1. PHAR201 Lecture 2 20121 Principles of DNA and RNA Structure PHAR 201/Bioinformatics I Philip E. Bourne Department of Pharmacology, UCSD Prerequisite Reading: Structural Bioinformatics Chapters 3 Thanks to Helen Berman for many slides

2. PHAR201 Lecture 2 20122 We start with DNA

3. PHAR201 Lecture 2 20123 History 1946 – DNA is the main constituent of genes (Avery) 1950 – First X-ray pictures of DNA (Franklin) 1953 – DNA structure revealed (Watson and Crick) 1970 onwards - Multiple conformations and structures, initially from fibers 1973 - X-ray structure confirms double helix (Rich) 1974 - t-RNA structure (Kim) 1980 – Structure of first complete turn of B (“normal”) DNA (Dickerson)

4. PHAR201 Lecture 2 20124 What Have we Learnt from These Structures? Hydration, ionic strength and sequence all impact the type of structure We see single stranded helices, double, triple and quadruple Alone DNA and RNA does not crystallize easily, hence strands are short – eg 10-mer (unless complexed) Contrast this to the ribosome (1FFK)

5. PHAR201 Lecture 2 20125 NOTE: Components Sugar Base Phosphate 5’ to 3’ direction T->U in RNA RNA - extra –OH at 2’ of pentose sugar DNA - deoxyribose Numbering Single vs double strands DNA more stable Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 792. DNA and RNA Structure

6. PHAR201 Lecture 2 20126 NOTE: Pyrimadines and Purines T->U in RNA Names Numbering Bonding character Position of hydrogen Tautomers Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 18. The 5 Bases of DNA and RNA Purines Pyrimadines

7. PHAR201 Lecture 2 20127 Keto vs enol (OH) Different hydrogen bonding patterns Saenger, Wolfram. Principles of Nucleic Acid Structure. Springer-Verlag New York Inc., 1984, p. 113. Tautomeric Structures

8. A:T and G:C pairs are spatially similar 3 H-bonds vs 2 (GC rich?) Sugar groups are attached asymmetrically on the same side of the pair Leads to a major and minor grove Bases are flat but the hydrogen bonding leads to considerable flexibility Base stacking is flexible Geometry of Watson Crick Base Pairs Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 797. 8PHAR201 Lecture 2 2012

9. 9 Hydrogen bonding of WC base pair Mechanisms of recognition The canonical Watson-Crick base pair, shown as the G-C pair. Positions of the minor and major grooves are indicated. The glycosidic sugar-base bond is shown by the bold line; hydrogen bonding between the two bases is shown in dashed lines. Definition of Major and Minor Groove

10. PHAR201 Lecture 2 201210 Base Stacking is a Major Defining Feature of DNA Morphology Dependant on: –Nature of the bases and base pairs –Stacking interactions Explains sequence dependant features Important for understanding molecular recognition

11. PHAR201 Lecture 2 201211 Base Morphology The base-pair reference frame is constructed such that the x-axis points away from the (shaded) minor groove edge. Images illustrate positive values of the designated parameters. Reprinted with permission from Adenine Press from (Lu, et al., 1999).

12. PHAR201 Lecture 2 201212 Backbone Conformation Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 807.

13. PHAR201 Lecture 2 201213 A Beta-nucleoside Ring is never flat – has 5 internal torsional angles The pucker is determined by what is bound A variety of puckers have been observed Pucker has a strong influence on the overall conformation

14. PHAR201 Lecture 2 201214 Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 808. The Ribose Ring is Never Flat

15. PHAR201 Lecture 2 201215 Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 27. The Glycosidic Bond Connects ribose sugar to the base Anti Syn

16. PHAR201 Lecture 2 201216 Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 808. Change in sugar conformation affects the backbone C2’-Endo C3’-Endo C3’ C2’

17. PHAR201 Lecture 2 201217 A DNA B DNA..and the position of the bases relative to the helix axis

18. PHAR201 Lecture 2 201218 Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 34. Canonical B DNA

19. PHAR201 Lecture 2 201219 Canonical B DNA First determined experimentally by fiber diffraction (Arnott) C2’-endo sugar puckers High anti glycosidic angles Right handed – 10 base pairs per turn Bases perpendicular to the helix axis and stacked over the axis Overall bending as much as 15 degrees (result of base morphologies – twist and roll) – {machine learning – sequence vs overall conformation?} Over 230 structures 25 with base mis-pairing – only cause local perturbations Strong influence of hydration along spine http://ndbserver.rutgers.edu/index.html

20. PHAR201 Lecture 2 201220 Major vs Minor Groove – distinctly different environments – important for recognition and binding Major –Richer in base substituents Minor –Hydrophobic H atoms of ribose groups forming its walls

21. PHAR201 Lecture 2 201221 Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 97. Spine of Hydration

22. PHAR201 Lecture 2 201222 Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 36. A DNA

23. PHAR201 Lecture 2 201223 Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 800. Canonical A DNA

24. PHAR201 Lecture 2 201224 Canonical A DNA C3’-endo sugar puckers – brings consecutive phosphates closer together 5.9A rather than 7.0 Glycosidic angle from high anti to anti Base pairs twisted and nearly 5A from helix axis Helix rise 2.56A rather than 3.4A Helix wider and 11 base pairs per repeat Major groove now deep and narrow Minor grove wide and very shallow

25. PHAR201 Lecture 2 201225 Z-DNA Helix has left-handed sense Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure. Narrower, more elongated helix than A or B. Major "groove" not really groove Narrow minor groove Conformation favored by high salt concentrations, some base substitutions, but requires alternating purine-pyrimidine sequence. N2-amino of G H-bonds to 5' PO: explains slow exchange of proton, need for G purine. Base pairs nearly perpendicular to helix axis GpC repeat, not single base-pair –P-P distances: vary for GpC and CpG –GpC stack: good base overlap –CpG: less overlap. Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation Conformations: –G; syn, C2'-endo –C; anti, C3'-endo

26. PHAR201 Lecture 2 201226 Z-DNA

27. PHAR201 Lecture 2 201227 Z-DNA Convex major groove Deep minor groove Alternate C then G Spine of hydration

28. PHAR201 Lecture 2 201228 Drug complexes to DNA Bound to the base pair – double helix can accommodate this Bound in the minor grove – show base specificity Cis-platinum drugs

29. PHAR201 Lecture 2 201229 Quadruplex DNA 1NP9 Jmol

30. PHAR201 Lecture 2 201230 Saenger, Wolfram. Principles of Nucleic Acid Structure. Springer-Verlag New York Inc., 1984, p. 333. tRNA 1EVV jmol Invariant L-shape

31. PHAR201 Lecture 2 201231 Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 148. tRNA H bonds between distant regions

32. PHAR201 Lecture 2 201232 The Ribosome Complex of protein and RNA Small 30S subunit – controls interactions between mRNA and tRNA Large 50S subunit – peptide transfer and formation of the peptide bond

33. PHAR201 Lecture 2 201233 Putting it all Together – Major Categories of DNA Binding Proteins Jones et al. 1999 JMB 287(5) 877 Protein residues that make no contacts with the DNA are colored blue, those contacting the sugar- phosphate backbone are colored red, and those making base contacts are colored yellow. (a) Proteins with a single binding head: T4 endonuclease V (1vas), PU.1 ETS domain (1pue). (b) Proteins with a double binding head: lambda repressor (1lmb), papillomavirus-1 E2 DNA-binding domain (2bop). (c) Proteins with an enveloping mode of binding: NF-kB (1nfk),EcoRI restriction endonuclease (1eri).