1

MCB 110:Biochemistry of the Central Dogma of MB

Prof. NogalesPart 3. Membranes, protein secretion, trafficking and signaling

Part 2.RNA & protein

synthesis.Prof. Zhou

Part 1.DNA replication,

repair andgenomics

(Prof. Alber)

MCB 110:Biochemistry of the Central Dogma of MB

Part 2.RNA & protein

synthesis.Prof. Zhou

Prof. NogalesPart 3. Membranes, protein secretion, trafficking and signaling

Part 1.DNA replication,

repair andgenomics

(Prof. Alber)

2

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 onthe inside and the sugar-phosphate backbone on the outside.

2. Each chain runs 5’ to 3’ (by convention).

Profound implications: complementary strands suggested mechanismsof 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 strandsare antiparallel andcomplementary.Backbone outside;H-bonded basesstacked inside.

2. DNA strands are directional

1. Nucleotide = sugar-phosphate + base

4. The strands form a double helix

3

Nucleic-acid building blocksnucleoside

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

4

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

5

Average structure of dsRNA (like A DNA)

“side” view

“End” view

3’

5’

5’

3’

Minor grooveshallow and wide

Major groove deepand narrow(distortions neededfor proteins tocontact 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

6

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 middle2. 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).

7

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

8

Slide and helical twist

Slide = translation along the long (C6-C8) axis of the base pair

Regular DNA variations

B-like A-like

9

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

10

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 topropeller twist canbe alleviatedby positive roll(bottom left) orchanges in helicaltwist (right)

N atoms close

N atoms separated

Δ roll Δ helical twist

11

Different patterns of H-bond donors andacceptors 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 tospecific and nonspecific DNAs

Nonspecific DNA

Symmetric operator Natural operator

Bent DNA

Straight DNA

12

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

13

E. coli catabolite activator protein (CAP)

Stabilizes kinks in the DNA

Human TATA binding protein binds in theminor groove and stabilizes large bends

Twist along the DNA

DNAbent

14

Human TATA binding protein binds in theminor 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

15

Base flipping in DNA repair enzymes

Human AlkylAdenine DNAGlycosylase

Phage T4A Glycosyl

Transferase,AGT

What causes bases to flip out?

16

What cause bases to flip out?

Thermal fluctuations

Fluctuations include denaturation

T

+

Native Denatured

Tm = 50/50 native/denatured

17

Tm depends on?

Tm depends on?

DNA LengthBase compositionDNA Sequence

Salt concentrationHydrophobic and charged solutes

Bound proteinsSupercoiling density

18

Length dependence of DNA stability

Frac

tion

den

atur

ed

Temperature °C

10 20 30No further increase> ~50 base pairs

Tm depends on G+C content

Why?

19

Tm depends on G+C content

Why? GC bps contain 3 H-bonds and stack better.

Calculated base stacking energies

AT worst

GC best

20

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 NH2C

Other conditions that change Tm

OO

Stabilize (why?)

Destabilize (why?)

}

}

21

Two formulas for oligonucleotide Tm1. 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.

22

Chemical structures of 4 baseseach in DNA and RNA

RNAonly

DNAonly DNA and RNA

Ribo-AGUC chain

Chain is directional. Convention: 5’ 3’.

Chemical schematic One-letter code

23

Six backbone dihedral angles (α−ζ)per nucleotide

Is ssDNA floppy or rigid?

Two orientations of the bases: Anti and syn

24

“Fiber diffraction” pattern revealeddimensions and helix of “B” DNA

Experiment

X-rays

DNAfiber

X-rayfilm

Conclusion: Helix with 10 bp/repeat and 3.4 Å between bps

Average structure of “B” DNABall-and-stick Space filling

“side” view

“End” view

25

Average structure of “B” DNABall-and-stick Space filling

“side” view

“End” view

Minor groove (narrow)

and

Major groove (wide)

Equal twist/bp (36°)10 bp/turn

5’ 3’

R-handedHelix

Anti-parallelstrands

Average structure of “A” DNABall-and-stick Space filling

“side” view

“End” view

3’

3’5’

Minor groove (shallow and wide)

Major groove (deep and narrow)

Twist/bp ~32.7°~11 bp/turn

5’

Bases tilted~20°