MCB 110:Biochemistry of the Central Dogma of MB
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Transcript of MCB 110:Biochemistry of the Central Dogma of MB
MCB 110:Biochemistry of the Central Dogma of MB
Prof. Nogales
Part 3. Membranes, protein secretion, trafficking and signaling
Part 2.RNA & protein
synthesis.Prof. Zhou
Part 1.DNA
replication, repair and genomics
(Prof. Alber)
MCB 110:Biochemistry of the Central Dogma of MB
Part 2.RNA & protein
synthesis.Prof. Zhou
Prof. Nogales
Part 3. Membranes, protein secretion, trafficking and signaling
Part 1.DNA
replication, repair and genomics
(Prof. Alber)
DNA structure summary 1
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.
2. Each chain runs 5’ to 3’ (by convention).
Profound implications: complementary strands suggested mechanisms of replication, heredity and recognition.
MissingStructural variation in DNA as a function of
sequenceTools to manipulate and analyze DNA (basis for
biotechnology, sequencing, genome analysis)
DNA schematic (no chemistry)
3. Duplex strands are antiparallel and complementary. Backbone outside;H-bonded bases stacked inside.
2. DNA strands are directional
1. Nucleotide = sugar-phosphate + base
4. The strands form a double helix
Nucleic-acid building blocks
nucleoside
nucleotide
glycosidicbond
Geometry of DNA bases and base pairs!
C G T A
H-bonds satisfiedSimilar widthSimilar angle to glycosidic bondsPseudo-symmetry of 180° rotation
Major groove and minor groove definitions
Major groove Major groove
Minor groove Minor grooveSubtended by the glycosydic bonds
Opposite the glycosydic bonds
Comparison of B DNA and A DNA (formed at different humidity)
bp/turnBase tiltMajor grooveMinor grooveP-P distance
10smallwide
Narrow6.9 Å
1120°
narrow & deepwide & shallow
5.9 Å
Major groove(winds around)
Minor groove(winds around)
3.4- 3.6 Å
Bps near helix axis Bps off helix axis
Average structure of dsRNA (like A DNA)
“side” view
“End” view
3’
5’
5’
3’
Minor groove shallow and wide
Major groove deep and narrow (distortions needed for proteins to contact bases)
Twist/bp ~32.7°~11 bp/turn
Bases tilted
DNA structure varies with sequence1. “Dickerson dodecamer” crystal structure2. Twist, roll, propeller twist and displacement3. Variation in B-DNA and A-DNA
Proteins recognize variations in DNA structure
DNA stabilityDepends on sequence & conditionsForces that stabilize DNA: H-bonds, “stacking”,
and interactions with ions and water
DNA structure and stability
Crystal structure of the “Dickerson dodecamer”
Synthesize and purify 12-mer: d(CGCGAATTCGCG) = sequenceCrystallizeShine X-ray beam through crystal from all anglesRecord X-ray scattering patternsCalculate electron density distributionBuild model into e- density and optimize fit to predict the dataDisplay and analyze model
Experiment -- 1981
ResultsB-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/turn4. 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
AT6. Hydration: water, water everywhere on the outside
(not shown).
Nomenclature for helical parameters
Propeller twist: dihedral angle of base planes.
Displacement: distance fromhelix axis to bp center
Slide: Translation along the C6-C8 line
Twist: relative rotation aroundhelix axis
Roll: rotation angle of mean bp plane around C6-C8 line
Tilt: rotation of bp plane aroundpseudo-dyad perpendicularto twist and roll axes
Slide
Propeller twist, roll and slide
No roll or propeller twist
20° propeller twist
Slide = -1 Å to avoid clash *
Or roll = 20 ° and slide = + 2Å topromote 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 alleviatedby 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)
Minor groove side (S)
Most differences inH-bond donors andacceptors occur inthe major groove!
Sequence-specificrecognition usesmajor-groove contacts.
Seeman, Rosenberg & Rich (1976),Proc Natl Acad Sci USA 73, 804-8.
Lac repressor headpiece binds differently to specific and nonspecific DNAs
Nonspecific DNA
Symmetric operator Natural operator
Bent DNA
Straight DNA
E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helixLacI tetramer
E. coli lac repressor tetramer binds 2 duplexes
Headpiece
Hinge helix
NH2
N-subdomain
C-subdomain
Tetramerization helixRepressor 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
DNAbent
Human TATA binding protein binds in the minor groove and stabilizes large bends
View into the saddle End view
DNA
TBP TBP
DNA bending by E. coli AlkA DNA glycosylase
Leu125 insertedinto the DNA
duplex!
66° bend
Base flipping in DNA repair enzymes
Human AlkylAdenine DNAGlycosylase
Phage T4A
Glycosyl Transfera
se,AGT
What causes bases to flip out?
What cause bases to flip out?
Thermal fluctuations
Fluctuations include denaturation
T
+
Native Denatured
Tm = 50/50 native/denatured
Tm depends on?
Tm depends on?
DNA LengthBase composition
DNA SequenceSalt concentration
Hydrophobic and charged solutesBound proteins
Supercoiling density
Length dependence of DNA stability
Fract
ion
den
atu
red
Temperature °C
10
20
30
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
AT worst
GC best
Tm depends on ionic strength
High KCl stabilizes duplex DNAWhy?
Mg2+ ionsPolyamines: 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
Other conditions that change Tm
OO
Stabilize (why?)
Destabilize (why?)
}
}
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.
Duplex stability depends on length (to a point)and base composition (GC content)
Summary1. DNA structure varies with sequence.2. Propeller twist, helix twist, roll, slide, and
displacement (local features) vary in each base step.3. These differences alter the positions of interacting
groups relative to ideal DNA.4. Structural adjustments maximize stacking.5. Proteins can read out base sequence directly and
indirectly (e.g. H2O, PO4 positions, structure and motions).
6. Proteins can trap transient structures of DNA.7. Duplex stability varies with sequence, G+C > A+T8. High salt, Mg2+, polyamines increase duplex
stability.9. DMSO and formamide decrease duplex stability. 10. Stability increases with oligonucleotide length up to
a point.