1. 10/09/08Biochemistry: Nucleic Acid Struct II Nucleic Acid Structure II Andy Howard Introductory Biochemistry 9 October 2008

2. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 2 of 47 What we’ll discuss Folding kinetics Supercoils Nucleosomes Chromatin and chromosomes Lab synthesis of genes tRNA & rRNA structure

3. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 3 of 47 Getting from B to Z Can be accomplished without breaking bonds … even though purines have their glycosidic bonds flipped (anti -> syn) and the pyrimidines are flipped altogether!

4. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 4 of 47 Summaries of A, B, Z DNA

5. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 5 of 47 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

6. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 6 of 47 Intercalating agents Generally: aromatic compounds that can form  -stack interactions with bases Bases must be forced apart to fit them in Results in an almost ladderlike structure for the sugar-phosphate backbone locally Conclusion: it must be easy to do local unwinding to get those in!

7. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 7 of 47 Instances of inter- calators

8. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 8 of 47 Denaturing and Renaturing DNA See Figure 11.17 When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of the DNA double helix Stacked base pairs in native DNA absorb less light When T is lowered, the absorbance drops, reflecting the re-establishment of stacking

9. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 9 of 47 Heat denaturation Figure 11.14 Heat denaturation of DNA from various sources, so-called melting curves. The midpoint of the melting curve is defined as the melting temperature, T m. (From Marmur, J., 1959. Nature 183:1427–1429.)

10. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 10 of 47 GC content vs. melting temp High salt and no chelators raises the melting temperature

11. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 11 of 47 How else can we melt DNA? High pH deprotonates the bases so the H- bonds disappear Low pH hyper-protonates the bases so the H-bonds disappear Alkalai is better: it doesn’t break the glycosidic linkages Urea, formamide make better H-bonds than the DNA itself so they denature DNA

12. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 12 of 47 What happens if we separate the strands? We can renature the DNA into a double helix Requires re-association of 2 strands: reannealing The realignment can go wrong Association is 2nd-order, zippering is first order and therefore faster

13. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 13 of 47 Steps in denaturation and renaturation

14. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 14 of 47 Rate depends on complexity The more complex DNA is, the longer it takes for nucleation of renaturation to occur “Complex” can mean “large”, but complexity is influenced by sequence randomness: poly(AT) is faster than a random sequence

15. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 15 of 47 Second-order kinetics Rate of association: -dc/dt = k 2 c 2 Boundary condition is fully denatured concentration c 0 at time t=0: c / c 0 = (1+k 2 c 0 t) -1 Half time is t 1/2 = (k 2 c 0 ) -1 Routine depiction: plot c 0 t vs. fraction reassociated (c /c 0 ) and find the halfway point.

16. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 16 of 47 Typical c 0 t curves

17. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 17 of 47 Hybrid duplexes We can associate DNA from 2 species Closer relatives hybridize better Can be probed one gene at a time DNA-RNA hybrids can be used to fish out appropriate RNA molecules

18. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 18 of 47 GC-rich DNA is denser DNA is denser than RNA or protein, period, because it can coil up so compactly Therefore density-gradient centrifugation separates DNA from other cellular macromolecules GC-rich DNA is 3% denser than AT-rich Can be used as a quick measure of GC content

19. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 19 of 47 Density as function of GC content

20. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 20 of 47 Tertiary Structure of DNA In duplex DNA, ten bp per turn of helix Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state Enzymes called topoisomerases or gyrases can introduce or remove supercoils Cruciforms occur in palindromic regions of DNA Negative supercoiling may promote cruciforms

21. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 21 of 47 DNA is wound Standard is one winding per helical turn, i.e. 1 winding per 10 bp Fewer coils or more coils can happen: This introduces stresses that favors unwinding Both underwound and overwound DNA compact the DNA so it sediments faster than relaxed DNA

22. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 22 of 47 Linking, twists, and writhe T=Twist=number of helical turns W=Writhe=number of supercoils L=T+W = Linking number is constant unless you break covalent bonds

23. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 23 of 47 Examples with a tube

24. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 24 of 47 How this works with real DNA

25. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 25 of 47 How gyrases work Enzyme cuts the DNA and lets the DNA pass through itself Then the enzyme religates the DNA Can introduce new supercoils or take away old ones

26. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 26 of 47 Typical gyrase action Takes W=0 circular DNA and supercoils it to W=-4 This then relaxes a little by disrupting some base-pairs to make ssDNA bubbles

27. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 27 of 47 Superhelix density Compare L for real DNA to what it would be if it were relaxed (W=0): That’s  L = L - L 0 Sometimes we want  = superhelix density = specific linking difference =  L / L 0 Natural circular DNA always has  < 0

28. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 28 of 47  < 0 and spools The strain in  < 0 DNA can be alleviated by wrapping the DNA around protein spool That’s part of what stabilizes nucleosomes

29. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 29 of 47 Cruciform DNA Cross-shaped structures arise from palindromic structures, including interrupted palindromes like this example These are less stable than regular duplexes but they are common, and they do create recognition sites for DNA-binding proteins, including restriction enzymes

30. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 30 of 47 Cruciform DNA example

31. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 31 of 47 Eukaryotic chromosome structure Human DNA’s total length is ~2 meters! This must be packaged into a nucleus that is about 5 micrometers in diameter This represents a compression of more than 100,000! It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

32. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 32 of 47 Nucleosome Structure Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond) Nonhistone proteins are regulators of gene expression

33. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 33 of 47 Histone types H2a, H2b, H3, H4 make up the core particle: two copies of each, so: octamer All histones are KR-rich, small proteins H1 associates with the regions between the nucleosomes

34. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 34 of 47 Histones: table 11.2 Histone#lys, #argMr, kDaCopies per Nucleosome H159, 321.21 (not in bead) H2A13, 1314.12 (in bead) H2B20, 813.92 (in bead) H313, 1715.12 (in bead) H411, 1411.42 (in bead)

35. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 35 of 47 Nucleosome core particle

36. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 36 of 47 Half the core particle Note that DNA isn’t really circular: it’s a series of straight sections followed by bends

37. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 37 of 47 Histones, continued Individual nucleosomes attach via histone H1 to seal the ends of the turns on the core and organize 40-60bp of DNA linking consecutive nucleosomes N-terminal tails of H3 & H4 are accessible K, S get post-translational modifications, particularly K-acetylation

38. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 38 of 47 Chromosome structure: levels Each of the first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 10 4

39. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 39 of 47 Synthesizing nucleic acids Laboratory synthesis of nucleic acids requires complex strategies Functional groups on the monomeric units are reactive and must be blocked Correct phosphodiester linkages must be made Recovery at each step must high!

40. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 40 of 47 Solid Phase Oligonucleotide Synthesis Dimethoxytrityl group blocks the 5 ' -OH of the first nucleoside while it is linked to a solid support by the 3 ' -OH Step 1: Detritylation by trichloroacetic acid exposes the 5 ' -OH Step 2: In coupling reaction, second base is added as a nucleoside phosphoramidate

41. Figure 11.29 Solid phase oligonucleotide synthesis. The four-step cycle starts with the first base in nucleoside form (N-1) attached by its 3'-OH group to an insoluble, inert resin or matrix, typically either controlled pore glass (CPG) or silica beads. Its 5'-OH is blocked with a dimethoxytrityl (DMTr) group (a). If the base has reactive -NH 2 functions, as in A, G, or C, then N- benzoyl or N-isobutyryl derivatives are used to prevent their reaction (b). In step 1, the DMTr protecting group is removed by trichloroacetic acid treatment. Step 2 is the coupling step: the second base (N- 2) is added in the form of a nucleoside phosphoramidite derivative whose 5'-OH bears a DMTr blocking group so it cannot polymerize with itself (c).

42. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 42 of 47 Solid Phase Synthesis Step 3: capping with acetic anhydride blocks unreacted 5’-OHs of N-1 from further reaction Step 4: Phosphite linkage between N-1 and N-2 is reactive and is oxidized by aqueous iodine to form the desired, and more stable, phosphate group

43. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 43 of 47 Activation of the phosphoramidate

44. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 44 of 47 Secondary and Tertiary Structure of RNA Transfer RNA Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem Only one tRNA structure (alone) is known Phenylalanine tRNA is "L-shaped" Many non-canonical base pairs found in tRNA

45. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 45 of 47 tRNA structure: overview

46. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 46 of 47 Amino acid linkage to acceptor stem Amino acids are linked to the 3'- OH end of tRNA molecules by an ester bond formed between the carboxyl group of the amino acid and the 3'-OH of the terminal ribose of the tRNA.

47. 10/09/08 Biochemistry: Nucleic Acid Struct II p. 47 of 47 Yeast phe- tRNA Note nonstandard bases and cloverleaf structure