L2 - DNA recognition 1 Flashcards
what is a good tertiary structure for DNA binding/recognition
helix-term-helix motif
biologists can tell function if a protein just by seeing shape - if a protein has a hth probably a DNA binding protein
what might long sequnces if DNA need to be ‘read’
multiple interactions
= means that shape/fold of DNA binding proteins is key
what does the catabolite activator protein (Cyclic AMP Receptor Protein) do
= CAP
turns genes on when glucose conc is low in bacteria
in order to work must bind very tightly to genes
what is CHIP seq* and whart does it show when done on CAP protein
measures how often a protein us bound across the genome
taller the spike = protein is commomnly/often bound o this gene
CAP regulates 100 genes - BUT more than 100 spikes ?
= shows CAP binds other places non-specifically
what do DNA binding proteins bond to
Intergenic regions
- between genes = promoters
What is affinity
affinity is how tightly a protein binds
- mesured by Kd
measures at equilbrium how much protein is bound and how much DNA is free
show the quation for Kd (affinity) and what a high and low kd mean
Kd = [P][D] P + D = PD
——–
[PD]
Kd is the discosociutaion constant
low Kd = more specific
specifcity
how tightly a protein binds to a site RELATIVE to another site
Ratio
measures affinity for 2 sites and compares
- high affinity for multiple sites = low specificity
band shift assay
measures affinity
DNA probe added with proteins to acrylamide gel
free DNA moves faster than bound DNA
= if protein has a high affinity for DNA probe/sequnce the band will be higher /move less
= kd is protein conc when half of DNA is bound
= a low kd means hifgh adffinity as less protein is needed to bind to 50% of DNA
what do the base pairs have that are read
specific functional groups
chemical signature reae by binding proteins like CAP
why would DNA biundintg proteins being positively charfged affect there affinity BUT not their specificity
allows them to ionically bind to negatively charged phosphate backbbone
BUT this is the same for all sites on DNA = not specific
what are the 4 determiners that affect the chemical signature and thefore what proteins canb bind
H-bond acceptor
H-bond donor
Methyl group (does not participate in H-bonding)
hydrogen - in a grpup that doesnt H bond
why is the major groove considered to contain more indormation than the minor
The two grooves (are formed because the two strands of DNA are not symmetrically aligned
Asymmetry creates one wider groove and one narrower
- major groove is more exposed and shows ALL 4 chemical features of EACH Bp
- minor is less exposed and only shows 3
major is thefdore preffered to bind to as shows diversity of info per base pair
how do you calculate probability of a specific sequnence appearing in DNA
there is a 1/4 chance a base will be the one you want
can calculate probability of a specifc sequnence
(1/4)n = to the power of n
a binding protein cannot recognise a sequnce longer than 10 base pairs naturally
so how do you do it ?
amino acids can recgonise more than 1 bp at a time or in base pair steps ahead of them
different teriary structures overcome problem
alpha helix suited to recognition in major groove
uinserted into groove and then side chains point outwards at regular intervals
