Nucleic Acid Structure II - PowerPoint PPT Presentation

nucleic acid structure ii n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Nucleic Acid Structure II PowerPoint Presentation
Download Presentation
Nucleic Acid Structure II

play fullscreen
1 / 47
Nucleic Acid Structure II
180 Views
Download Presentation
brigham
Download Presentation

Nucleic Acid Structure II

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Nucleic AcidStructure II Andy HowardIntroductory Biochemistry9 October 2008 Biochemistry: Nucleic Acid Struct II

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

  3. 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! Biochemistry: Nucleic Acid Struct II

  4. Summaries of A, B, Z DNA Biochemistry: Nucleic Acid Struct II

  5. 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 Biochemistry: Nucleic Acid Struct II

  6. 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! Biochemistry: Nucleic Acid Struct II

  7. Instances of inter-calators Biochemistry: Nucleic Acid Struct II

  8. 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 Biochemistry: Nucleic Acid Struct II

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

  10. GC content vs. melting temp • High salt and no chelators raises the melting temperature Biochemistry: Nucleic Acid Struct II

  11. 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 Biochemistry: Nucleic Acid Struct II

  12. 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 Biochemistry: Nucleic Acid Struct II

  13. Steps in denaturation and renaturation Biochemistry: Nucleic Acid Struct II

  14. 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 Biochemistry: Nucleic Acid Struct II

  15. Second-order kinetics • Rate of association: -dc/dt = k2c2 • Boundary condition is fully denatured concentration c0 at time t=0: • c / c0 = (1+k2c0t)-1 • Half time is t1/2 = (k2c0)-1 • Routine depiction: plot c0t vs. fraction reassociated (c /c0) and find the halfway point. Biochemistry: Nucleic Acid Struct II

  16. Typical c0t curves Biochemistry: Nucleic Acid Struct II

  17. 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 Biochemistry: Nucleic Acid Struct II

  18. 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 Biochemistry: Nucleic Acid Struct II

  19. Density as function of GC content Biochemistry: Nucleic Acid Struct II

  20. 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 Biochemistry: Nucleic Acid Struct II

  21. 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 Biochemistry: Nucleic Acid Struct II

  22. 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 Biochemistry: Nucleic Acid Struct II

  23. Examples with a tube Biochemistry: Nucleic Acid Struct II

  24. How this works with real DNA Biochemistry: Nucleic Acid Struct II

  25. 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 Biochemistry: Nucleic Acid Struct II

  26. 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 Biochemistry: Nucleic Acid Struct II

  27. Superhelix density • Compare L for real DNA to what it would be if it were relaxed (W=0): • That’s L = L - L0 • Sometimes we want = superhelix density= specific linking difference = L / L0 • Natural circular DNA always has  < 0 Biochemistry: Nucleic Acid Struct II

  28.  < 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 Biochemistry: Nucleic Acid Struct II

  29. 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 Biochemistry: Nucleic Acid Struct II

  30. Cruciform DNA example Biochemistry: Nucleic Acid Struct II

  31. 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 Biochemistry: Nucleic Acid Struct II

  32. 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 Biochemistry: Nucleic Acid Struct II

  33. 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 Biochemistry: Nucleic Acid Struct II

  34. Histones: table 11.2 Biochemistry: Nucleic Acid Struct II

  35. Nucleosome core particle Biochemistry: Nucleic Acid Struct II

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

  37. 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 Biochemistry: Nucleic Acid Struct II

  38. Chromosome structure: levels • Each of the first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 104 Biochemistry: Nucleic Acid Struct II

  39. 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! Biochemistry: Nucleic Acid Struct II

  40. 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 Biochemistry: Nucleic Acid Struct II

  41. Figure 11.29Solid 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 -NH2 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. 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 Biochemistry: Nucleic Acid Struct II

  43. Activation of the phosphoramidate Biochemistry: Nucleic Acid Struct II

  44. 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 Biochemistry: Nucleic Acid Struct II

  45. tRNA structure: overview Biochemistry: Nucleic Acid Struct II

  46. 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. Biochemistry: Nucleic Acid Struct II

  47. Yeast phe-tRNA • Note nonstandard bases and cloverleaf structure Biochemistry: Nucleic Acid Struct II