BMB 170 Lecture 10 Nucleic Acids, October 26th

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1 BMB 170 Lecture 10 Nucleic Acids, October 26th Today - Basics and structure Doodle Poll!!! EM of T2 coliphage - Kleinschmidt et al (1962) BBA 61:857-64

2 The basic bases in DNA and RNA RNA only DNA only

3 Nucleic-acid building blocks Nucleoside Nucleotide Glycosidic bond

4 Single strand of RNA Chain is directional with the convention being 5 3

5 Six backbone dihedral angles (α ζ)

6 Chargaff s Rule Erwin Chargaff ~1950 Enzymatically hydrolyzed DNA from many sources and compared ratios of bases

7 Tautomers : Keto and amino forms occur >99.99% of the time under physiological conditions. D = H-bond donor; A = H-bond acceptor

8 Pair interactions Lot s of pairs with at least 2 h-bonds (28 possible) Only 2 in DNA 20 observed in RNA

9 Adenine Crystals Fig Common base pairs

10 Sugar pucker RNA DNA

11 Rosalind Franklin Photograph 51 A physical chemist who refined methods for DNA fiber diffraction first identifying A and B forms of DNA. Died at 37 from cancer Conclusion: Helix with 10 bp/repeat and 3.4 Å between bps

12 DNA structure determination Rosalind Franklin Watson & Crick A B Franklin & Gosling Acta Cryst (1953) 6:673 Watson & Crick Nature (1953) 171:737 Nobel in Chemistry 1962 (w/ M. Wilkins)

13 Science Museum - London Morgan Beeby, Imperial College

14 Geometry of Base Pairing Major groove Opposite the glycosydic bonds C G T A Minor groove H-bonds satisfied Similar width Similar angle to glycosidic bonds Pseudo-symmetry of 180 rotation

15 BP on axis Comparison of B-DNA and A-DNA BP off axis Maj Min B-DNA BP/Turn 10 Rise/base3.4Å Helical twist 36 Base tilt 6 P i Backbone Out P i -P i 6.9Å Diameter ~20Å Major groove wide Minor groove narrow Min Maj A-DNA Å Interior 5.9Å ~26Å narrow/deep wide/shallow

16 Sequence-specific recognition of double helical nucleic acids by proteins Seeman et al (1976) PNAS 73:804-8 Major groove: all 4 Minor groove: GC/CG vs AT/TA. Seeman et al proposed that you need two H-bonds for discrimination (bidentate interactions). vdw Major Minor

17 Dickerson B-DNA (Caltech!) First structure of DNA double helix 19 bend/12 bp Core GAATTC: B-like with 9.8 bp/turn Flanking CGCG more complex, but P-P distance = 6.7 Å (like B) BPs not flat Propeller twist 11 for GC 17 for AT Very hydrated Wing et al Nature (1980) 28:755 (1bna)

18 DNA parameter descriptors Relative to helix Propeller twist: dihedral angle of base planes Displacement: distance from helix axis to bp center Twist: relative rotation around helix axis Roll: rotation angle of mean bp plane around C8-C6 line Tilt: rotation of bp plane around pseudo-dyad perpendicular to twist and roll axes Fig. 3.17

19 Helical parameters of the dodecamer C1/G24 G12/C13 Range Å

20 Effects of parameters Fig. 3.18

21 Calculated base stacking energies Can vary quite a lot Accommodating base geometries affects stacking energy ~3-10 kcal/mole (slightly stronger than an H-bond) Florián, Sponer and Warshel (1999) J. Phys Chem B 103:884

22 Tm depends on G+C content

23 Tm depends on ionic strength High KCl stabilizes duplex DNA

24 Predicting secondary structure Calculate all of the energies involved Penalties for loops and mismatches Dependent on solvent considerations Tinoco et al Nature (1971) 230:362

25 Central 3 Major 16S Ribosomal RNA 5 3 Minor 1542 bases in E. coli Often several copies in a genome Highly conserved Used to classify genus First model from 100 genomes (Noller lab) Woese et al NAR (1980) 8:2275

26 Current models much more refined Better free energy minimization and phylogenetic comparisons DNA parameters relatively defined/rna pretty good A number of algorithms Not good at pseudoknots NUPACK (Pierce lab) mfold (Zuker lab) now UNAFold mfold.rna.albany.edu/? q=unafold-man-pages

27 Using nucleic acids as design tools Pierce lab Winfree lab Rothemund lab

28 Self-assembly of polyhedra He et al (2008) Nature 452:

29 Reconfigurable topologies Uses DNA origami to generate möbius strips Strand displacement can yield novel structures Han et al (2010) Nature Nano 5:712-7

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32 G-tetrads Fig. 3.29, 3.30 & 3.31 RNA quadruplex UGGGGU (1j8g)

33 RNA structure (A-form) RNA Steric clashes force A-form to dominate Can form complicated tertiary structure Large complexes Spliceosome Ribosome Lot s of structures Small RNA pieces trna Ribozymes Self splicing/cleaving Introns (261), hammerhead, HDV, hairpin Ribosomes (catalytic RNA?) 30S (1500), 23S (3400), 5S (120) Signal Recognition Particle Spliceosome components Min Maj Reviewed in Chen & Varani FEBS Journal (2005) 272:

34 trna Links genetic code to amino acid code Predicted by Frances Crick The Sequence Hypothesis assumes that the specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and that this sequence is a (simple) code for the amino acid sequence of a particular protein The Central Dogma the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible The Adaptor Hypothesis One would expect, therefore, that whatever went on to the template in a specific way did so by forming hydrogen bonds. It is therefore a natural hypothesis that the amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part which actually fits on to the RNA. In its simplest form one would require twenty adaptors, one for each amino acid Crick "On Protein Synthesis Symp Soc Exp Biol (1958)12: 138

35 trna secondary structure All trnas are 73 to 93 nucleotides 7-15% of the bases are modified The cloverleaf Many trnas, all have same overall structure Receptor Stem Anti-codon Stem RajBhandary & Chang JBC (1962)

36 Examples of modified bases

37 More modifications Fig AnQcodon of Ile-tRNA 2 CATfor codon AUA

38 Race to the trna structure (1974) 3Å resolution Suddath, Quigley, McPherson, Sneden, Kim, Kim & Rich Nature (Mar 1974) 248:20 3Å resolution Robertus, Ladner, Finch, Rhodes, Brown, Clark & Klug Nature (Aug 1974) 250:546

39 31st July, 1974 Dr. Alex Rich, Department of Biology Massachusetts Institute of Technology Cambridge Mass / U.S.A. Dear Alex, Does your name stink. Aaron was convinced that once you had weedled out the details of his structure you would attempt to publish it as your own, This is exactly what has happened, I real& that you had already gone some distance along the same lines, but the fact remains that you said nothing aijout this at all in public at the Madison meeting and that Kim obtained the details of the Cambridge structure from Robertus at the Gordon Conference. There is absolutely nothing to suggest that you would have actually published a revised structure at this time except for the knowledge you obtained of the Can&rids structure, Moreover you did not even have the elementary courtesy to ackknowledge the Cambridge work. In addition to use your special influence with Science to rush into publication is quite inexcusable. Unless you are prepared to make a suitable apology in public I must tell you that your visits to Cambridge in future will not he welcomed, F, H. C: Crick copy R. Sinsheimer.

40 trna Structure trna Phe Rhodes lab at the LMB 15 year old xtals (1evv) Jovine et al JMB (2000) 301:401

41 Fig & 3.53 trna folding

42 Detailed interactions Interaction between D- and T- loops Sharp turns in the trna structure (Anticodon, T- loop, D-loop, 9-11 U- turn) Fig & 3.55

43 Modified bases Conserved Not all the roles are clear yet Often aids in stabilizing long range interactions m 5 U Ψ m 1 A

44 Mg 2+ stabilizes tertiary structure Tertiary structure brings lots of negative charge together Divalent metal ions do the trick Note the hexavalent coordination Jovine et al JMB (2000) 301:401

45 Fig Base triples

46 Fig & 3.43 JuncQons

47 Hammerhead ribozyme Fig & 3.59

48 Reaction Hypothetical transition state

49 Tetrahymena group I intron P4-P6 domain (1gid) First ribozyme described (Tom Cech - Nobel) Cech et al Cell (1981) 27: Self catalyzes removal of intron Stable tertiary domain of P4-P6 Doudna & Cech Labs: Cate et al Science (1996) 273:

50 Fig & 3.63 Structure (from the text)

51 Hepatitis Delta Virus (HDV) ribozyme double pseudoknot Top view 2 structure schematic U1A protein cocrystals Ferré-D Amaré, Zhou & Doudna Nature (1998) 395:567 (1cx0)

52 Pseudoknot structure A pseudoknot structure contains more than one stem-loops where at least one is intercalated into another. Fig & 3.47 Biotin bound pseudoknot aptamer (1f27)

53 Central 3 Major 5 3 Minor Tetraloops Helices are capped by a limited pool of 4 residue sequences General rule for stable helix capping Woese et al PNAS (1990) 87:8467

54 RNA tetraloop C U motif G U Hyperabundunt 4 nucleotide terminal loops 3 Classes UNCG GNRA CUYG 256 possible but 16 dominate! A A 3 A G 5 5 Tuerk et al PNAS (1988) 85:1364 Woese et al PNAS (1990) 87:8467 1j5e 3

55 RNA tertiary interactions Several large RNA structures Ribozymes Self splicing/cleaving Introns (261), hammerhead, HDV, hairpin Ribosome (catalytic RNA?) 30S (1500), 23S (3400), 5S (120) Structural motifs Surprisingly few long range motifs

56 Motifs from P4-P6 A-platform Ribose zipper Fig & 3.65

57 A-minor motif First noted in P4-P6 domain, also found in ribosome Long range stabilizing interaction Minor groove bulge Exposed A-platform Triplet GAAA tetraloop docks in minor groove Stabilized by π-stacking Adenines can measure minor groove Steitz & Moore Labs: Nissen et al PNAS (2001) 98: gid

58 A-minor interacqons from 23S rrna Fig. 3.49

59 Minor groove packing Adenine stabilized (most typical) Phosphate ridge to minor groove (usually stabilized by guanine N2s) End on mode, unpaired purine mediates helices at right angles Examples taken from the 30S ribosome

60 Directed in 1971 by Robert Alan Weiss for the Department of Chemistry of Stanford University and imprinted with the "free love" aura of the period, this short film conqnues to be shown in biology class today. It has since spawn a series of similar funny azempts at vulgarizing protein synthesis. Narrated by Paul Berg, 1980 Nobel prize for Chemistry. hzps://