Robert McKenna
Structural biologist
Office:
LG179 UFBI
email:
[email protected]
Nucleic Acid-Binding Proteins (13-14)
-Clearly of great biological importance since these proteins
are involved in replication or expression of genetic information.
-Of great interest are those that bind to specific sequences:
Helical structure
deoxyribonucleic acid (DNA)
Tertiary structure
ribonucleic acid (RNA)
Before we can understand specific interactions we need to
understand DNA and RNA structures.
The DNA structure:
linear polymer of monomeric units, the nucleotides
Three components:
A base
A sugar
A Phosphate group
DNA contains the bases:
A,G,C,T
whereas RNA contains the bases:
A,G,C,U. Thymine is 5-methyluracil
Watson-Crick Model:
Hydrophilic phosphate-deoxyribose backbone on the outside.
Bases (Paired by H-bonding) - are stacked on one another
- planes perpendicular to the helix axis.
T
A DNA Base Pairs C
G
0.29 nm
0.28 nm
5
6
4
3
6
1
2
1
2
7
5
4
3
0.30 nm
C1’
7
5
8
9
4
6
6
C1’
3
1
2
0.30 nm
1
2
C1’
0.29 nm
1.08 - 1.11 nm
1.08 nm
5
8
9
4
3
N
C1
-There are THREE forms of ds DNA
which exist under different conditions
A DNA
Dehydrated
Nonphysiological
conditions
B DNA
Fully hydrated
as in vivo
physiological
conditions
Z DNA
G-C base
pairs only
Light = Base pairs, Dark = sugar-phosphate backbone.
HELICES
Helical staircase grooves are same width and depth.
In DNA
Edges of bases are wider in the major groove than in the minor
groove, because of asymmetrical attachment of the base pairs to
the sugar- phosphate backbone.
Bulky sugar-phosphate backbone
on edges of helices > grooves within
which the bases are exposed.
Bulky sugar-phosphate backbone on edges of helices >
grooves within which the bases are exposed.
Bases
B
DNA HELICES
B
A
Sugar-phosphate
Helical axis
B-DNA - Helical axis runs through the center of each base pair
which are stacked almost perpendicular to the axis. Major and
minor grooves are the same depth, but major groove is wider.
A-DNA - Helical axis is shifted from the center of the bases into the
major groove. Base pairs are not perpendicular to the axis.
They are tilted 13° to 19°. Major groove is deep, minor groove
is shallow.
SECONDARY STRUCTURE : DNA HELICES
1.
B Form - Major form found in cells (described by W&C).
Properties
a.
Right handed helix coiled around a common axis
b.
Chains antiparallel
c.
Hydrophobic bases inside; Hydrophilic sugar-P outside
d.
Dimensions:
23 Å Diameter
3.4Å Between bases
~ 10 bases per turn : 34Å
e.
H-bonding
A=T GºC
f.
g.
Base sequence is unrestricted.
Major and minor groove - same depth, different width.
B Form
3’
5’
Bases
5
Sugar-P backbone
4
1
3
2
3.4nm
3’
5’
Double helix is stabilized by
Hydrophobic interactions, base stacking
H-bonding between bases
Specific Base sequence recognition in B-DNA
M
M
m
m
M
m
Interactions of proteins with the
sugar-phosphate backbone are non-specific.
N and O atoms at the edges
of base pairs can made
H-bonds to side-chains of
proteins.
H in C and methyl group in
T > sequence-specific
recognition sites.
2. A form
-H2O
a. Dehydrated form è A form.
b. Right handed helix - bases lie to the outside
and are tilted (13-19°) relative to the helix axis.
Axis shifted into the major groove.
d. Major groove is deep, minor groove shallow.
c. ds RNA adopts A form structures.
3. Z form
ACGCGCGTAC
a. Higher energy form, left handed helix
b. Alternating Py - Pu segments (+in A and B form)
c. Helix stabilized by
- High salt
- DNA binding proteins
- Methylation of C residues
d. More open form, may be important for gene
regulation/ genetic recombination
Zigzag
Narrow
minor
groove,
no major
groove.
Protein-DNA Interactions:
DNA-protein complexes
DNA-binding motifs
THREE commonly found
HELIX-TURN-HELIX
ZINC FINGER
LEUCINE ZIPPER
Helix-Turn-Helix (HTH)
Example Structure of Lambda Cro
66 aa (per monomer), functions as dimer
each monomer 3 b-strands and 3 a-helices
HTH motif (a2 and a3)
Dimer interaction
is with the b-strand 3
Example Structure of Lambda Repressor
236 aa (per monomer), functions as dimer
each monomer 2 domains
N-terminal domain binds DNA (92 aa)
Similar to Cro (with b-strands replaced
with a-helices)
HTH motif (a2 and a3)
Dimer interface between a5
Importance of HTH and helix a 3 in DNA recognition:
Dimeric nature > places a 3 helix 3.4 nm apart
allows recognition of major groove of DNA (palindromic sequence)
Common features of HTH mofit
Helix 2
Helix1
Residue 9 Glycine
Residue 4 & 15 buried
Residue 8 &10 partially buried
Residues 3-7 &15-20 helical
Residue 5 no branched side chain
HTH encoded in homeoboxes in Eukaryotes
(60 aa) transcription factors
Example POU structure
Known to work in pairs
Zinc finger families
First discovered transcription factor TFIIIA (Xenopus)
344 aa ( 9 repeats of 30aa -fingers)
Zinc important in structural stability
Families of Zinc fingers
Classic:
2 cysteine and
2 histidine residues
Usually have several hydrophobic residues
Examples of Zinc fingers
Base recognition
Leucine Zipper:
First discovered in yeast transcription factor GCN4
Helical wheel plot of
sequence showed every 7th
residue leucine
> a helix with leucine on
one side
Example a) GCN4, b) Max, c) Fos, d) jun,
N-terminus DNA recognition
sequence )
function as dimer
palindromic sequences
scissors grip model
A helical motif follows curvature of DNA major grove
Many DNA binding proteins use the a-helix to bind to specific DNA
sequences
Recognize the bases in the major groove of the DNA
(donor/acceptor pattern)
Note: there are other motifs that recognize DNA in a more open form