MCB 110:Biochemistry of the Central Dogma of MB
Part 1.
DNA
replication,
repair and
genomics
(Prof. Alber)
Part 2.
RNA &
protein
synthesis.
Prof. Zhou
Part 3. Membranes, protein secretion, trafficking and signaling
Prof. Nogales
MCB 110:Biochemistry of the Central Dogma of MB
Part 1.
DNA
replication,
repair and
genomics
(Prof. Alber)
Part 2.
RNA &
protein
synthesis.
Prof. Zhou
Part 3. Membranes, protein secretion, trafficking and signaling
Prof. Nogales
DNA structure summary 1
1.
2.
W & C (1953) modeled average DNA (independent of sequence) as
an:
anti-parallel, right-handed, double helix with H-bonded base pairs
on the inside and the sugar-phosphate backbone on the outside.
Each chain runs 5’ to 3’ (by convention).
Profound implications: complementary strands suggested
mechanisms of replication, heredity and recognition.
Missing
Structural variation in DNA as a function of sequence
Tools to manipulate and analyze DNA (basis for
biotechnology, sequencing, genome analysis)
DNA schematic (no chemistry)
1. Nucleotide =
sugar-phosphate
+ base
2. DNA strands
are directional
3. Duplex strands
are antiparallel and
complementary.
Backbone outside;
H-bonded bases
stacked inside.
4. The strands
form a double
helix
Nucleic-acid building blocks
nucleoside
glycosidic
bond
nucleotide
Geometry of DNA bases and base pairs!
C
G
T
A
H-bonds satisfied
Similar width
Similar angle to glycosidic bonds
Pseudo-symmetry of 180° rotation
Major groove and minor groove definitions
Major groove
Major groove
Opposite the glycosydic bonds
Minor groove
Minor groove
Subtended by the glycosydic bonds
Comparison of B DNA and A DNA (formed at different humidity)
Bps near helix axis
Bps off helix axis
Major groove
(winds around)
3.4- 3.6 Å
Minor groove
(winds around)
bp/turn
Base tilt
Major groove
Minor groove
P-P distance
10
small
wide
Narrow
6.9 Å
11
20°
narrow & deep
wide & shallow
5.9 Å
Average structure of dsRNA (like A DNA)
3’
5’
“side”
view
Bases tilted
5’
3’
“End”
view
Minor groove
shallow and wide
Major groove
deep
and narrow
(distortions
needed for
proteins to
contact bases)
Twist/bp ~32.7°
~11 bp/turn
DNA structure and stability
DNA structure varies with sequence
1. “Dickerson dodecamer” crystal structure
2. Twist, roll, propeller twist and displacement
3. Variation in B-DNA and A-DNA
Proteins recognize variations in DNA structure
DNA stability
Depends on sequence & conditions
Forces that stabilize DNA: H-bonds, “stacking”,
and interactions with ions and water
Crystal structure of the “Dickerson dodecamer”
Experiment -- 1981
Synthesize and purify 12-mer: d(CGCGAATTCGCG) = sequence
Crystallize
Shine X-ray beam through crystal from all angles
Record X-ray scattering patterns
Calculate electron density distribution
Build model into e- density and optimize fit to predict the data
Display and analyze model
Results
B-DNA!!
The structure was not a straight regular rod.
There were sequence-dependent variations
(that could be read out by proteins).
Two views of the Dickerson dodecamer
1. Double helix: Anti-parallel strands, bps “stacked” in the
middle
2. Not straight (19° bend/12 bp, 112 Å radius of curvature)
3. Core GAATTC: B-like with 9.8 bp/turn
4. Flanking CGCG more complex, but P-P distance = 6.7 Å (like
B)
5. Bps not flat. Propeller twist 11° for GC and 17° for AT
Nomenclature for helical parameters
Propeller twist: dihedral angle
of base planes.
Displacement: distance from
helix axis to bp center
Slide: Translation along the
C6-C8 line
Slide
Twist: relative rotation around
helix axis
Roll: rotation angle of mean bp
plane around C6-C8 line
Tilt: rotation of bp plane around
pseudo-dyad perpendicular
to twist and roll axes
Propeller twist, roll and slide
Slide = -1 Å to avoid clash *
No roll or propeller twist
20° propeller twist
Or roll = 20 ° and slide = + 2Å to
promote cross-chain purine stacking
Slide and helical twist
Slide = translation along the long (C6-C8) axis of the base pair
Regular DNA variations
B-like
A-like
Helical parameters of the dodecamer
C1/G24
G12/C13
Range
4.9-18.6° 32.2-41.4°
8.1-11.2
3.14-3.54 Å
Helical parameters of the dodecamer
C1/G24
G12/C13
Range
4.9-18.6° 32.2-41.4°
8.1-11.2
3.14-3.54 Å
Helical parameters of the dodecamer
C1/G24
G12/C13
Range
4.9-18.6° 32.2-41.4°
8.1-11.2
3.14-3.54 Å
Base “stacking” maximizes favorable interactions
Clashes due to
propeller twist can
be alleviated
by positive roll
(bottom left) or
changes in helical
twist (right)
N atoms close
N atoms separated
 roll
 helical twist
Different patterns of H-bond donors and
acceptors bases in different base pairs (gray)
Major groove side (w)
Most differences in
H-bond donors and
acceptors occur in
the major groove!
Sequence-specific
recognition uses
major-groove
contacts.
Minor groove side (S)
Seeman, Rosenberg & Rich (1976),
Proc Natl Acad Sci USA 73, 804-8.
Lac repressor headpiece binds differently to
specific and nonspecific DNAs
Symmetric operator
Natural operator
Bent DNA
Straight DNA
Nonspecific DNA
E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helix
LacI tetramer
E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helix
Repressor tetramer
loops DNA
E. coli catabolite activator protein (CAP)
Stabilizes kinks in the DNA
Human TATA binding protein binds in the
minor groove and stabilizes large bends
Twist along the DNA
DNA
bent
Human TATA binding protein binds in the
minor groove and stabilizes large bends
TBP
TBP
DNA
View into the saddle
End view
DNA bending by E. coli AlkA DNA glycosylase
66° bend
Leu125 inserted
into the DNA
duplex!
Base flipping in DNA repair enzymes
Human Alkyl
Adenine DNA
Glycosylase
Phage T4
A Glycosyl
Transferase
,AGT
What causes bases to flip out?
What cause bases to flip out?
Thermal fluctuations
Fluctuations include denaturation
Native
Denatured
+
Tm = 50/50
native/denatured
T
Tm depends on?
Tm depends on?
DNA Length
Base composition
DNA Sequence
Salt concentration
Hydrophobic and charged solutes
Bound proteins
Supercoiling density
Fraction denatured
Length dependence of DNA stability
10
20
30
Temperature °C
No further increase
> ~50 base pairs
Tm depends on G+C content
Why?
Tm depends on G+C content
Why? GC bps contain 3 H-bonds and stack better.
Calculated base stacking energies
GC best
AT worst
Tm depends on ionic strength
High KCl stabilizes duplex DNA
Why?
Other conditions that change Tm
}
Mg2+ ions
Stabilize (why?)
Polyamines: spermidine and spermine
+
+
+
NH3-CH2-CH2-CH2-NH2-CH2-CH2-CH2-CH2-NH3
NH3-CH2-CH2-CH2-NH2-CH2-CH2-CH2-CH2-NH2-CH2CH2-CH2-NH3
+
+
+
+
DMSO
H3C
formamide
CH3
HC NH2
C
O
O
} Destabilize (why?)
Duplex stability depends on length (to a point)
and base composition (GC content)
Two formulas for oligonucleotide Tm
1. Tm = (# of A+T) x 2 + (# of G+C) x 4
2. Tm= 64.9 +41 x ((yG+zC-16.4)/
(wA+xT+yG+zC)) where w, x, y, z are the
numbers of the respective nucleotides.
Summary
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
DNA structure varies with sequence.
Propeller twist, helix twist, roll, slide, and displacement (local
features) vary in each base step.
These differences alter the positions of interacting groups
relative to ideal DNA.
Structural adjustments maximize stacking.
Proteins can read out base sequence directly and indirectly
(e.g. H2O, PO4 positions, structure and motions).
Proteins can trap transient structures of DNA.
Duplex stability varies with sequence, G+C > A+T
High salt, Mg2+, polyamines increase duplex stability.
DMSO and formamide decrease duplex stability.
Stability increases with oligonucleotide length up to a point.