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.