MCB Notes Part 2

 Why is Transcription Significant

o Transcription is the regulatory connection between the cell genome and the actual functions / molecules that are
produced. Cell function and activity is largely determined by proteins and protein state - the control of these
factors is dictated by transcription.
 Overview of Transcription
o Transcription utilizes RNA polymerases to split apart the DNA strand such that ribonucleotide triphosphates
can be paired accordingly with the template strand (noncoding strand), such that the sequence matches the
coding strand
 RNA Polymerases, General Characteristics
1. Does not require any primer
2. Low fidelity (10^5 higher than DNA pol): acceptable; 1) redundancy in RNA codons allows for substitution 2)
introns are removed may be in region doesn’t matter 3) single amino acid doesn’t affect protein 4) many copies
made 5) nonsense mediated decay - premature stop codons immediately detected and selected for decay
3. The nucleotide at the 5’ end maintains its PPPi, all subsequent RNTP lose PPi
4. Break of the phosphodiester bonds in PPi drives the elongation by pyrophosphatase
5. 5’ - 3’ direction of growth
6. requires DNA template, Mg2+ ions, ribonucleotide triphosphates RNTPs
 RNA Polymerases
o Initiation, elongation, termination
 Initiation
 Binding RNA Pol to the promoter region
 RNA Pol separates DNA strands at that point, adds a few RNTP while remaining bound
to the promoter
 Undergoes conformational change and begins to move
 Elongation
 Moves along the DNA strands creating a unzipped bubble as it moves; DNA rejoins
behind the bubble, the RNA template is threaded through the pol itself
 Termination
 Terminator sequence
 Pol dissociates
o Types of Polymerases
 Prokaryotes only have a single type
 Eukaryotes have 3 for different types of RNA
 RNA Pol I = rRNA : 1 gene 200 copies
 RNA Pol II = mRNA for proteins some snRNA : >20,000 genes
 RNA Pol III = tRNA and regulatory snRNA, rRNA 30-50 genes
 RNA Pol IV / V (plants only) = regulatory RNA
 Amanitin from death cap - toxic to Pol II; o Amanitin poison from mushroom - destroys Pol II (1
ug/mL) (10 ug/mL for Pol III); freezes transcriptional state to study mRNA, pauses RNAP
 Core enzyme is conserved !
o E.coli Core Enzyme
 2 alpha subunits, 1 Beta, 1 Beta’, 1 omega (w), 1 sigma
 alpha subunits - upstream in negative region; regulatory binding, has the alpha CTD (C-terminal
domain) which is basically a long tail that sticks off
 omega - promoter recognition
 Beta for making phosphodiester bonds
 Beta’ for binding DNA
o Eukaryotes have corresponding subunits and additional
 2 alpha = Rpb 3, 11
 Beta’ , Beta = Rpb1, Rpb2
 omega = Rpb 6
 Rpb 1 contains the important CTD tail that extends far
off the main protein complex
 Repeated 7 amino acid sequence about 26-27 times in yeast, 52 times in humans
 Rich in Tyr, Ser, Thr which can undergo phosphorylation
 Phosphorylated patterns recruit other modification enzymes
STEPS in Transcription
o Promoter recognition by sigma factors
 Join with other subunits to form the RNA Pol holoenzyme
 E.Coli
 Regonition of conserved sequences at -35 and -10
 Typically a TATA at -10 for sigma 70
 Slight differences between different genes but similar
 f)
 The degree of sequence match to CONSENSUS SEQUENCE determines the high
promoter binding affinity and consequent gene expression
 o So will it not always find it? Does the strength of the sigma binding affinity to
a difference consesnsus sequence correlate to gene expression ?
 Different sigma factors promote different things
o Sigma 70 - general housekeeping
o Sigma 32 - heat shock
o Sigma 38 - stress response
o Sigma 28 - motility
o Sigma 54 - Nitrogen metabolism (used a -12 and -24 binding !)
 Sigma containing holoenzyme ‘scans’ until it comes to a sequence then binds and sigma
factor leaves
 Technique: DNA Footprinting
o Purpose: detects regions of protein binding to DNA
o Method:
 DNA strands are labeled with 32 phosphorus on ONE END
 DNAase I restriction enzyme is mixed with DNA with and without
the binding protein of interest; a low DNAase concentration is used
such that only one cut made per strand
 The two samples are run on separate lanes on a gel to spread out
the fragments based on length
 If the protein bound, some fragments will not be found in the
sample 2; this ‘empty’ region is the footprint and corresponds to
the region of protein binding
 Detection with autoradiography which detects only labeled strands
o Limitations;
 Not very sensitive
 Can’t be used to study protein protein interaction - only 1 footprint
even for multiple proteins if on top of each other
 Polymerase has tight non-specific DNA binding without the sigma factor
o Why doesn’t it just bind to DNA without the sigma factor and start copying?
 Eukaryotes
 Contain a variety of more complicated general transcription factors
o A general transcription factor both recognizes promoter sequences and can
serve later functions in generating transcript
o Examples: RNA Pol II has TFIIA - TFIIH
 All of these assemble TOGETHER and serve different functions to
create the pre-initiation complex
 TFIIB binding to the BRE (TFIIB Recognition Element)
 DPE - Downstream promoter element
o TBP - TATA Binding Protein
 TATA box - A highly conserved sequence promoter that works for
ANY polymerase
 TAT(A/T)A(A/T)
o Typical Promoters for Pol II
 Promoters: BRE TATA INR (initation element) DPE
 Trans Fac.: TFIIB TBP
o Step-wise assembly process - one thing at a time
 TFIID - contains multiple proteins; TBP (tata box binding protein
makes sequence specific contact with tata), TAF (TBP associate
factors); TAFS also make sequence specific contacts and may
extend to the other initaiation sites: extends to DPE
 TFIIB - contacts D and BRE
 TFIIA - contacts B and D
 Quasi stable D A B complex
 Pol II + TFIIF (already bound to Pol)
 TFIIE/TFIIH
 Housekeeping genes
o Often don’t contain a TATA box
o CpG island promoter
 intiates at several alternative sites in 100-1000 bp region
containing high levels of C/G that prevent good binding to histone
octamers leaving free for Pol II and transcription factor binding
 GC rich means increased rigidity and can’t wrap histone
 Goes both directions: nonsense and sense strands
 sense strand
 can go in either direction ? whats the point of that?
o Elongation
 Transcription factors have resulted in Pol complex bound to the promoters but in closed complex
 1) Needs to open 14 bp region to become ‘open’ complex
 Pol II uses helicase activity of the TFIIH requires ATP
  No ATP required for POL I / III or bacterial except for those with sigma 54
 Rifampicin blocks the RNA transcript by binding right in the exit channel so only a
transcript 2-3 nucleotides long can form; used as an antibiotic

 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