Protein-DNA Interactions
? Equilibria and free energies
? Estimate binding site occupancy
? Forces driving protein-DNA interactions
? Lambda repressor
? Methods to study protein-DNA interactions
A A
A A
D
D
A
A
AA
D M H
1
A AD M
Major
A-T
AA DMT-A
Minor
A-T
T-A
AA
AA
A A D HG-C
C-G
G-C
C-G
Major
H D A A
A AD
A AD
Minor
The 4 Major Groove Patterns Are Distinct
2
A A
A A
D
D
A
A
AA
D M H
1
Hyrdophobic Groups on Bases
3
Helix-Turn-Helix Motif
34 Ao
3 41
2
5
N-term
3’
4
Specificity Determinants? Generic Interactions?
5
6
>104
>103
Gln33 Networks with Gln44
Ser
? ?G =
4.5 kcal
Leu
? ?G =
>6.2 kcal
7
Conclusion
? DNA binding specificity from major groove
interactions
? Mutation in one AA affects neighbors
-->Protein-DNA interfaces contain an intricate
Network of hydrogen bonds
B DNA 434 Complex 9
4 bases don’t
contact protein
But very
Important!
Filter
Binding
vacuum
%
Bou
nd
Protein [nM]
0 20 40 60 80 100
100
50
Kd = 20 nM
10
Immunoprecipitaiton
%
Bou
nd
Protein [nM]
0 20 40 60 80 100
100
50
Kd = 20 nM
10
A
Sepharose
Gel-Shift
%
Sh
ifte
d
Protein [nM]
0 20 40 60 80 100
100
50
Kd = 20 nM
[Protein]
11
DNAse I
Footprinting
%
Pr
ote
cte
d
Protein [nM]
0 20 40 60 80 100
100
50
Kd = 20 nM
[Protein]
+DNAse I
12
Cooperative DNA binding
By the ? repressor
13
Interference Footprinting
DNA + MMS
+ Saturating [Protein]
Gel Shift
14
Electron Microscopy
15
YFPTilt
+Roll
Tilt
-Roll
19
Tilt
Roll
Interaction of TBP (TATA Binding Protein) With DNA
Major Groove
Minor Groove
17
Protein-Induced DNA Bending From Positive Roll
18
5’ 3’
Pyrimidine-Purine Steps Have Little Base Stacking
5’
3’ 3’
5’C
GA
T
19A (Lecture 1)
For advanced reading:
Nucleic Acids:
Structures,Properties,and Functions
V.A,Bloomfield
D.M,Crothers
I,Tinoco
8
Gln
? ?G =
5.2 kcal
Lys
? ?G =
4.1 kcal