Exam 1 Flashcards
All content (minus mendelian genetics and evolution)
Condensins
induce chromosome compaction/condensation during mitosis and meiosis
how do you supercoil something that is linear?
put it in a loop
cohesins
associate with chromosomes after S phase to keep sister chromatids together until anaphase
what are SMC complexes and where are they found?
- Structural maintenance of chromosomes complexes
- Cohesins and condensins
how are DNA loops in euk and bacterial chromosomes constrained?
topologically:
euk: loops of DNA attached to chromosomal scaffolds after extracting histones
bacteria: condensed chromosomal DNA in loops
Topologically associating domains (TAD)
- domains that are crammed together in chromatin organization
- transcriptionally active domain in euchromain
- have active genes present in phase-separated molecular condensates
- increase local [RNA polymerase, transcription factors]
- formed by interaction of unstructured regions of proteins and some long noncoding RNAs (lncRNA)
euchromatin vs heterochromain
euchromatin: loosely packed DNA (accessible), transcriptionally active genes, acetylation
heterochromatin: tickly packed DNA (inaccessible), transcriptionally inactive genes, methylation
acidic vs alternative chaperones
acidic- promote in vivo assembly
alternative- for histone variants
what is nucleosome remodeling catalyzed by?
ATPases
How is nucleosome assembly and organization dynamic?
7
- ATPases
- Histone variants (centromers, X-chrom activation)
- Acidic and alternative chaperones
- modification and remodeling (reversible)
- epigenetics- modifications inherited
- bromodomain proteins (acetylation)
- chromodomain proteins (methylations)
what sequences have a greater chance of being accessible?
in a nucleosome-free region or near the edge of nucleosomes
CAF-1 and NAP-1
acidic chaperone proteins that facilitate binding to histones
H2AZ and H3.3
- replace H2 and H3 in euchromatin via histone chaperones
- inhibits nucleosome-nucleosome interaction
H2AZ deletion in mammals
embryonic death (because deals with development)
MacroH2A1
concentrated on inactive X-chromes of females, but depleted on transcriptionally active chromatin
H2AX
involved in DNA repair and recombination
CENPA
- H3 histone variant
- associates with centromeres, deletion is final
what are chromatin remodeling complexes?
move nucleosomes around and ATP-driven
SWI/SNF
- bromodomains associate with histones
- activate transcription
- ATP driven
chromatin remodeling complexes
ISWI
- NO domain associates with histones
- transcription repression
- ATP driven
chromatin remodeling complexes
Mi2/NURD
- chromodomain associates with histones:
- transcription repression
- ATP driven
what is needed to move histones?
ATP
what does micrococcal nuclease do?
- digests DNA
- basically an on/off switch
formaldehyde crosslinking
- cross-link nucleosomes to DNA within chromatin
- basically glues things together
HAT’s
- transcription activation
- bromodomain with histones (interacts with acetylated histones)
- histone acetyltransferases
How are histone tails modified?
H2A: P, A, U
H2B: P, A, M, U
H3: P, A, M
H2B: P, A, M
P: phosphorylation
A: acetylation
M: methylation
U: ubiquination (only in C terminal)
histone deacetylases (HDACs)
transcription repression
what do modifications recruit?
effector factors that recognize particular modified amino acids
Gcn5
- YEAST ortholog
- HAT
most actively-transcribed DNA lacks……..
histone H1
Histone Code
- DNA transcription is largely regulated by post-translational modifications
- maintained by chaperones and is essential for epigenetic inheritance
how are histone modification maintained after replication?
- after rep, new daughter duplex lack histones H2A-H2B
- parental (marked) H3-H4 bind RANDOMLY to both daughter strands, along with new (unmarked) H3-H4
- old and new H2A-H2B reassemble randomly on both daughter strands along with old and new H3-H4
- epigenetic marks spread to adjacent nucleosomes by recruiting histone-modifying enzymes that recognize the existing pattern and modifying the new histones to match
how can males live with only one X while females have two?
gene dosage compensation- in female mammals, one X is randomly inactivated in embryo cells (Lyonization)
why is it said that female mammals are mosaics?
in cats, gene for orange vs black coat is on X chromosome
X inactivation in hetero females –> tortoiseshell or calico phenotype of orange & black patches
all toroisesehll & calico cats are female or Kleinfelter (XXY)
XIST
- non-coding RNA
- X-chromosome inactivation via DNA methylation and nucleosome modification
- permanent in somatic cells
- not expressed in normal cells
bar bodies ……… in germ lines
reset
what did Meselon and Stall identify
confirmed semiconservative using gradient centrifugation
1st gen: old,new + old,new
2nd gen: 2 old,new + 2 new strands
what is RNA primer made by and how long is it?
primase
11-13 ntd
how long are the okazaki fragments in eukaryotes and bacteria?
eurkaryotes: 100-300 ntd
bacteria & phage: 1-2KB
replicon
total length of DNA replicated from one origin
plasmid stability assays
allow for identification of both cis -acting sequences and trans-acting factors needed for DNA replication
shuttle plasmids
- can replicate in more than one type of cell, such as bacteria and mammalian cells
- used to transfer cloned genes between different organism
why do bacteria not have telomerase?
don’t need it because have circular DNA
what do plasmid stability assays do?
allows infentification of both cis-acting sequences and transpacting factors necessary for DNA replication
cis-acting factor
- oriC: where the genome replication can initiate (replicator)
- part of the DNA itself
trans-acting factor
- DnaA: can initate replication (initiator)
- diffusable through the DNA
are cis and trans-acting factors enough to start DNA synthesis?
No, also need primer
what does Seq A do?
binds to hemi-methylated DNA and prevents DnaA from binding oriC again
how is
why do histone proteins migrate anomalously?
histones don’t migrate because they are very charged
what does B-mercaptoethanol do in SDS gels?
breaks disulfide bonds
epitope tags for protein detection and isolation
GST- binds to glutathione
6HIS- 6 histidines in a row: binds to nickel chelate resin
peptide (epitope) tags: FLAG, myc, HA: bind to specific antibodies
GFP: small protein
TAP: sequentially uses 2 different epitopes
what are epitopes?
tags for protein detection and isolation
antibody binding sites
TAP tags
tandem affinity purification tags
very low background of non-specific interactions
- purify target protein using IgG beads, which bind protein A, which is removed by TEV protease
- purify target and interactors a second time using Calmodulin beads
ChIP
- proteins-DNA interactions
- can only do 1 gene at a time
- PCR, uses formaldehyde
what can ChIP-exo identify?
- protein-DNA interactions
- near base pair precision from exonuclease
- Exonucleases degrade the DNA from the ends, leaving more precise boundaries
-
Chromatin Endogenous Cleavage-Seq (ChEC-seq)
accessibility of chromatin regions, how tightly or loosely DNA is packaged
1. micrococcal nuclease (MNase) attached to protein of interest (YFP) via a flexible linker (completly inactive until Ca2+ ions are added)
2. introduce YFP-MNase into live cells
3. permeabilize cell membrane and add Ca2+
4. DNA near (but not bound by) YFP cleaved by MNase
5. isolate and sequence small fragments of DNA
omics
Genomics: DNA sequencing
Transcriptomics: RNA-seq
Proteomics: Protein/complex purification and MS
Metabolimics: MS and NMR
what was used in typical large genome projects?
promoters as landmarks
size of human map unit
1,000,000 bp
how many map units in humans is more than all DNA of E. coli
4 map units
high-resolution recombination mapping
landmarks for anchoring sequence information
1cM = 1 Mb DNA
1cm = 1% chance of recombination during meiosis
how identical are humans at sequence level?
99.9
how many SNPs are between any 2 individuals?
3 million
FISH
- fluorescent in-situ hybrid
- cloned DNA with fluorescent dye
- hybridize to denatured metaphase or polytene chromosomes
- chromosome locations
physical maps
based on bp, not recombination
maps of purified pieces of genome (cloned DNA)
clones with large inserts are most useful
overlapping clones are assembled into contigs
Contigs
long continuous stretches of chromosome DNA
BAC sequences
what is the purpose of integrating genetic and physical maps?
to know which chromosome is which
order of markers is the SAME on genetic and physical maps
physical distance (base pairs) is NOT THE SAME as map distance in % recomb
evidence that physical evidence (bp) IS NOT the same as map distance in % recomb
- frequency of recombination can differ 100 fold
- recombination rates are influenced by chromatin structure
- in humans, there are 30,000 recombination hot spots spaced every 50-100 kb
genome sequencing
one consensus sequence per chromosome
<1 error/ 10,000bp
usually 10 independent reads of each ntd
what are the 2 basic genome sequencing strategies
ordered clone sequencing- clones make up a physical map; requires the mapping of each chromosome prior to DNA splitting.
whole genome shotgun sequencing- randomly sequenced clones are assembled; best suited for small (bacterial) genomes; gaps filled by primer walking
ordered clone-by-clone genome sequencing
- overlaps allow fragments to be assembled
- requires the mapping of each chromosome prior to DNA splitting.
first draft of the human genome sequence?
2001
took 13 years and $100 million
how many bp and genes are in genome?
3 billion bp
20,000 genes
how many bp per cell?
60 billion
what % of the human genome codes for enzymes?
0.015
haplotypes
DNA variants thatare inherited together because they are close to each along a single chromosome
(shared group of polymorphisms that are very close together, so stick together)
homolog definition
genes related by descent from a common ancestral DNA sequence
ex: hemoglobin and myoglobin
2 types of homologs
ortholog- gene in different species that evolved from common ancestor. retain the same function during evolution; ancestors and progeny
paralog- genes related by duplication within a genome. May evolve new functions; cousins
C-value paradox
- DNA value per nucleus
- lack of correlation between genome size and developmental, metabolic, or behavioral complexity
what % of genome is transcribed?
more than 80%
what % of genome accounts for introns?
30%
microRNAs and long noncoding RNA
regulate gene expression at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels
what things have acquired key roles in gene regulation, development, and diseases?
highly repetitive elements, remains of transposable elements, and pseudogenes (inactive genes)
basically, dead genes acquire function
synteny
- retain the same function during evolution organisms of relatively recent divergence
- between species there are regions in the same order
- for estabilishing orthology of genomic regions in different species
what are hominids?
extint members of the human lineage (not our ancestors, but cousins)
human-specific changes
most proteins have only 1-2 amino acid differences (30% are identical)
changes in conserved noncoding sequences (regulatory regions)
1. brain development (GADD45G)
2. ability to speak and use language (FOXP2)
3. decreased sensitivity to smoke derived toxins (AHR)
what is measured in a northern vs southern vs western
blot?
northern: RNA
southern: DNA
western: proteins
3 requirements for linear chromosome stability and inheritance
telomeres: protect chromosome ends and allow their complete DNA replication
centromeres: facilitate segregation during mitosis and meiosis
origins of replications
telomerase
TER RNA
TERT
telomerase: ribonucleoprotein enzyme (protein + essential RNA)
TER RNA: template for addition of G-rich telomeric repeats to the chromosome 3’ end
TERT: reverse transcriptase that copies the TER template
how are the ends of chromosomes preserved during DNA replication?
telomerase has its own primer (uses DNA as the primer), uses reverse transcriptase because copies DNA
telomeres promote T-loop structure, which protects DNA ends and blocks DNA damage response
what can telomere shortening lead to?
DNA damage response
cell cycle arrest
deletional recombination
telomere fusion
dangers of telomerase inhibition and activation
inhibition: limited proliferation potential (stem cell disease like aplastic anemia)
activation: unlimited proliferation potential (cancer)
what do telomeropathies cause?
mutations in core telomerase subunits or accessory factors –> premature telomere shortening –> stem cell disease/ failure of stem cells to proliferate
mutations in telomere binding proteins –> cell death or genome instability
centromeres
- DNA sequences/regions where sister chromatids adhere to each other most strongly before anaphase
- assemble at kinetichore, (where spindle microtubules attach to the chromosome (trans-acting factors)
cis-acting factors vs trans-acting factors
cis: affect gene expression on same piece of DNA
trans: diffuse through DNA and affect different genes
chromosome disjunction
cohesins- ring-like proteins that prevent premature separation
separase- cleaves cohesin at anaphase
what is anaphase driven by?
APC and activator Cdc20 –> triggers activation of separase and degradation of cyclins
less CDK activity –> exit from M phase
by how much do chromosomes compact?
7000 fold
length of DNA wrapped around histones
length of linker DNA
146 ALWAYS
around 50 bp (varies)
nucleosomes
146 bp of DNA (-) wrapped around histone octamers (+)
what makes up nucleosome core?
H2A
H2B
H3
H4
made up of Lys and Arg
core is highly conserved because highly specialized
core has a well-ordered crystalline structure
what does DNA wrapped around nucleosome consist of?
relatively straight 10 bp segments that are connected by bends and the DNA is slightly underwound
A=T vs GC regions in nucleosomes
2 or more A=T at 10 bp spacing will tend to position nucleosomes
GC tracts inhibit nucleosome placement
arrange DNA to where nucleosomes want or dont want to park there
what mediates histone-DNA and histone-histone interactions?
Histone-fold motif
tails of histones
extend from core octamer
unstructured and available for interaction and modification
how many DNA turns around core octamer?
1.65 turns
who has the least neanderthal DNA?
humans from Africa
by how much does chromosome compact from nucleosomes?
6 fold linear compaction
what does linker histone H1 do?
goes at the end and compacts nucleosomal arrays
what do N-terminal tails of histones do?
interact with adjacent nucleosomes compacting chromatin
Whole-genome shotgun sequencing (WGS)
sequence a large number of overlapping DNA fragments in parallel (reads)
uses computers to assemble into largers contigs
primer walking to get scaffolds
how are polynucleotide chains conneted in DNA and RNA?
phosphodiester bonds
DNA’s relation to:
2’ -OH
3’ -OH
2’ : DNA lacks
3’: where things are added on to
why use DNA for long-term storage of genetic info?
because DNA is not rapidly hydrolyzed under basic conditions, unlike
RNA because of the 2’ -OH
RNA sense vs antisense
RNA: top strand, same as sequence message
antisense: bottom/coding stand, complementary to message
often only top/sense strand is written
chargaff rules
suggested base pairing and replication mechanism
A=T, G=C
B-DNA
- right-hand double helix
- 20 A wide
- 3.4 A vertical rise/ bp
- 10.5 bp/turn
what is DNA stabilized by?
hydrophobicity, H-bonds, base stacking
why does DNA have T and not U?
C deaminates to U
U is removed from DNA by uracil-DNA glycosylase
why is 5’ methyl C often a hot spot for mutation?
5’ methyl C deamintes to T
recognition element
a-helix protein can easily bind to major grove IFFFF it has the correct pattern of H-bonding
R-group amino acids that can also make hydrogen bonds with base edges
- polar and hydrophilic R-groups
- Asn, Gln, Glu, Lys, Arg
how are polynucleotide chains flexible?
rotation around glycosidic bond
sugar pucker
- pentose pops up; large impact on chain geometry
- C-2’ endo predominant in DNA
- C-3’ endo predoimant in RNA
propeller twist
- allows bases to stack in a way that excludes more water
- constrained by H-bonds
approx 3.2 for A=T
6.7 for G=- C
GC rich regions vs AT rich regions
naturally “bent” DNA
GC rich: have WIDER minor groove (GC greater twist than AT)
AT rich: have NARROWER minor groove
sharp kinks at boundary of regions with greater propeller twist
eliminates need for certain regulatory proteins
A-DNA
- short, fat, tilted, different pucker
- predominates at low moisture
- has low water content
- naturally found in RNA/DNA and RNA/RNA helices (because RNA does 3’ pucker)
how do extremophiles survive of 80 C and pH3?
by adopting the complete DNA in the A-form and therby aids protein to encapsulate DNA
when does Z-DNA form?
- at high [salt]: poly d(GC); poly d(AC)+poly d(GT)
- physiological conditions: poly d(GC) mainly form B-DNA
- methylation of carbon 5 of G (m5G): shifts equilibrium to favor Z-DNA by binding a hydrophobic patch
- underwinding
predicted effect of B–> Z shift?
radical change in gene expression
what happens if you put methyl hydrophobic pocket?
can more easily transition into Z-DNA
which DNA occur in nature?
All 3: B, A, Z
hairpins and cruciforms
- SS DNA can readily form hairpins but cruciforms usually not favored under phisiological conditions
- SS binding proteins prevent hairpins during DNA replication
triplexes vs quadraplexes
trip: “Hoogsteen” pairing; 3-stranded proteins
quad: 4 GC rich strands; form on telomeres with assistance of proteins
how does RNA fold?
- very compact –> less exposure to water (good)
- structures can be predicted by having the most negative deltaG
- greater structural flexibility of RN allows G=U (wobble) base pairs
pseudoknots
- make different sets of proteins byshifting reading frames in RNA
- cause ribosomal frame shifting in HIV, to allow production of the reverse transcriptase needed for viral replication
RNA features that increase secondary structure stability
- unique recognition sites for aminoacyl-tRNA synthetases ribosomal proteins
- A-form: closer phosphates
- 2’ -OH hydrogen bonds
- coordination with metals
- direct to oxygen on an adjacent ribose
- via a water, between a 2’ OH and a phosphate oxygen
RNA tertiary structures
- vast array, bind ligands and catalyze chemical reactions
- self-splicing introns
- riboswitches (can act different based on of its bound to a drug)
- form peptide bond in robosomes (done by RNA chemistry, not any proteins)
DNA hybrid formation
DS DNA melted and re-annealed
higher Tm: higher UV absorbance and lower viscosity
increased absorption = hyperchromic effect
what does Tm depend on?
- GC content
- [salt] (if low, repels)
- pH (low= purines bases fall) (high= disrupts H-bonds)
- chaotropic agents
- for short sequences, length is importnt
high salt= phosphate shielded
low salt = phosphates not shielded –> ripped apart
between dsRNA and dsDNA, which is more stable?
dsRNA
what nucleic acids can form hybrids?
from different species (alive or extinct)
southern vs northern blotting
southern: separate DNA with restriction enzyme on native non-denaturing agarose gel (look at complex structure)
northern: separate RNA on denaturing gel (to have single strands so migration is based on size and not structure)
DNA or RNA on solid supports must be denatured before hybridization with labeled probe
restriction enzyme for cutting ranges of sizes to see better in gel
stringency of hybridization
- conditions used during a nucleic acid hybridization that determines how closely a probe sequence must match the target sequence to bind
- how close are you to Tm? how much salt youre gonna put and at what temp?
DNA supercoiling
- when ends are fixed like in circular bacterial chromosome or the loop domains of eukaryotic chromosomes
- underwinding: negative supercoiling –> facilitates strand separation (predominant in DNA)
- overwinding: positive supercoiling
linkage number
linkage= writhing # + twisting #
= number of times DNA strands twist about each other (a fixed number)
supercoiling alterations
- topoisomerases alleviate the stress of replication and transcription by introducing/ relaxing supercoils –> allows DNA to maintain an underwound state
- palindromic sequences allow cruciform DNA
Type 1 topoisomerase
- break 1 strand of DNA, pass unbroken strand through, and religate broken ends
- changes linking number by 1 (delta Lk=1)
- reaction cycle involves formation of an enzyme bridge that prevents uncontrolled relaxation of DNA
- does NOT require ATP
Type 2 topoisomerase
- break both strands of DNA, pass unbroken strand through, and religate broken ends
- changes linking number by 2 (deltaLk=2)
- requires ATP
bacterial DNA gyrase
introduces negative supercoils
Eukaryotic type 2 topoisomerase
do not introduce - supercoils
can relax + and - supercoils and untangle DNA by allowing one strand of DNA to pass through another
how do topoisomerases increase/decrease underwinding?
by changing linking number (Lk)
topoisomerase inhibitor
Ciprofloxacin- for bacterial infections including Anthrax. Blocks DNA passage
Topotecan- antitumor agent, block human topo1
electrophoretic mobility of linear DS DNA is determined by …………..
length
because charge of nucleic acids comes from phosphodiester backbone
1% vs 2% gel
1%: if small, go right through and stack at the bottom
2%: avg pore size is smaller, so small stuff will get stuck where supposed to
why cant you go higher than 0.5% gel?
because at some point, friction does not make that much difference
standard DNA gel electrophoresis vs pulse-field electrophoresis
standard: can resolve fragments up to 50kb using 0.5% gels, which are very soft
pulse-field: separates DNA up to 10 Mb, using 1% gel, which are much easier to work with
- DNA slowly zig-zags down the gell
- every time the current shifts direction, DNA must re-orient to align with field before it can migrate
- small DNA reorients more quickly and thus moves faster
what is used to visualize DNA with UV light?
- ethidium bromide (EtBr): detection limit 0.5 to 5.0 ng/band; toxic
- GelRed: less toxic because unlike EtBr, does NOT cross cell membranes
both intercalate between the basepairs of DNA
blot hybridization
- detect specific DNA and RNA sequences
- charge on nucleic acids allows them to bind + charged surface (like nitrocellulose)
housekeeping genes
- used as controls on Northern blots
- PECAM-1 (mRNA)
- GAPDH (mRNA)
recombinant DNA technology
- get DNA segment to be cloned (restriction enzyme and size selection after electrophoresis; direct synthesis)
- select DNA vector that can self-replicate (usually plasmid with antibiotic resistance gene)
- join 2 DNA fragments covalently (DNA ligase, Gibson assembly)
- transform recombinant DNA into a host (typically E. coli)
- select hosts that have recombinant DNA
restriction enzymes
- have different recognition sites and cut DNA differently
- chop up foreign DNA if it comes with the wrong pattern
what does cleavage of palindromic sequence generate?
DNA with complementary ends
cloning vectors key features
origin of replication (high vs low copy #)
selectable marker (antibiotic resistance)
insertion site for foreign DNA (polylinker)
Gibson assembly
glues things together without need for compatible (sticky) ends in a single isothermal reaction
1. exonuclease chews 5’ to 3’
2. single strand regions anneal
3. gaps filled by pol 1 and ligase
DNA libraries
recombinant/cloned DNAs, each with same vector but different inserts
2 types
1. genomic library: entire genome is represented
2. CDNA library: expressed RNAs from particular cell or tissue-type are represented
what does reverse transcriptase (RT) do?
generates complementary DNA (cDNA) from RNA template
Hairpin primed 2nd strand cDNA synthesis
- reverse transcriptase form loop that can prime 2nd strand synthesis
- forms due to endogenous RNase H activty of AMV reverse transcriptase
- simple, but unpredictable because the 2nd strand priming event can occur randomly along the mRNA template
- must cleave loop with S1 nuclease
template switching RT
when RT reaches the end of mRNA it often adds a few Cs (non-templated)
these can bind to G residues of a Template Switching (TS) oligo
RT can then extend across the TS oligo giving common sequence on the 3’ of all the transcripts
PCR elements
DNA template, primers complementary to ends of target, dNTP, thermostable DNA polymeaser (pol)
PCR steps
- assemble reaction mix minues pol on ice
- add polymerase and start first melt cycle
- anneal at temp that only allows primers to bind correct sequence (usually 5C below primer Tm)
- elongate (72 C for 1 min/kb)
- repeat (heat, anneal, elongate) 30-35x
- long final elongation to finish all ends
heat-stable DNA polymerase
Taq!!!!!: can remain active after every heating up step; does NOT have proofreading activity and thus makes mistakes
Physion: have proofreading activity and 50x lower error rate
what does CODIS show?
highly polymorphic regions in chromosomes
when does pre-replication complex form?
- during G1
- it is “licensed”: low CDK activity
- cant fire it
why does formation and activation of pre-RC occurs in separate parts of the cell cycle?
different levels of CDK activity
(low @ G1 and G2; high @ S)
DNA polymerase architecture
- 3’ –> 5’ exonuclease at the palm
- incoming nucleotides come between thumb and fingers; if it’s in right form, closes
DNA polymerase open vs closed form
closed: orients substrates properly for catalysis
open: much slower rate of catalysis; time for wrong base to dissociate
how are DNA polymerase mistakes proofread?
- polymerase mispairs dC with dT
- polymerase repositions the mispaired 3’ terminus into the 3’ –> 5’ exonuclease site
- exonuclease hydrolyzes the mispaired dC
- 3’ terminus repositions back to the polymerase site
- polymerase adds the correct nucleotide, dA
what catalyzes polymerase and exonucleases?
2 metal ions and the DNA
NO AMINO ACIDS
what defines helicase polarity?
the tracking strand, NOT complement
E. Coli replication
- elongation (Okazaki fragment synthesis)- helicase stimulates primase
- RNA priming- primer-template junction stimulates clamp loader
- clamp loader loads clamp
- clamp recriots and increases processitivity of polymerase –> Okazaki fragment maturation
- polymerase stimulates helicase and polymerase dissociates
SS binding proteins
protect SS DNA and remove 2nd structure
what removes 5’ –> 3’ exonuclease
subtilism (useful in making blunt ends nd making DS DNA from SS)
yeast two-hybrid analysis
protein-protein interactions
determine whether interaction between two proteins is enough to form a functional bridge between the DNA binding and activation domains of a yeast transcription factor
EMSA
to detect protein-DNA complexes
electorphoresis with a non-denaturing gel
