Protein-DNA Interactions Flashcards
What are the two types of protein-DNA interactions?
- non-specific interactions with DNA (indirect readout)
- specific interactions with DNA bases (direct readout)
direct + in direct interactions determines specificity and strength
Interactions with the major grove can
provide more specificity because of the
higher number of specific molecular contacts (HB acceptor/donor, methyls for hydrophobic interactions)
What are non-specific interactions? Examples?
- don’t discriminate what base
- HB or charged contact to phosphate bb
- HB oxygen in DNA deoxyribose
- stacking interaction between aromatic aa residues and ribose ring
Indirect contacts can happen through…
a bridging water molecule
non-specific interactions can occur through minor groove of DNA because…
Can access bb
Specific interactions are often H-bonding to acceptor or donor groups by …
amino acid side chains TO bases
discriminate what base is contacted
The specificity of protein-DNA interactions are influenced by…
- nature of contact between protein and DNA (specific vs non specific)
- length of target site
- how proteins interact with the substrate (dimer doubles specificity)
restriction endonuclease enzymes have very specific contacts because
they are homodimers, make the same interactions directly with DNA on both palindromic sequences
What is a common motif in DNA binding proteins?
helix-turn-helix
- a-helix fits into major groove of B-DNA
why do we use non-denaturing gel?
don’t want to disrupt protein complexes
why do we use polyacrylamide rather than agarose?
- better resolution
- substrates are short DNA fragments
how could you determine the Kd (binding affinity) on an EMSA gel?
- binding affinity of protein to DNA
- where bands of free DNA and protein-DNA look same intensity (50% binding)
What is the K(D) equation?
[protein][ligand(DNA)]/[protein-ligand(DNA)] = [k(d)]/[k(a)]
tells you th minimal amount of a protein required for binding
kd = dissociation rate | ka = association rate
What more information do you get from DNAseI footprinting compared to EMSA?
see multiple locations where the protein binds (rather than a yes or no)
DNAaseI footprinting gel can be converted to a
isotherm binding curve
compare protein concentration added vs fraction bound
disadvantages of DNAseI footprinting?
- time consuming
- no info on importance of specific positions within footprinted region
- digestion can be sterically hindered and often report footprints that are larger than they actually are
advantages of DNAseI footprinting?
- measures protein-DNA interactions under equilibrium conditions
- report info on individual bind sites
disadvantages of EMSA?
non-equilibrium method – can miss weak or unstable binding reactions
Advantages of Chemical footprinting?
- very high resolution
- provides single nucleotide resolution
Disadvantages of Chemical footprinting?
- if sites are far apart, multiple gels required
- free radical hydroxyl used could attack other areas -> appears to be no binding even if there is
Analysis of protein-DNA interactions can provide insight into
- Affinity of interactions
- Region of DNA contacted by protein
- Information about molecular contacts made by bases
What is a sequence logo?
The height of the individual letter is proportional to the frequency of the letter within each column of the alignment
- Positions with high information (more bits) are likely critical for binding
if a position can accomodate every possible base,
if a possible can only accomodate one base,
there is high entropy (more variability) and no information
there is low entropy and maximal information
