N ucleic acid structure and chemistry

1. Nucleic acid structure and chemistry Andy HowardIntroductory BiochemistryMonday 20 October 2014 Nucleic Acid Structure and Chemistry

2. Nucleic acid structure and function • Nucleic acid bases, nucleosides, and nucleotides have characteristic structural and chemical properties, both on their own and as polymers Nucleic Acid Structure and Chemistry

3. Nucleosides and deoxynucleosides Oligomers and polymers Duplexes & helicity DNA structure Characterizations B, A, and Z-DNA Dynamics Function DNA sequencing RNA:structure & types mRNA tRNA rRNA Small RNAs What we’ll discuss Nucleic Acid Structure and Chemistry

4. iClicker quiz, 1st question 1. Which type of ion-channel gating have we not discussed? • (a) ligand-gating • (b) substrate-gating • (c) magnetic-field gating • (d) voltage-gating • (e) we discussed all of these Nucleic Acid Structure and Chemistry

5. iClicker quiz, question 2 2. We would expect the central pore of a cation channel to be lined with • (a) hydrophobic residues • (b) asp and glu residues • (c) lys and arg residues • (d) cofactors • (e) none of the above. Nucleic Acid Structure and Chemistry

6. iClicker quiz, question 3 3. Which of the following atom types are absent in nucleotides? • (a) P and S • (b) S and K • (c) N and O • (d) P and H • (e) all of these atom types are found in nucleotides. Nucleic Acid Structure and Chemistry

7. Purine nucleosides • Drawn in amino and lactam forms Nucleic Acid Structure and Chemistry

8. Purine deoxynucleosides Nucleic Acid Structure and Chemistry

9. Conformations around the glycosidic bond • Rotation of the base around the glycosidic bond is sterically hindered • In the syn conformation there would be some interference between the sugar ring and the 2-position of the base • Therefore pyrimidines are always anti, and purines are usually anti • Furanose and base rings are roughly perpendicular Nucleic Acid Structure and Chemistry

10. Glycosidic bonds • This illustrates the roughly perpendicular positionings of the base and sugar rings Nucleic Acid Structure and Chemistry

11. Solubility of nucleosides and lability of glycosidic linkages • The sugar makes nucleosides more soluble than the free bases • Nucleosides are generally stable to basic hydrolysis at the glycosidic bond • Acid hydrolysis: • Purines: glycosidic bond fairly readily hydrolyzed • Pyrimidines: resistant to acid hydrolysis Nucleic Acid Structure and Chemistry

12. Chirality in nucleic acids • Bases themselves are achiral • Four asymmetric centers in ribofuranose, counting the glycosidic bond. • Three in deoxyribofuranose • Glycosidic bond is one of those 4 or 3. • Same for nucleotides:phosphates don’t add asymmetries Nucleic Acid Structure and Chemistry

13. Mono-phosphorylated nucleosides • We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: • They are nucleotides • Adenosine 5’-monophosphate = AMP = adenylate • GMP = guanylate • CMP = cytidylate • UMP = uridylate Nucleic Acid Structure and Chemistry

14. pKa’s for base N’s and PO4’s Nucleic Acid Structure and Chemistry

15. UV absorbance (G&G fig. 10.8) • These aromatic rings absorb around 260 Nucleic Acid Structure and Chemistry

16. Deoxynucleotides • Similar nomenclature • dAMP = deoxyadenylate • dGMP = deoxyguanylate • dCMP = deoxycytidylate • dTMP (= TMP) = deoxythymidylate = thymidylate Nucleic Acid Structure and Chemistry

17. Di and triphosphates • Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety Nucleic Acid Structure and Chemistry

18. Cyclic phosphodiesters • 3’ and 5’ hydroxyls are both involvedin -O-P-O bonds • cAMP and cGMP are the important ones(see earlier in the course!) Cyclic AMP Nucleic Acid Structure and Chemistry

19. Oligomers and Polymers • Monomers are nucleotides or deoxynucleotides • Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose • It’s logical to start from the 5’ end for synthetic reasons Nucleic Acid Structure and Chemistry

20. Typical DNA dinucleotide • Various notations: this is pdApdCp • Leave out the p’s if there’s a lot of them! Nucleic Acid Structure and Chemistry

21. DNA structure • Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA • Explained [A]=[T], [C]=[G] • Specific H-bonds stabilize double-helical structure: see fig. 10.17 Nucleic Acid Structure and Chemistry

22. What does double-stranded DNA really look like? • Picture on previous slide emphasizes only the H-bond interactions; it ignores the orientation of the sugars, which are actually tilted relative to the helix axis • Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix Nucleic Acid Structure and Chemistry

23. Sizes (cf. fig. 11.8, table 11.1) • Diameter of the double helix: 2.37nm • Length along one full turn:10.4 base pairs = pitch = 3.40nm • Distance between stacked base pairs = rise = 0.33 nm • Major groove is wider and shallower;minor groove is narrower and deeper Nucleic Acid Structure and Chemistry

24. What stabilizes DNA? • Variety of stabilizing interactions • Stacking of base pairs • Hydrogen bonding between base pairs • Hydrophobic effects (burying bases, which are less polar) • Charge-charge interactions:phosphates with Mg2+ and cationic proteins Courtesy dnareplication.info Nucleic Acid Structure and Chemistry

25. How close to instability is it? • Pretty close. • Heating DNA makes it melt: fig. 11.20 • pH > 10 separates strands too • The more GC pairs, the harder it is to melt DNA thermally • Weaker stacking interactions in A-T • One more H-bond per GC than per AT Nucleic Acid Structure and Chemistry

26. Do the differences between RNA and DNA matter? Yes! • Deoxy sugars don’t degrade as readily! • DNA has deoxythymidine, RNA has uridine: • cytidine spontaneously degrades to uridine • dC spontaneously degrades to dU • The only dU found in DNA is there because of degradation: dT goes with dA • So when a cell finds dU in its DNA, it knows it should replace it with dC or else synthesize dG opposite the dU instead of dA Nucleic Acid Structure and Chemistry

27. Ribose vs. deoxyribose • Presence of -OH on 2’ position makes the 3’ position in RNA more susceptible to nonenzymatic cleavage than the 3’ in DNA • The ribose vs. deoxyribose distinction also influences enzymatic degradation of nucleic acids • I can carry DNA in my shirt pocket, but not RNA Nucleic Acid Structure and Chemistry

28. Base composition for DNA • As noted, [A]=[T], [C]=[G] because of base pairing • [A]/[C] etc. not governed by base pairing • Can vary considerably (table 10.3) • E.coli : [A], [C] about equal • Mycobacterium tuberculosis: [C] > 2*[A] • Mammals: [C] < 0.74*[A] Nucleic Acid Structure and Chemistry

29. DNA secondary structures • If double-stranded DNA were simply a straight-legged ladder: • Base pairs would be 0.6 nm apart • Watson-Crick base-pairs have very uniform dimensions because the H-bonds are fixed lengths • But water could get to the apolar bases • So, in fact, the ladder gets twisted into a helix. • The most common helix is B-DNA, but there are others. B-DNA’s properties include: • Sugar-sugar distance is still 0.6 nm • Helix repeats itself every 3.4 nm, i.e. 10 bp Nucleic Acid Structure and Chemistry

30. Properties of B-DNA • Spacing between base-pairs along helix axis = 0.34 nm • 10 base-pairs per full turn • So: 3.4 nm per full turn is pitch length • Major and minor grooves, as discussed earlier • Base-pair plane is almost perpendicular to helix axis From Molecular Biology web-book Nucleic Acid Structure and Chemistry

31. Major groove in B-DNA • H-bond between adenine NH2 and thymine ring C=O • H-bond between cytosine amine and guanine ring C=O • Wide, not very deep Nucleic Acid Structure and Chemistry

32. Minor groove in B-DNA • H-bond between adenine ring N and thymine ring NH • H-bond between guanine amine and cytosine ring C=O • Narrow but deep From Berg et al.,Biochemistry Nucleic Acid Structure and Chemistry

33. Cartoon of AT pair in B-DNA Nucleic Acid Structure and Chemistry

34. Cartoon of CG pair in B-DNA Nucleic Acid Structure and Chemistry

35. What holds duplex B-DNA together? • H-bonds (but just barely) • Electrostatics: Mg2+ –HPO4- • van der Waals interactions • - interactions in bases • Solvent exclusion • Recognize role of grooves in defining DNA-protein interactions Nucleic Acid Structure and Chemistry

36. Helical twist (fig. 11.10a) • Rotation about the backbone axis • Successive base-pairs rotated with respect to each other by ~ 32º Nucleic Acid Structure and Chemistry

37. Propeller twist (G&G fig. 11.11b,c) • Improves overlap of hydrophobic surfaces • Makes it harder for water to contact the less hydrophilic parts of the molecule Nucleic Acid Structure and Chemistry

38. A-DNA (figs. 11.11b) • In low humidity this forms naturally • Not likely in cellular duplex DNA,but it does form in duplex RNA & DNA-RNA hybrids because the2’-OH gets in the way of B-RNA • Broader • 2.46 nm per full turn • 11 bp to complete a turn • Base-pairs are notperpendicular to helix axis:tilted 19º from perpendicular Nucleic Acid Structure and Chemistry

39. Z-DNA (fig. 11.11c) • Forms in alternating Py-Pu sequences and occasionally in PyPuPuPyPyPu, especially if C’s are methylated • Left-handed helix rather than right • Bases zigzag across the groove Nucleic Acid Structure and Chemistry

40. Getting from B to Z • Can be accomplished without breaking bonds • … even though the Z-DNA purines have their glycosidic bonds flipped (anti syn) and the pyrimidines are flipped altogether! Nucleic Acid Structure and Chemistry

41. Summaries of A, B, Z DNA Nucleic Acid Structure and Chemistry

42. DNA is dynamic • Don’t think of these diagrams as static • The H-bonds stretch and the torsions allow some rotations, so the ropes can form roughly spherical shapes when not constrained by histones • Shape is sequence-dependent, which influences protein-DNA interactions Nucleic Acid Structure and Chemistry

43. What does DNA do? • Serve as the storehouse and the propagator of genetic information:That means that it’s made up of genes • Some code for mRNAs that code for protein • Others code for other types of RNA • Genes contain non-coding segments (introns) • But it also contains stretches that are not parts of genes at all and are serving controlling or structural roles • Avoid the term junk DNA! Nucleic Acid Structure and Chemistry

44. Supercoiling • Refers to levels of organization of DNA beyond the immediate double-helix • We describe circular DNA as relaxed if the closed double helix could lie flat • It’s underwound or overwound if the ends are broken, twisted, and rejoined. • Supercoils restore 10.4 bp/turn relation upon rejoining Nucleic Acid Structure and Chemistry

45. Supercoiling and flat DNA Diagram courtesy SIU Carbondale Nucleic Acid Structure and Chemistry

46. iClicker quiz, 4th question 4. What positions of a pair of aromatic rings leads to stabilizing interactions? • (a) Parallel to one another • (b) Perpendicular to one another • (c) At a 45º angle to one another • (d) Both (a) and (b) • (e) All three: (a), (b), and ( c) Nucleic Acid Structure and Chemistry

47. iClicker question 5 • 5. Which has the highest molecular mass among the compounds listed? • (a) cytidylate • (b) thymidylate • (c) adenylate • (d) adenosine triphosphate • (e) they’re all the same MW Nucleic Acid Structure and Chemistry

48. iClicker quiz question 6 6. Shown is a lactim form of which nucleic acid base? • Uracil • Guanine • Adenine • Thymine • None of the above Nucleic Acid Structure and Chemistry

49. iClicker quiz question 7 7. Suppose someone reports that he has characterized the genomic DNA of an organism as having 29% A and 22% T. How would you respond? • (a) That’s a reasonable result • (b) This result is unlikely because [A] ~ [T] in duplex DNA • (c) That’s plausible if it’s a bacterium, but not if it’s a eukaryote • (d) none of the above Nucleic Acid Structure and Chemistry

50. Sanger dideoxy method • Incorporates DNA replication as ananalytical tool for determining sequence • Uses short primer that attaches to the 3’ end of the ssDNA, after which a specially engineered DNA polymerase replicates it • Each vial includes one dideoxyXTP and 3 ordinary dXTPs; the dideoxyXTP will be incorporated but will halt synthesis because the 3’ position is blocked. • See figs. 11.3 & 11.4 for how these are read out Nucleic Acid Structure and Chemistry