2. 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

3. 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

4. DNA structure summary 1
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)

5. DNA schematic (no chemistry)
1. Nucleotide =
sugar-phosphate
+ base
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

6. Nucleic-acid building blocks
nucleoside
glycosidic
bond
nucleotide

7. 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

8. Major groove and minor groove definitions
Opposite the glycosydic bonds
Minor groove
Minor groove
Subtended by the glycosydic bonds

9. Comparison of B DNA and A DNA (formed at different humidity)Å
Bps near helix axis
Bps off helix axis
Major groove
(winds around)
Minor groove
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

10. Average structure of dsRNA (like A DNA)3’ 5’
Minor groove shallow and wide
Major groove deep
and narrow
(distortions needed for proteins to contact bases)
“side”
view
Bases tilted 5’ 3’
Twist/bp ~32.7°
~11 bp/turn
“End”
view

11. 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

12. Crystal structure of the “Dickerson dodecamer”
Experiment
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).

13. Two views of the Dickerson dodecamer
Double helix: Anti-parallel strands, bps “stacked” in the middle
Not straight (19° bend/12 bp, 112 Å radius of curvature)
Core GAATTC: B-like with 9.8 bp/turn
Flanking CGCG more complex, but P-P distance = 6.7 Å (like B)
Bps not flat. Propeller twist 11° for GC and 17° for AT
Hydration: water, water everywhere on the outside (not shown).

14. 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

15. 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

16. Slide and helical twist
Slide = translation along the long (C6-C8) axis of the base pair

17. Regular DNA variations
B-like
A-like

18. Helical parameters of the dodecamer
C1/G24
G12/C13
Range ° ° Å

19. Helical parameters of the dodecamer
C1/G24
G12/C13
Range ° ° Å

20. Helical parameters of the dodecamer
C1/G24
G12/C13
Range ° ° Å

21. 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

22. 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.
Seeman, Rosenberg & Rich (1976),
Proc Natl Acad Sci USA 73,
Minor groove side (S)

23. Lac repressor headpiece binds differently to specific and nonspecific DNAs
Symmetric operator
Natural operator
Bent DNA
Straight DNA
Nonspecific DNA

24. E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helix
LacI tetramer

25. E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helix
Repressor tetramer
loops DNA

26. E. coli catabolite activator protein (CAP)
Stabilizes kinks in the DNA

27. Human TATA binding protein binds in the minor groove and stabilizes large bends
Twist along the DNA
DNA
bent

28. Human TATA binding protein binds in the minor groove and stabilizes large bends
TBP
TBP
DNA
View into the saddle
End view

29. DNA bending by E. coli AlkA DNA glycosylase
Leu125 inserted
into the DNA
duplex!
66° bend

30. Base flipping in DNA repair enzymes
Human Alkyl
Adenine DNA
Glycosylase
Phage T4
A Glycosyl Transferase,AGT

31. What causes bases to flip out?

32. What cause bases to flip out?
Thermal fluctuations

33. Fluctuations include denaturation
Native
Denatured +
Tm = 50/50
native/denatured T

34. Tm depends on?

35. Tm depends on? DNA Length Base composition DNA Sequence
Salt concentration
Hydrophobic and charged solutes
Bound proteins
Supercoiling density

36. Length dependence of DNA stability
No further increase
> ~50 base pairs 10 20 30
Fraction denatured
Temperature °C

37. Tm depends on G+C content
Why?

38. Tm depends on G+C content
Why? GC bps contain 3 H-bonds and stack better.

39. Calculated base stacking energies
GC best
AT worst

40. Tm depends on ionic strength
High KCl stabilizes duplex DNA
Why?

41. Other conditions that change Tm}
Mg2+ ions
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 formamide
H3C CH3 HC NH2 C
Stabilize (why?) }
Destabilize (why?) O O

42. Two formulas for oligonucleotide Tm
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= x ((yG+zC-16.4)/
(wA+xT+yG+zC)) where w, x, y, z are the
numbers of the respective nucleotides.

43. Summary 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.

44. Chemical structures of 4 bases each in DNA and RNA
only
DNA
only
DNA and RNA

45. Ribo-AGUC chain Chain is directional. Convention: 5’ 3’.
Chemical schematic One-letter code
Chain is directional. Convention: 5’ 3’.

46. Six backbone dihedral angles () per nucleotide
Is ssDNA floppy or rigid?

47. Two orientations of the bases: Anti and syn

48. “Fiber diffraction” pattern revealed dimensions and helix of “B” DNA
Experiment
X-rays
DNA
fiber
X-ray
film
Conclusion: Helix with 10 bp/repeat and 3.4 Å between bps

49. Average structure of “B” DNA
Ball-and-stick Space filling
“side”
view
“End”
view

50. Average structure of “B” DNA
Ball-and-stick Space filling
5’ ’
“side”
view
Minor groove (narrow)
and
Major groove (wide)
R-handed
Helix
Anti-parallel
strands
“End”
view
Equal twist/bp (36°)
10 bp/turn

51. Average structure of “A” DNA
Ball-and-stick Space filling 3’ 5’
Minor groove (shallow
and wide)
Major groove (deep
and narrow)
“side”
view
Bases tilted
~20° 5’ 3’
Twist/bp ~32.7°
~11 bp/turn
“End”
view