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more bp/turn means that?
it is more loose
how to create negative supercoils
by unwinding DNA and resealing it.
how to create positive supercoils
breaking DNA, overwinding DNA, and resealing
what are the two forms of underwound DNA?
- in relaxed with loose section
what is the linking number
- always positive integer (b/c need to have full turn in order to reseal it)
- number of times one strand wraps around the other
- it cannot change unless DNA is cut (one or both strands)
T for Twist
- total bp/ (bp/turn)
- if the number of bp/turn increase (T decrease)
what effect does unwinding DNA does for bp/turn?
increase bp/turn, decrease in twist
what is writhe
number of supercoils/number of turns of the double helix axis around the superhelical axis
everytime you unwind by 1
you get 1 supercoil
how does S value (sedimentation value) change with supercoiling?
increases as supercoiling increases
what is the effect of EtBr
unwinds DNA (bp/turn increases)
what happens when you unwind DNA without cutting it?
- create supercoils
- cannot change linking number unless DNA is broken
- neg Twist (when you increase the bp/turn), which means positive Writhe if L does not change.
when DNA is being replicated
positive supercoils are created (not breaking DNA)
function of TOP 1
- do not require ATP
- use energy in SC to relieve SC
- nick one strand to allow for rotation and then reseal after removing one SC.
- change in L by 1 at a time
function of TOP II
- change L by 2
- need ATP
- breaks both strands, one strand threads through to relieve 2 supercoils.
bacterial gyrase adds what
- two negative supercoils
- they are also TOP II
3 tricks of TOP II
- remove neg supercoils
- tie knots
- decatenate ccDNA (ATP)
why is decatenation important?
after DNA replication, daughter chromosomes must be decateneated
what kind of supercoil does eukaryotic chromosomes have?
naturally negative supercoiled
how can we use Ethidium to see what kind of supercoil the DNA is?
- negative supercoil with high s value: as you increase in Ethidium, you get relaxed plasmid (migrate slower, low s value) and then you get positive supercoil (high s value)
- if it was originally positive then DNA would be more twisted and have a higher s value
what is quantitative real time PCR? (QPCR)
- used to monitor eery round of increase in particular fragment
- when piece of DNA is amplified, quencher and flurophore separated, flurophore emits light
nuclease hypersensitive sites (HSS)
- developmentally regulated (they come and go.
- they are site specific
- located in promoter regions
Glucocorticoid receptor (GR) bind hormone
- binds GRE
- CBP (coactivator) is recruited by GR. CBP is a HAT.
- chromatin remodeling complex comes in
- exposes TATA binding regin
- GTF bind once opened and transcription begins after phosphorylation of CTD
how does bromodomain act enzymatically
within the subunit of TFIID (TAF250), which binds modified histones (acetylated), acetylation causes more acetylation to unzip region
what are chromatin remodeling enzymes?
- SWI/SNP (swiff/sniff)
- they contain ATPase in part of complex (to rip off histone or to alter chromatin structure)
how can TATA box be exposed/remodeled?
- conformational change of DNA (remodeled Nucs) to reveal TATA using SWI/SNF+ATP
function of FACT
rip all histones out of DNA, naked nucleosomal DNA, then use DNAse
function of SWR1
- histone exchange
- H2A/H2B remodeler and assembly factor
- pop out H2AZ/H2AB dimer
- H2AZ specific
what pathways does enhancer affect?
- RNAPII and mediator
- TAFs and TBP
- histone modification complex
- chromatin remodeling complex
what does HP1 bind with
- h3 methK9 (chromodomain)
- bind up to 3 methyl groups, which does not change charge, but makes more hydrophobic
sir protein bind
silent information protein bind H4 deaceylated lysine tails.