Protein-DNA Interactions

Question 1

What are the two types of protein-DNA interactions?

Answer 1

1. non-specific interactions with DNA (indirect readout)
2. specific interactions with DNA bases (direct readout)

direct + in direct interactions determines specificity and strength

Question 2

Interactions with the major grove can
provide more specificity because of the

Answer 2

higher number of specific molecular contacts (HB acceptor/donor, methyls for hydrophobic interactions)

Question 3

What are non-specific interactions? Examples?

Answer 3

• 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

Question 4

Indirect contacts can happen through…

Answer 4

a bridging water molecule

Question 5

non-specific interactions can occur through minor groove of DNA because…

Answer 5

Can access bb

Question 6

Specific interactions are often H-bonding to acceptor or donor groups by …

Answer 6

amino acid side chains TO bases

discriminate what base is contacted

Question 7

The specificity of protein-DNA interactions are influenced by…

Answer 7

1. nature of contact between protein and DNA (specific vs non specific)
2. length of target site
3. how proteins interact with the substrate (dimer doubles specificity)

Question 8

restriction endonuclease enzymes have very specific contacts because

Answer 8

they are homodimers, make the same interactions directly with DNA on both palindromic sequences

Question 9

What is a common motif in DNA binding proteins?

Answer 9

helix-turn-helix
- a-helix fits into major groove of B-DNA

Question 10

why do we use non-denaturing gel?

Answer 10

don’t want to disrupt protein complexes

Question 11

why do we use polyacrylamide rather than agarose?

Answer 11

• better resolution
• substrates are short DNA fragments

Question 12

how could you determine the Kd (binding affinity) on an EMSA gel?

Answer 12

• binding affinity of protein to DNA
• where bands of free DNA and protein-DNA look same intensity (50% binding)

Question 13

What is the K(D) equation?

Answer 13

[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

Question 14

What more information do you get from DNAseI footprinting compared to EMSA?

Answer 14

see multiple locations where the protein binds (rather than a yes or no)

Question 15

DNAaseI footprinting gel can be converted to a

Answer 15

isotherm binding curve
compare protein concentration added vs fraction bound

Question 16

disadvantages of DNAseI footprinting?

Answer 16

• 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

Question 17

advantages of DNAseI footprinting?

Answer 17

• measures protein-DNA interactions under equilibrium conditions
• report info on individual bind sites

Question 18

disadvantages of EMSA?

Answer 18

non-equilibrium method – can miss weak or unstable binding reactions

Question 19

Advantages of Chemical footprinting?

Answer 19

• very high resolution
• provides single nucleotide resolution

Question 20

Disadvantages of Chemical footprinting?

Answer 20

• 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

Question 21

Analysis of protein-DNA interactions can provide insight into

Answer 21

• Affinity of interactions
• Region of DNA contacted by protein
• Information about molecular contacts made by bases

Question 22

What is a sequence logo?

Answer 22

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

Question 23

if a position can accomodate every possible base,

if a possible can only accomodate one base,

Answer 23

there is high entropy (more variability) and no information

there is low entropy and maximal information