosome remodeling
Recruitmentment of factors by histone tail
modification codes
o Deactylation and acetylation provide a gene activation and
repression mechanism
Activation
UAS - upstream activation sequence ; enhancer in yeast
Recruits a modular activator protein
GCN4 (lysine zipper) modular protein with activator
domain bound to Gcn5 which is a HAT complex
which relaxes the surrounding chromatin structure
the TATA box is nearby the UAS allowing binding by
TBP of TFIID starts assembly of PIC
Repression
URS (upstream repressor sequence)
Recruits a modular repressor protein Ume6 with
lysine zipper DBD and repressor domain that
recruits Rpd3
HDAC histone deacetylase complex
o Covalent Histone modification important for gene expression
Methylation (K9) promotes heterochromatin formation and
gene silencing
Acetylation (K9) and methylation (K4) gene expression
Acelation and phosphorylation associated with gene
expression
Epigenetic !
K9 involves in two different mechanisms
Acetylation K9 removes + charge, loosely binds (-)
DNA
Enzymes: HAT (histone acetyl transferase), HDAC
(histone deactylase complex), HMT (histone methyl
transferase), histone lysine demethylatse
(coactivators - work with targeting proteins)
Why is adding 3 methyl groups so effective?
o ATP dependent chromatin remodeling
Coactivators
Eukaryotes
ySwi/Snf, hSwi/Snf, hACT:RSF
helicase / ATPase component
ATP dependent “sliding” causing by Swi/Snf
Can measure with DNA footprinting? Could measure
such a sliding effect with DNA footprinting
Can also assess with restriction enzymes. Access to
restriction sites change after remodeling.
o How this fits in to overall order of events
Gene activation protein
Chromatin remodeling complex relaxed state
Histone modification enzymes
Sequence specific activators and enhancers
Mediator
General transcription factors + polymerases
Other activators rearrangement to activated complex
Mediators
o Activator domains connected to DNA binding domains of activators can’t
connect directly to the RNA polymerase and PIC
o A mediator is used to ‘translate’ between several activators and PIC
o Mediator is huge, each activator is important
o Example:
TTR - transthyretin is expressed only in liver hepatocytes
Activators / regulatory sequences
Promoter proximal: HNF1, HNF3, C/EBP, HNF4
Enhancers: AP1, C/EBP/HNF4
Only HNF1 and HNF3 are unique to hepatocytes
ALL 5 required for activated and stable PIC
Classic ChIP method - chromatin immunoprecipitation method
o Goal: in living cells, interaction with chromatin template
o Formalydehyde fixing
o Proteins on DNA are covalently bound
o Sonication breaks into frangments
o Antibodies are bound to a solid bead - the antibody binds to the proteins
and preciptates these particular sequences
o Hydrolysis of crosslinks
o PCR reaction
ChIP-seq method
o Precipitated DNA fragment is sequenced fully
o We can look for every possible binding site and how much binds
Example global binding sites of p53
o Volume of peaks indicates strength of binding
o Sonication is not specific so many different lengths and cuts of strands
with gaussian distribution about the point of interest
o Example: Distribution of Pol II
Polymerase Pausing
Chip sequencing reveals high distribution of Pol II, NELF and DSIF around the
transcription sart; seems to be concentrated at 25 - 50 bp from start
NELF (Negative Elongation factor) and DSIF pause - adds cap
Continues productive elongation after P-TEFb (phosphatase Trans elong factor b)
adds phosphate to serine 2 on CTD of Rbp 1
o P-TEFb also phosphorylation the negative factors NELF - it leaves
o DSILF upon phosphorylation goes from negative to positive factor
o Other proteins besides P-TEFb
o ELL ½ have anti pausing function (not pausing is intrinsic to
polymerases)
o FACT
HIV
HIV Tat protein activates HIV-1 transcription elongation
o May produce apoptosis in bystander cells
During initial HIV-1 transcription RNA transcript becomes a loop hairpin when the
polymerase pauses
Tat binds to this loop (called TAR hairpin) and recruits SEC - super elongation
complex in which a host of elongation related proteins
SEC contains ELL and p-TEFb (contains the kinases to phosphorylate NELF and
DSILF)
Tat promotes effective elongation of Polymerase to transcribe HIV DNA
Tat binds to the p-Tefb which is part of the Super Elongation Complex (in this case?)
SEC binds together a bunch of elongation factors: p-Tefb and TF11S
Leukemia
Chromosome translocation results in fusion protein
MLL is fused to ENL/AF9 - MLL normally a transcription factor
MLL genes - Mixed lineage leukemia
Normally SEC doesn’t come close to important genes ; regulated by MLL not that
strong
After translocation MLL gains the ability to recruit SEC !
Hox genes go out of control; 20 -- > 20,000 RNA transcripts
Regulation of Regulators
6 ways to regulate regulators
1. ligand binds activation (trp repressor by trp, CAP by CAMP)
2. activated by phosphorylation (Rho B or NtrC)
3. Stuck to the membrane (needs to be cleaved off)
4. Masking protein needs to be removed
5. Needs an additional protein (DNA binding domain + activator region
for UAS HAT recruitment)
6. Inhibitory protein must be removed to enter nucleus
Cyclic AMP inducible gene expression
o camp is SECOND MESSENGER
o Elevation in cytosolic cAMP level activates the transcription of specific
genes
o Much more complex signal transduction
Exterior of cell Gs protein picks up hormones and
neurotransmitters (type I regulator)
Activates Adenylyl cyclase which creates CAMP from ATP
The camp breaks off R protein from C making it an active
kinase and allows to enter nucleus (type 4/5 regulator)
C phosphorylates CREB (type 2 regulator) which binds to CRE
(camp responsive element) and recruits HAT which then starts
transcription (type 5)
o Gs protein coupled receptor
o Activates adenylyl cyclase cAMP from ATP
o Binds PKA regulatory subunits
o R subunits break off and releases the catalytic subunits
Cytosolic inhibitor protein activates NF-kB transcription factor
o NF-kB activator (contains -50 and -60) - important for inflammation
response; 150 genes
o Receptors for tnf-alpha and IL1 activates the kinase (infections)
o In order to import NF-kB into the nucleus have to get rid of the attached
inhibitor I NF-kBa
o Kinase phosphorylates the inhibitor I NF-kBa
o A Ubiquitin ligase then adds polyubiquitin to the inhibitor and the entire
inhibitor is broken down - degraded by a proteasome
o Nuclear localization signal NLS on p65 and p50 are exposed
o Then NF-kB then can enter the nucleus
o NF-kB responds to itself by turning itself off by activating I kba
Nuclear Receptor Superfamily Transcription Factors
o Ligands: steroid / thyroid, vitamin A and D; retinoid
o Are fat soluble and can penetrate the cellular membrane
o Steroid hormones
Cortisol - one of the stress hormones
Cortosol and Kennedy / Nixon
Steroid hormones derived from cholesterol
Estrogen and androgens are all steroid hormones
Anabolic steroids help build muscle mass
Testicular feminization - mutation in testosterone receptor
o Transcription factors in nuclear receptor superfamily
All different receptors for the steroid hormones
N - variable region --DNA binding domain -- Ligand binding
domain --C : recognize similar DNA binding sequence : the way
they interact with other factors (activation domain) is
completely different
Glucocorticoid receptor (GR) is good model receptor
Immunofluorescence: use amino acid to bind
fluorescent tag to particular proteins for location
visualization under microscope
Dex - dexamethasone, a synthetic member of
glucocorticoid class of homrones
Prescencence of DEX causes the GR protein to go
into the nucleus
The hormone binding domain alone is responsible
for this translocation
Experiment : bound galactosidase via fusion protein
to GR protein (antibody fluorescent tag targeted
galactosidase)
Model for GR
Initially masked by inhibitor protein; Hsp90 ; the
mask is on the ligand binding domain
Upon hormone binding to ligand binding domain
conformation change that removes inhibitor , goes
into the nucleus: 2 units binds to regulator sequence
with DBD and then the AD domain is free to recruit
other promoters or polymerases
Processing of pre-mRNA
Processing Overview
o Example: BETA globin
3 exons and 2 introns
starts transcribing earlier ! UTR - untranscribed region 5’ and 3’ UTR
o 1) Primary RNA transcript on the 5’ end - 5’ cap is added m7Gppp during Pol pausing
o 2) Poly A tail added
o 3) Intron excision; exon ligation Are there introns in prokaryotes?
o Mature beta globin transcript
o Cotranscriptional splicing - splicing of introns occurs before the primary transcript even created
o Example: Human dystrophin gene has 79 exons, requires 16 hours , makes sense for processing of intron
removal to occur before done
1) Capping of the 5’ end of nascent RNA transcripts with m7Gppp
o Why? 2 reasons: increases stability by protecting from 5-3’ exonucleases, enhances translation helping
with splicing in the cytoplasm and translation start sites
o When? After about 25 base pairs have been transcribes; occurs during pausing of Pol II; activated and
recruited by the 5’ serine phosphorylated CTD of rbp I subunit
o 4 steps
1) the 5’ end is initially a ribonucleotide triphosphate: a phosphohydrolase cleaves on the of
the phophates leaving behind 2
2) gaunyltransferase forms a phosphodiester linkage to a guanine (so 3 phosphates in
between)
3) guanine 7 methyl transferase methylates the 7’ nitrogen of the guanine with a methyl from
adenosyl methionine
4) additional 2’-O methyltransferases sometimes methylate the 2’ hydroxyls of the first 2
riboses
2) Poly A tail added
o Why? 3 reasons: prevents degradation by 3-5’ exonucleases, transport into cytoplasm, enhances
translation
o When? Enzymes recruited by the CTD tail (dragged along) until 2 signals emerge on the transcript
o 4 steps
1) Initial signal emerges: 2 component signal AAUAAA and a G/U rich region more
downstream, the actual cleavage site it in between these two
2) Binding of CPSF (Cleavage and poly A specificity factor) to AAUAAA, CStF (Cleavage
stimulatory factory) to G/U rich; cleavage factors CF1 / CFII at the cleavage site; the whole
thing causes the complex to kind of bend around
3) PAP Poly A Polymerases already binds to complex so when cleavage occurs it slowly starts
adding adenosine residues (template free elongation)
4) after tail is ~12 nucleotides long, PABPII (Poly A binding protein II) comes in an causes the
PAP to accelerate into faster speed
3) Splicing Reaction
o Signals to tell machinery where intron is
Consensus sequences
All introns start with GU
Branch point Adenosine in a conserved sequence - about 20 -50 bp from end with a
pyrimidine rich region (U,C)
All introns end with AG
Everything else is unnecessary (could only be ~40 bases everything is a conserved
region)
Often introns are huge - but the intron of one gene could be exon of another, overlap
of genetic information
Example: point mutation in the intron sequence causes B-thalassemia
G A creates an early AG so the intron ends early
Unfortunately, 5 codons away is a TAG stop codon
This is in the beta globin, a subunit of hemoglobin; no longer functional
o Splicing reaction
Transesterification 1: The 2’ OH group of the branch point adenosine attacks the
phosphodiester linkage of the beginning of the intron; forms the lariate loop and leaves behind
the highly reactive 3’ hydroxyl
Transesterification 2: The 3’ hydroxyl attacks the phosphodiester at the end of the intron
resulting in the spliced intron
Doesn’t require energy? Same number of bonds in the end
BUT - all have to be in PERFECT alignment to carry out the reaction
o Reaction Machinery - Spliceosome
5 different snRNP (small nuclear ribonucleotide proteins) containing 5 different snRNA with
associated proteins; called U1, U2,U4,U5,U6
1) U1 recognize the intron start, U2 recognizes Adenosine branch site
2) preassembled U4, U5, U6 tri snRNP recruited, currently not active; catalytic activity of the
U6 is masked by the U4 until things are properly arranged
3) U1 and U4 are kicked off - already know where everything is and don’t need masking
anymore; U2 and U6 are brought together - catalytically active form of the spliceosome
RNA helicase / RNA ATPase helps rearrange the RNA into the correct alignment of
the branch point adeonise relative to the 5’ and 3’ splice site
What do the snRNAs actually do?
U1 / U2 - base pairing with the transcript for recognition of exon sites
U2 / U6 - catalytic center - Ribozymes
U1 /U2 recognition of splice site
5’ end of the snRNA actually directly base pairs to the conserved sequences at the
intron start and around the branch point adenosine
Experiment: mutation in the intron starting sequence stops splicing but
compensatory mutation in the U1 snRNA resolves the problem
Regulation of Splicing
o Alternative splicing - different type of splicing patterns lead to different gene expression
Example: isophorms (different forms of same protein) of fibronectin in fibroblast and
hepatocytes
In fibroblasts includes the EIIIB / EIIIA in the firbonectin transcript - allows protein
to adhere to the extracellular matrix
Exon skipping in hepatocytes results in absence of EIIIA / EIIIB - firbonectin
circulates involved in blood clotting
o Detection of alternative splicing by northern blotting
Southern (DNA) Northern (RNA) Western (protein via antibody interaction)
Separate the RNA mixture via gel electrophoresis, transfer onto nitrocellulose via capillary
action, hybridize with a labeled DNA / RNA probe
o Regulation of Exon skipping for alternative splicing
Cross exon recognition complex
Bookended by the U2 and U1
In between are the ESE - exonic splicing enhancers that bring in SR proteins (contain a lot of
phosphorylated serine residues and Argenine )
SR have cooperative binding to each other and the U1 snRNP
SR recruit more proteins that bind to the end of the intron on the other side
U2AF65 - binds to pyrimidine rich sequence
U2AF35 - binds to the AG exons end
If SR isn’t recruited (maybe because of incomplete phosphorylation), exons are not recognized
and the exon is removed as part of its bordering introns
Coupling of processing: capping 7-Gppp and PolyA and splicing machinery coupled to the CTD tail
o Why?
Allows everything to be present at high concentration so when a signal emerges (intron start
end sites for cleavage) or polyA recognition sites mechanisms can proceed immediately
Complexation of different proteins to CTD actually stimulates elongation ! - e.g. the polymerase
can’t really move fast until everything is present
Post trancscriptional control in cytoplasmic mechanisms
RNA Interference
o Processing of small regulatory RNAs
Typically cut from double stranded RNA via some endonuclease
microRNA (imperfect pairing) vs. siRNA (perfect pairing with target and cleavage of target)
o MicroRNA processing (miRNA)
Step 1 in nucleus
Drosha and DGCR8 part of RNase III family cut a hairpin off from pri-miRNA via two
cuts in the double stranded
Exportin5 takes it out of the nucleus
In the cytoplasm, Dicer in complex with a double stranded RNA binding protein TRBP results
in dual cleavage to produce a sRNA of a precise length
21 - 25 bp strand with a 2 bp overhang at the 3’ end
Argonaute proteins are loaded with specific sRNA - only the guide strand, other strand
removed
Create the RISC complex - RNA induced silencing complex
Complete RISC may also contain GW182, Dicer, TRBP
o siRNA and miRNA block translation of specific mRNA
60% of human genes are regulated by ~1000 miRNAs (cell type specific expression)
Extensive base pairing with target (siRNA) - argonaute cuts the mRNA strand at site 10/11 of
miRNA
Partial homology between target and miRNA results in translational repression but not
destruction - this usually requires multiple sites of complementarity (multiple microRNAs
binding) in the 3’UTR ; ALLOWS MULTIPLE MECHANISMS OF REGULATION
Originally developed for viral defense
o P - bodies (processing bodies)
WHY is miRNA binding result in translational repression
Most animals miRNA results in translational repression (plant miRNA results in cleavage)
Enriched in RNA decay factors
Immunofluorescent staining to locate P -bodies via staining of Argonaut and GW182 with two
different colors
o knock-down of genes - a synthetic method for gene silencing
two methods
directly synthesize a short siRNA
directly introduce the shRNA for cutting by DICER
Introduce a plasmid containing a short hair pin (palindromic separated short loop
spacer) - can be further trimmed by dicer in vivo shRNA
Place into the cell to be taken up by RISC !
mRNA degradation
o lifespans are related to function
bacterial half lives are typically only a few minutes, eukaryote half lives could be many hours
signaling molecules (cytokines and hormone response ) and response regulators (MYC, FOS,
JUN) might have shorter half lives in eukaryotes (half life parallels function somewhat)
o Proteins can be synthesized by multiple ribosomes on a single transcript
Poly A binding protein PABPI and 5’ cap are connected by TRNASLATION initiation factors that
form a bridge !
ELF and PAPBP I
This enables a circular ‘polyribosome’
Therefore cap and poly A tail enhance translation by facilitating recycling of ribosome by
enabling it to jump onto the start again after reaching end of transcript and preventing
degradation
o 3 different methods of degradation
decapping of 7-methyl guanosine ppp then 5 - 3 exonuclease
DEADENYLATION - shortening of the poly A tail by deadenylase either decapping and 5-3
exo OR 3-5exo
endonuclease followed by 3 -5’ exo ‘ exosome’
o What determines the half life ?
o ARE mediated decay AMD
TTF / BRF binds to the AU rich Element ARE
A/U rich regions direct polyA tail removal
A/U rich region inside the 3’ UTR
Enzymes recruited by TTF and BRF
Dcp 1 / 2 decapping complex
XRN1 exonuclease 5-3 that rapidly destroys the strand
3’ -5’ exosome and endonucleases
deadenylation machinery
o Iron dependent regulation
Transferrin (TFR) receptor helps transport iron inside the cells (iron is bound to the
transferrin which is imported into the cell through endocytosis)
On the IRE regions there are ARE (AU rich regions !!!!!)
If iron levels are low, transferrin receptor mRNA is protected from degradation by two iron
regulatory proteins IRP1 IRP2 that bind to IRE (iron regulatory element)stem loop structure
in 3’ UTR
Presence of iron inhibits the iron receptor protein
o nonsense mediated decay (NMD)
P - bodies are the location where NMD occurs
PRC = premature termination codons ; normal stop codons are almost ALWAYS in the final
exon
Splicing machinery leaves a protein mark at the exon exon junction - EJC (exon junction
complex - ‘memory of the junction of two exons’), a premature termination is identified as
occurring upstream from a EJC
Enzymes involved
eRF3 - termination factor in the ribosome complex
Upf1 helicase domain, interacts with termination factor erF3
Upf2 / 3 interact with EJC
Entry into P body is mediated by Upf1 which hydrolyzes ATP and has helicase
activity