BMB- control of gene expression

features of base pairing

perpendicular to helical axis
exist as amino and keto tautomers
isomorphic

features of base stacking

provides stability due to hydrophobic effect
maximised by propellor twist of 16-18 degrees
favourable electrostatics and van der waals
10.5bp per turn

interstrand electrostatic repulsion

destabilises double helix

intrastrand electrostatic repulsion

favours extended conformation
can be counteracted by binding proteins to facilitate bending

major groove

information rich and accessible
arise from 120/240 degree angle between glycosidic bonds in each base pair
can read out base pairs from chemical information alone

TA rich effect on grooves

narrow minor groove and wide major groove

Z-DNA

left handed helix
only seen in some pyr-pur repeats
backbones zigzag due to syn position at purine N9-C1 bond

A-DNA

right handed helix
11bp per helical turn
tilted relative to axis
grooves similar width
major groove very deep - inaccessible
minor groove very shallow - information poor
found in dsRNA

base pairing in RNA

more H bonding potential due to 2'OH on ribose
can have non canonical base pairs - GU wobble
28 possible base pairs with more than 2 H bonds

functions of 3D structure in RNA

catalytic - ribosome and spliceosome
bind lignads - riboswitches
needs cations and proteins as cofactors

base triples - UAU

gives rise to triple helices
3' poly A forms triple helix with 2 internal U tracts = stable

pseudoknot

stem loop with complementary sequence outside the loop enclosing the loop
stabilised by co axial stacking of 2 helices

recognition of histone RNA

no poly A
stem loop structure recognised by proteases
exposed bases from ssRNA areas provide easy access to information

bacterial RNAP

single polymerase for all RNAs
alpha - assembly
beta - active site
omega - assemble
sigma - promoter recognition
cleft between alpha and beta with Mg co factor = active site
more core enzyme than holoenzyme in the cell - both versions present

experimental identification of promoters

in silica approach, EMSA, DNA foot printing, modi, CHiP

in silica approach

align genes with respect to +1 TSS
generate consensus sequences
strong promoter = similar to consensus

EMSA

seeing if protein interacts

1. mix end labelled DNA with pure protein or cell lysate
2. non denaturing gel - DNA and protein should run less far and get caught in pores
3. sequence specificity demonstrated by adding excess unlabelled random and wt DNA to show that wt competes for protein with labelled and random doesnt

DNA footprinting

where does protein binding protect DNA from attack

1. fluorescently label DNA at one end
2. incubate with and without protein
3. digest mildly with DNase - single hit conditions
4. high resolution denaturing gel - footprint gap in DNA ladder = binding site of protein that protected DNA from digestion

modification interference

what bases are important for binding

1. radiolabel end DNA
2. modify with ENU - phosphates or DMS - methylates purines
3. incubate DNA and protein
4. separate bound and unbound by EMSA
5. purify DNA and cleave at modified sites
6. denaturing gel - missing bands are where modification affected binding

CHiP

monitors DNA sites that specific proteins bind in vivo

1. cross link proteins to DNA with formaldehyde
2. fragment DNA
3. immunoprecipitation with Ab to the protein
4. wash
5. reverse cross links and purify DNA
6. sequence and assemble map
7. sequence analysis and motif identification

bacterial promoter recognition - cis elements

-10 box TATAATG binds sigma 2
-35 TTGACA binds sigma 4
16-18bp spacing highly conserved - boxes on same face
UP elements at highly active promoters - AT rich 20bp sequence, narrow minor groove recognised by alpha su C terminus

bacterial promoter recognition - trans factors

sigma
2 HTH motifs - one at -35 specific base readout and one at -10 reads after promoter melting
sigma 1.1 mimics DNA to bind sigma 4 in free sigma
in holoenzyme, sigma 1.1 occupies DNA binding cleft to reduce non specific binding - displaced upon promoter binding

overview of initiation

Holoenzyme binding, closed binary complex, open binary complex, initial transcribing complex, cycles of abortive initiation, ternary elongation complex

holoenzyme binds

reversible binding of sigma 4 to -35

closed binary complex

footprint = -11 to+ 3
promoter melting at -10: base flipping of A and T into specific binding pockets on sigma 2, unwinding of -11 to +3 sigma 1.1 ejects

open binary complex

after melting, footprint = -55 to +20

initial transcribing complex

first NTP binds with low affinity

cycles of abortive initiation

PDE bonds formed between NTPs aligned by RNAP
sigma 3.2 blocks exit channel
sigma released after 8-9 nt = helical turn formed
abortive initiation can lead to collapse of transcription bubble and release of oligo

ternary elongation complex

not sequence specific
stable and processive
sigma 2 and 4 disconnect
alpah CTD breaks from UE
9bp DNA:RNA hybrid duplex with an active site channel

overview of regulation of prokaryotic transcription

alternative sigma factors, activator and repressors (lac operon)

alternative sigma factors

7 types in E.coli
e.g. sigma 30 - heat shock is short term, outcompetes 70 by increased efficiency, recruits RNAP to protective protein promoters

induction vs repression

switched off gene until substrate is available = induction
when nutrient available, biosynthesis switched off = repression

lac operon - glucose and no lactose

repressor bound operator, no lac

lac operon - low glucose and low lactose

increased cAMP due to low glucose, detected by CAP, binds DNA, low level lac

lac operon - glucose and lactose present

no CAP activator, allolactose binds repressor and exposes operator for RNAP, weak recruitment due to sub optimal consensus

lac operon - low glucose and high lactose

released repressor, CAP activator binds, high RNAP recruitment
high levels of transcription

LacI repressor

dimer of dimers
recognises operator through N terminal HTH: docks into major groove and RH fits in major, SH stabilises RH

non specific DNA binding in lac operon

operator binding to repressor decreases to 3% when inducer present
repressor binds non specifically when inducer is present = increases speed of specific binding by reducing diffusion

CAP activator

HTH binds DNA
TA rich area - narrow minor groove recognised
cAMP activates CAP - brings RHs closer together
compensates for weak consensus

experimental evidence for core and linker histone

micrococcal nuclease treatment releases nucleosomes
digest between nucleosomes
extensive digestion = 147bp DNA associated with histone core
limited digestion = 200bp DNA associated with core and linker

structure of histone octamer

(H3.H4)2 tetramer + 2 x (H2A.H2B) dimers
tetramer interacts with ends and middle 60bp
dimers interact with 30bp between these
core fold = 3 alpha helices

functions of histone octamer

lysine and arginine residues - positively charged so bind DNA backbone and aid bending
PTM at N termini

nucleosome positioning

TA narrow minor groove and wide major groove
periodicity of TA between GC to facilitate bending

evidence of nucleosome positioning

CHiP seq of nucleosome and pol2
non expressed genes = decreased pol 2
expressed genes = increased pol 2
increased nucleosomes at +40 TSS = +1 nucleosome
nucleosomes decreased at promoter regions

evidence that higher level packaging = reduced expression - DNase I sensitivity

1. digest with DNase
2. purify to DNA + nucleosome
3. restriction digest known expressed genes
4. run on gel and prove for known sequences
   if fragment in tact = band visual
   add DNase = expressed gene digested faster as not bound to nucleosome

evidence that higher level packaging = reduced expression - DNase I hypersensitive sites

1. light DNase digestion
2. purify DNA
3. restriction digest
4. southern blot and probe - hypersensitive sites are nucleosome free regions and should run faster on gel

linker histone H1

positive charge termini
20bp protection
30nm fibre - not accessible to RNAP or DNase 1
can switch on/off condensation

PTM of histones

HaTs - acetylate lysine = neutralise positive charge = decompaction
acetylated lysine recognised by bromodomain in HaTs = reinforcement of decompaction
HDACs - reverse acetylation and recruited by repressors
methylation recruits repressor through their bromodomain

eukaryotic RNAP2 and RPB1 CTD

transcribes mRNA
10-12 su
RPB1 CTD = largest su: multiple heptad repeats, can be phos (hyper = elongation, hypo = initiation), serine 2 and 5, joined to pol 2 close to exit channel - recruits RNA processors

RNAP2 promoters

sharp/focused = TATA -25, INR +1
broad/dispersed = CpG islands

in vitro assay of basal transcription - what is needed for accurate initiation

1. incubate DNA template with NTPs and nuclear extract
2. hybridise mRNA from this with 32P labelled oligo
3. incubate with dNTPs and reverse transcriptase
4. analyse labelled DNA - shows TATA/INR needed to accurately initiate, pol2 alone lead to non specific TSS

overview - assembly of pre initiation complex

promoter binds TF2D
recruitment of RNAP2
recruitment of kinase
ATP hydrolysis

TF2D bind

TATA binding domain + TAFs (TBP associated factors that bind INR)

RNAP2 recruitment

TF2A binds and recruits TF2B
TF2F then pol2

recruitment of kinase

TF2E then TF2H - kinase

ATP hydrolysis

drives promoter melting
footprint -80 to +30

TF2D role

13 su
TBP binds TATA via beta sheet that binds narrow minor groove
flexible Phe residues of TBP between TA - widens minor groove by 80 degrees
TAF1 has bromodomain to decompact chromatin at INR

TF2F role

binds stably to pol 2
reduces non specific binding

TF2H role

recruited after pol 2
kinase and ATPase activity
phosphorylates RPB1 CTD at serine 5 - signal to start elongation
helicase - unwinds at TSS to create open complex

experimental evidence of enhancers - reporter gene assay

1. clone promoter into reporter plasmid
2. see if promoter drives transcription of reporter
3. 3' deletions = efficiency reduced
   5' deletions = transcription impacted well before TATA reached

evidence of promoters - beta globin promoter

-75 and -95 deletions gave larger drop in transcription efficiency than TATA mutations
SP1 and CEBP binding sites uncovered

evidence of MYL1 muscle specific enhancer

low levels of reporter transcription under promoter assay
measured reported activity in differentiated vs undifferentiated cells
SV40 positive control = strong enhancer = high expression in both cells
MYL1 enhancer = expression only in differentiated cells

general features of enhancers

larger than UE
array of TF binding sites
conserved
few vital sites
mutations at most points have an effect
transcribed to eRNAs
1-5 per gene

DNA binding domains of ATFs - overview

HTH, zinc fingers, basic leucine zipper, bHLH

HTH

H bonds from side chains on alpha helix of RH
3 H bonds per bp
forms heterodimers

Zinc fingers

beta-beta-alpha with zinc ion tetrahedrally coordinated between cys
Zn stabilises
RH docks in major groove
non palindromic sites

basic leucine zipper

4-5 leucine residues each 7 amino acids apart in coiled coil
dimerization region
DNA binding through basic N termini docking in major groove

bHLH

2 alpha helices separated by a loop
in myoD, Id is an inhibitor of differentiation - has dimerization region but not DNA binding helix

features of activation domains

not structured - adopt structure when bound target
identified by mutations in ATFs that impaired activity but not binding

targets of activation domains

nucleosome modifiers - HaTs through bromodomain
GTFs - TAFs in TFIID, enhancers can bind protein hub = DNA looping

evidence of DNA looping - SV40 beta globin

1. SV40 active promoter and beta globin gene on separate fragments with biotin tags at one end
2. combine both fragments with nuclear extract
3. avidin in extract binds biotin and links two fragments with large protein blockade and no transcription- can't be sliding or conformational change model, must be looping

evidence of DNA looping - 3C technique

looking for in vivo long range looping

1. cross link with formaldehyde - protein hub should connect DNA fragments in looping section
2. isolate DNA and digest with restriction enzyme site close to enhancer and promoter
3. ligate only cross linked fragments
4. reverse cross links and qPCR

mapping protein-RNA interactions overview

protein purification RNAiP, RNA purification, UV, GWS

protein purification - RiP

RNA immunoprecipitation
use Abs against POI
identify associated RNA- generate cDNA
amplify with PCR

RNA purification

tag RNA with biotin - synthesis with biotin UTPs
incubate with protein
recover with streptavidin beads bind biotin
SDS-PAGE and western blot

UV cross linking

irradiation of complexes with UV induces covalent bonds

genome wide approach to splicing

deep sequencing
systematically identify targets of RNABP
measure use of exons and introns - tissue specific
measure translation rate of all mRNAs
determine decay rates of mRNAs

features of 5' cap

not germline encoded
co transcriptionally added
enhances splicing of first intron
increases mRNA export
increases efficiency of translation initiation
stabilises from attack against exonucleases
binds via phosphate bridge

formation of cap

1. removal of gamma phosphate
2. addition of GMP from GTP +PPi released by guanylyl transferase
3. methylation at N7 on guanine by 7-methyl transferase = Cap0

features of cap formation

capping enzymes occur on same polypeptide
Cap bound by CBC in nucleus and other proteins in cytoplasm
capping is specific - only at triple phosphorylated ends

why are only RNAP2 transcripts capped

CTD of pol2 is unique
has linker followed by 7aa tandem repeats
52 repeats in mammals
highly conserved
has unstructured domain close to mRNA exit channel
CTD undergoes phosphorylation which allows interaction with capping enzymes

experimental evidence of capping - RPB1 CTD needed

1. transfect cells with alpha amantin resistant RNAP2 (usually inhibits)
2. wt cells with normal CTD and mutant with 47 repeats deleted
3. inhibit endogenous RNAP 2 with alpha amantin
4. separate capped and uncapped mRNAs and qunatify
5. mutant = defective in capping, CTD essential for capping

evidence of capping - enzyme binds phosphorylated CTD

1: mutant, wt and phosphorylated CTD, add radiolabelled GMP - only phosphorylated CTD gets capped
2: make CTD mutant yeast strain that can't be phosphorylated at serine 5, mutant doesn't grow, fuse mammalian capping enzyme to CTD = mutant can now grow
shows serine 5 phosphorylation is essential

features and functions of poly A

added after cleavage of pre-mRNA
not germline encoded
shortened during export
gradually shortened in cytoplasm due to aging
protects against 3' exonucleases
activates translation

trans factors in poly A

CPSF = cleavage specific poly A factor - binds AAUAAA
CStF = cleavage stimulation factor - binds GU region, only involved in cleavage
both bind PCTD

alternative poly A sites

e.g. sex lethal RNABP in drosophila
only present in females
in males poly A is accessed by CStF in Gu region
in females, SXL binds and competes with CStF at GU region, so distal poly A site used = translational repression

experimental evidence of poly A - pol2 doesn't terminate at discrete sites

1. purify nuclei
2. incubate with NTPs and labelled UTP - no new initiation of transcription, only elongation of pre initiated transcripts
3. isolate labelled RNA and cleave into fragments
4. hybridise labelled RNA to DNA probes corresponding to 3'UTR - hybridisation at many sites - termination not at one site

evidence of poly A lengths

1. synthesis of radiolabelled RNA
2. add nuclear extracts and labelled ATP
3. shows increasing length on gel due to poly A over time and precursor decreases

evidence that cleavage and poly A are independent

1. nuclear extract + ATP + P labelled RNA = cleavage and poly A
2. nuclear extrace + ddATP + P labelled RNA = cleavage only
3. Pre cleaved mRNA + nuclear extract + ATP = no cleavage but poly A

evidence of poly A consensus

mutate U to G of AAUAAA = no poly A
add As to consensus = more poly A
shows 2 distinct steps = adding first ~10 residues dependent on consensus, then polymerising further independent of consensus

genome wide view of poly A

HTS
purify mRNAs
fragment
purify poly A with oligo dT beads
sequence

experimental discovery of splicing

adenovirus produces capped, spliced and poly A mRNAs = good mdoel

1. incubate mRNA with dsDNA
2. mRNA replaces one strand of DNA to form loop
3. visualisation - map RNA genome = tails of unhybridized RNA: 3' due to poly A, 5' hybridised to distal sites on genome
   showed mRNA is mosaic of DNA

cis elements of splicing - in intron

5' site = GU
3' site = AG
branch point = adenine not base paired before PPT
PPT = near 3' site

trans factors of splicing - snRNPs

snRNPs = small ribonucleo protein particles - contain small nuclear RNA and U protein
U1/2/4+6 - 4+6 base paired regions and can associate with U5

proteins that interact with snRNPs

U2AF
SR proteins

interaction of cis and trans factors in splicing

U1 binds 5' site - bp
U2 binds branch - bp
U2AF65 binds PPT
U2AF35 binds 3' site

evidence of splicing interactions

mutations in conserved sequences prevent splicing or move it to cryptic sites
removal of first 8 nts of U1 snRNA blocks splicing
complementary mutations are restorative

steps of splicing

1. lariat formation - 2'OH of branch point attacks phosphate at 5' SS, 5' exon cleaved and 2-5' lariat formed
2. intron released as lariat - 3'OH of 5' exon attacks phosphate at 3'SS, exons ligated and intron released

in vitro analysis of splicing over time

1. incubate P labelled RNA with ATP and nuclear extract
2. take samples over time course
3. run on gel
4. expose to film - over time, lariat intron increases, intermediate intron +exon 2 increases then decreases, pro-mRNA decreases, exons together increases, exon 1 alone increases then decreases

structure of the spliceosome

60S RNP complex
only assembles with substrate present
consists of pre-mRNA, U proteins, snRNPs and other proteins