Nucleic Acids Structures 1-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 Structures Nucleosome, G-quadruplex 4- Secondary and tertiary Structure of RNA

Polymeric Structure Of Nucleic Acids Links 3’-O of preceding nucleotide to 5’ of next nucleotide 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 conformation 1) R. Franklin DNA fibers X-ray diffraction data Spacing between Phosphates = 3.4A Helical Pitch = 34A Maltese Cross Indicates 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 pucker conformation Information that Watson and Crick used to propose the double helix model: keto enol Bragg’s Law: 2dsin  = n 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 Helical Pitch = 34 A (10 residues/turn) Essential features of the model that proved correct: 1) Antiparallel right-handed double helix 2) Strands are linked by complementary sets of donors and acceptor groups on bases The original model for DNA structure Watson and Crick (1953) Nature 171,

complementary sets of donors and acceptor groups on bases

Watson-Crick Model The Dickerson Dodecamer 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 Helix Definitions Bases Orientation

Pseudo Dyad Axis Base pairs seen from above the helix (helical projection) N3N3 N1N1 N O dR H H N3N3 N1N1 N7N7 N9N9 O H N H H Major Groove >180° Minor Groove <180° Helical Axis

H N N3N3 N1N1 N7N7 N9N9 dR H CH 3 N1N1 N3N3 O H O dR T A N1N1 N3N3 N H O N3N3 N1N1 N7N7 N9N9 O N H H H H G C N3N3 N1N1 N O H H N3N3 N1N1 N7N7 N9N9 O H N H H G C CH 3 N1N1 N3N3 O H O dR H N N3N3 N1N1 N7N7 N9N9 H A T CH 3 N1N1 N3N3 O H O T N3N3 N1N1 N7N7 N9N9 O H N H H G dR N3N3 N1N1 N O H H N3N3 N1N1 N7N7 N9N9 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 AB H20H20 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 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’ endo B-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 H 2 0 is essential in the transition A B DNA AB H20H20 This concept is controversial and will not be detailed further

Z-DNA Left handed Helix jagged backbone Occurs in DNA sequences with stretches of consecutive G-C base pairs Requires high salt in vitro G nucleotides: Switch C2’endo -> C3’ endo anti -> Syn C nucleotides: No change 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 conformation changes during the B->Z transition – it does not accurately shows the Z structure

Glycosidic bond Anti /Syn conformations Anti and Syn conformations are defined based on the torsion angle of the glycosidic bond 4' 1' 9 4 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' 9 4 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 BZ B Z ABZ-DNAs Backbone Profiles Helical Projections

AB Z

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 g Consequences for: DNA Replication, DNA Repair Transcription

Double-Stranded DNA is wrapped around nucleosomes in eukaryotic cells PDB ID: 1AOI

G-quadruplex structures in telomeric DNA: case of (T2G4) repeats Na + Example of intrinsic DNA 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 segments within the same molecule

Secondary and Tertiary Structure of transfer RNA (tRNA) PDB ID = 3TRA

Secondary Structure of the M1 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 range base-pairing Two major observations: “A” form tolerates the 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” ?

CH 3 N1N1 N3N3 O H O dR H N N3N3 N1N1 N7N7 N9N9 H H N N3N3 N1N1 N7N7 N9N9 H CH 3 N1N1 N3N3 O H O dR Minor groove Major groove A A H A D A A A H A D A A T T A Minor groove Major groove Conclusion: DNA binding proteins can differentiate A-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-bonds Donors (D) Acceptors (A) and Hydrophobic groups (H) Recognition of Specific sequences by DNA-binding proteins

N1N1 N3N3 N H O dR N3N3 N1N1 N7N7 N9N9 O N H H H H N3N3 N1N1 N O H H N3N3 N1N1 N7N7 N9N9 O H N H H A A D A A D A A D A A D G C G C Conclusion: DNA binding proteins can differentiate G-C base pairs from C-G base pairs if they bind from the major groove side, but not from the minor groove side Minor groove Major groove Minor groove Major groove Patterns of H-bonds Donors (D), Acceptors (A), and Hydrophobic groups (H) available for recognition Recognition of Specific sequences by 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 Wavelength (nm) Denatured DNA Native DNA Relative Absorbance Hyperchromic Effect: SS DNA > native DNA “melting curves” for two different DNA molecules (red and blue) show different “melting points” = 2 different Tms DNA molecule

Increasing Conformational Entropy 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: The Tm of a DNA molecule is a linear function of its G-C content/this is not because of higher energy of 3 H-bonds (GC) vs 2 (AT) Effect of G-C content on Stability is due to higher stacking of G-C base pairs compared to AT base pairs  G BP ≅ contribution of stacking to the stability of base pair