Nucleic Acids Structures1-Discovery of DNA structure

2- A, B and Z conformations of dsDNA/dsRNA

• Not treated:-DNA topology-DNA Sequencing

5- Principles of DNA Recognition by sequence-specific DNA binding proteins

6- Principles of Nucleic Acids Denaturation

3- DNA tertiary StructuresNucleosome, G-quadruplex

4- Secondary and tertiary Structure of RNA

Polymeric StructureOf Nucleic Acids

• Links 3’-O of precedingnucleotide to 5’ of nextnucleotide

• 1 negative charge per residue

->5’-3’polarity

Watson and Crick (1953)

Rosalind Franklin(1950 or 1951)Chargaff. 1950: “It is, however, noteworthy

-whether this is more than accidental, cannot yet be said-that in all deoxypentose nucleic acids examined thus far the molar ratios of total purines to total pyrimidines,and also of adenine to thymine and of guanine to cytosine, were not far from 1”.

Watson and Crick (1953): “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”.

2) bases are in the keto conformation1) R. Franklin DNA fibers X-ray diffraction data

Spacing between

Phosphates = 3.4A

Helical Pitch = 34A

Maltese CrossIndicates an

Helical pattern

3) Chargaff’s rules:(G+C)/(A+T) can vary But (G+A)/(C+T) = G/C = A/T =1

4) Density measurements:~2 polymers/helix

5) C2’ endo sugar puckerconformation

Information that Watson and Crick used to propose the double helix model:

keto enol

Bragg’s Law: 2dsin Q = nl used to interpret X-ray diffraction pictures

Bragg's Law

Bragg’s law indicates an inverse relationship between diffraction angle and actual distances between repeated features in crystal/fiber

= repeated atomic features in the crystal or fiber

Rise/residue= 3.4 A

HelicalPitch= 34 A(10

residues/turn)

Essential features of the model thatproved correct:

1) Antiparallel right-handed double helix

2) Strands are linked by complementary sets ofdonors and acceptor groupson bases

The original model for DNA structureWatson and Crick (1953)

Nature 171, 964-967

complementary sets of donors and acceptor groups on bases

Watson-Crick Model

The DickersonDodecamer

X-ray structure(CGCGAATTCGCG)

A comparison of the Watson-Crick

model (1953)and of the first

B-DNA structure solved (1980)

PDB ID:1BNA

DNA Double HelixDefinitions

Bases Orientation

Pseudo Dyad Axis

Base pairs seenfrom above the helix(helical projection)

N3

N1

N

OdR

H

H

N3

N1

N7

N9

dR

O

H

N H

H

Major Groove>180°

Minor Groove<180°

Helical Axis

HN

N3

N1

N7

N9

dR

H

CH3

N1

N3

O

H

OdR

TA

N1

N3

N H

OdR

N3

N1

N7

N9

dR

O

N

H

H

H

H

GC

N3

N1

N

OdR

H

H

N3

N1

N7

N9

dR

O

H

N H

H

G C

CH3

N1

N3

O

H

OdR

H N

N3

N1

N7

N9

dR

H

AT

CH3

N1

N3

O

H

O

T

N3

N1

N7

N9

O

H

N H

H

G

dR

dR

N3

N1

N

OdR

H

H

N3

N1

N7

N9

dR

O

H

N H

H

G C

Isostericity of Watson-Crick Base Pairs (and non isostericity of non WC base pairs)

Example of a G-T non WC base pair

A

B

A B

H20

Ethanol

PDB ID = 115D

PDB ID:1BNA

Major differences : - A DNA is shorter than B DNA: 1 helix turn is 28.6A vs 34 A for B DNA. This is due to the 3’ endo sugar pucker in A

- The Bases of A-DNA are shifted away from the helical axis. This results in a deep major groove and in a shallow minor groove. There is a 6 A hole in a helical projection.

Sugar Pucker

C3’endo

C2’endo

Planar

BA

helical projection

B- DNA A- DNA

Sugar pucker C2'-endo C3'-endo

Rise/residue 3.4 Å 2.6 Å

Residues/turn 10.5 11

Helical twist 34˚ 33˚

Diameter 20 Å 26 Å

Tilt 6˚ 20˚

Propellor twist 12 ̊ 15 ̊

Exact values need not to be remembered…

Sugar puckering: C2’ endo or C3’ endo

7 Å 5.9 Å

Distance between Consecutive Phosphates:

dsRNA or A-DNA : C3’ endoB-DNA: C2’ endo

A-DNA B-DNA

Base tilting in A-DNA

Base pairs are more tilted in A-DNA.

A water spine (green dots)has been proposed to exist

in the minor groove of B-DNA that would stabilize the B-form

H20 is essentialin the transition

A <--> B DNA

A B

H20

This concept is controversialand will not be detailed further

Z-DNALeft handed Helix

jagged backbone

Occurs in DNAsequences with stretches of consecutiveG-C base pairs

Requires high saltin vitro

G nucleotides:Switch C2’endo -> C3’ endoanti -> Syn

C nucleotides:No change

www.mun.ca/biology/scarr/A_B_Z_DNA.html

PDB ID:1DCG

Nucleotides flipping and grooves in Z-DNA

Major

Minor

Major

Minor

Z-DNA

B-DNA Note: this simplified diagram only summarizes the conformationchanges during the B->Z transition – it does not accurately shows the Z structure

Glycosidic bond Anti /Syn conformationsAnti and Syn conformations are defined based on the torsion angle of the glycosidic bond

4'1'

94

The sequence of atoms chosen to define the torsion angle to define anti/syn conformation is: O4'-C1'-N9-C4 for purines - O4'-C1'-N1-C2 for pyrimidines.

4'1'

9 4

4'1'

1 2

Anti A/G: C1’-O4’ and N9-C4 are pointing away from each other

Anti C/T: C1’-O4’ and N1-C2 are

pointing away from each other

Syn A/G: C1’-O4’ and N9-C4 are

pointing in same direction

Syn- A

4'1'

9

4 4'

1'9

4

4'1'

94 4'

1'9

4

Anti A/G: C1’-O4’ and N9-C4 are pointing

away from each other

Syn A/G: C1’-O4’ and N9-C4 are

pointing in same direction

Anti /Syn conformations in pseudo-3D

A B Z

B ZABZ-DNAs

BackboneProfiles

HelicalProjections

A B Z

www.mun.ca/biology/scarr/A_B_Z_DNA.html

• There are conformations other than A/B/Z e.g.: conformations intermediate between A and B

Also Tertiary conformation of DNA

Why Study DNA Structure ?

• Structure and Sequence Recognition by DNA binding proteins

• Some non B-DNA structures are biologically relevant - dehydrated living forms- dsRNA is A form (see PDB: 2KYD)- DNA/RNA duplex (replication, transcription) is A form- Z-DNA might be associated with promoter elements, regulatory sequences

Binding of histones to DNA through electrostatic interactions:Histones are + charged, DNA is - charged

Double-Stranded DNA is wrapped around nucleosomes in eukaryotic cells

http://www.bio.miami.edu/dana/104/nucleosome.jpg

Consequences for: DNA Replication,DNA RepairTranscription

Double-Stranded DNA is wrapped around nucleosomes in eukaryotic cells

http://www.chem.ucsb.edu/~molvisual/dna_biochem.html

PDB ID:1AOI

G-quadruplex structures in telomeric DNA:case of (T2G4) repeats

Na+

Example of intrinsicDNA Tertiary Structure

PDB ID:156D

Secondary and Tertiary Structure of RNA

Single strandedness nature of RNA makes it able to“fold” on itself and base-pair with complementary segmentswithin the same molecule

Secondary and Tertiary Structure of transfer RNA (tRNA)

PDB ID = 3TRA

Secondary Structure of theM1 RNA, a component of RNase P(see RNA processing chapter)

1-Abundance of G:U base pairs

Secondary and Tertiary Structure of RNA: See other examples

in the RNA Processing and Translation Chapters

2-Pseudoknot:long rangebase-pairing

Two major observations:

“A” form toleratesthe geometry of G:U base pairs

Can you read ?(the sequence of this DNA)

Sequence-Specific Recognition of DNA by proteins:What do proteins “see” ?

CH3

N1

N3

O

H

OdR

H N

N3

N1

N7

N9

dR

H

HN

N3

N1

N7

N9

dR

H

CH3

N1

N3

O

H

OdR

Minorgroove

Majorgroove

A A

H AD

A

A A

HAD

A

AT

TAMinor

groove

Majorgroove

Conclusion: DNA binding proteins can differentiateA-T base pairs from T-A base pairs if they bind

from the major groove side, but not from the minor groove side

Distribution of H-bondsDonors (D) Acceptors (A)and Hydrophobic groups (H)

Recognition of Specific sequencesby DNA-binding proteins

N1

N3

N H

OdR

N3

N1

N7

N9

dR

O

N

H

H

H

H

N3

N1

N

OdR

H

H

N3

N1

N7

N9

dR

O

H

N H

H

A A

D

AA

D

A A

D

A AD

GC

G C

Conclusion: DNA binding proteins can differentiateG-C base pairs from C-G base pairs if they bind

from the major groove side, but not from the minor groove side

Minorgroove

Majorgroove

Minorgroove

Majorgroove

Patterns of H-bondsDonors (D), Acceptors (A),and Hydrophobic groups (H)available for recognition

Recognition of Specific sequencesby DNA-binding proteins

DS DNA (Helix) What influences the equilibrium ?(important because DNA is “opened”during replication and transcription)

2 SS DNAs (“random coils”)

In favor of double-stranded DNA

- Hydrogen bonds between strands (minor)

- Base stacking Interactions (major)

In favor of single-stranded DNA

- Electrostatic Repulsion between strands

- Entropic considerations:Increased entropy for ssDNA vs dsDNA

Experimental Studies of DNA denaturation

UV spectroscopic analysis of SS (denatured) vs DS (native) DNA

180 200 220 240 260 280

Wavelength (nm)

Denatured DNA

Native DNA

Relative Absorbance

Hyperchromic Effect:SS DNA > native DNA

“melting curves” for two differentDNA molecules (red and blue) show different “melting points” = 2 different Tms

DNA molecule

Increasin

g C

on

form

ation

al En

trop

y

Increasing Entropy

( 1 -> 2 molecules)

DNA melting is a cooperative process: this explains the sigmoid denaturation curves

The Tm of a DNA molecule is a linear function of its G-C content

Yakovchuk P et al. Nucl. Acids Res. 2006;34:564-574

The Tm of a DNA molecule is a linear function of its G-C content/this is not because of higher energyof 3 H-bonds (GC) vs 2 (AT)

Effect of G-C content on Stability is due to higherstacking of G-C basepairs compared to AT base pairs

DG BP ≅ contribution of stackingto the stability of base pair