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 The basic bases in DNA and RNA

RNA DNA only only Nucleic-acid building blocks

Nucleoside

Nucleotide Glycosidic bond Single strand of RNA

Chain is directional with the convention being 5’ → 3’ Six backbone dihedral angles (α−ζ) Chargaff’s Rule

• Erwin Chargaff ~1950 • Enzymatically hydrolyzed DNA from many sources and compared ratios of bases “Tautomers”: Keto and amino forms occur >99.99% of the time under physiological conditions.

D = H-bond donor; A = H-bond acceptor Pair interactions

• Lot’s of pairs with at least 2 h-bonds (28 possible)

• Only 2 in DNA

• 20 observed in RNA Common base pairs

Adenine Crystals Fig. 3.25-3.27 Sugar pucker

RNA DNA 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 DNA structure determination

Rosalind Franklin Watson & Crick

A B

Watson & Crick Nature (1953) 171:737 Franklin & Gosling Acta Cryst (1953) 6:673 Nobel in Chemistry 1962 (w/ M. Wilkins) Science Museum - London Morgan Beeby, Imperial College Geometry of Base Pairing Major groove Opposite the glycosydic bonds

C T G A

Minor groove

• H-bonds satisfied • Similar width • Similar angle to glycosidic bonds • Pseudo-symmetry of 180° rotation Comparison of B-DNA and A-DNA BP on axis BP off axis

Min Maj

B-DNA A-DNA BP/Turn 10 Min Maj 10.9 Rise/base3.4Å 2.9Å Helical twist 36˚ 31˚ Base tilt 6˚ 20˚ Pi Backbone Out Interior Pi-Pi 6.9Å 5.9Å Diameter ~20Å ~26Å Major groove wide narrow/deep Minor groove narrow wide/shallow Sequence-specific recognition of double helical nucleic acids by proteins Seeman et al (1976) PNAS 73:804-8

• Major groove: all 4 vdw • Minor groove: GC/CG vs AT/TA. • Seeman et al proposed that you need two H-bonds for discrimination (bidentate interactions).

Major Minor 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) 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 Helical parameters of the dodecamer

C1/G24

G12/C13

Range 4.9-18.6° 32.2-41.4° 8.1-11.2 3.14-3.54 Å Effects of parameters

Fig. 3.18 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 Tm depends on G+C content Tm depends on ionic strength

High KCl stabilizes duplex DNA 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 Central 16S Ribosomal 3’ Major RNA

• 1542 bases in E. coli – Often several copies in a genome • Highly conserved • Used to classify genus 5’ • First model from 100 3’ Minor genomes (Noller lab)

Woese et al NAR (1980) 8:2275 Current models much more refined • Better free energy • minimization and NUPACK (Pierce lab) phylogenetic – http://www.nupack.org comparisons • DNA parameters • mFold (Zuker lab) – now relatively defined/RNA UNAFold pretty good – http:// mfold.rna.albany.edu/? • A number of algorithms q=unafold-man-pages • Not good at pseudoknots Using nucleic acids as design tools

• Pierce lab • Winfree lab • Rothemund lab Self-assembly of polyhedra

He et al (2008) Nature 452:198-201 Reconfigurable topologies • Uses DNA origami to generate möbius strips • Strand displacement can yield novel structures

Han et al (2010) Nature Nano 5:712-7

G-tetrads

Fig. 3.29, 3.30 & 3.31 RNA quadruplex UGGGGU (1j8g) RNA structure (A-form) • RNA – Steric clashes force A-form to dominate – Can form complicated tertiary structure • Large complexes – Spliceosome – Ribosome Min Maj • 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

Reviewed in Chen & Varani FEBS Journal (2005) 272:2088-97 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 tRNA secondary structure Receptor • All tRNAs are 73 to 93 Stem nucleotides • 7-15% of the bases are modified • The cloverleaf • Many tRNAs, all have same overall structure

Anti-codon Stem

RajBhandary & Chang JBC (1962) 598-608 Examples of modified bases More modifications

Fig. 3.32

Ancodon of Ile-tRNA2 CATfor codon AUA Race to the tRNA structure (1974)

3Å resolution 3Å resolution Suddath, Quigley, McPherson, Robertus, Ladner, Finch, Rhodes, Brown, Sneden, Kim, Kim & Rich Nature (Mar Clark & Klug Nature (Aug 1974) 250:546 1974) 248:20 31st July, 1974

Dr. Alex“Rich, Department of Biology Massachusetts Institute of Technology Cambridge Mass. 02139 / 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 ack- knowledge 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 . tRNA Structure

tRNAPhe Rhodes lab at the LMB 15 year old xtals (1evv)

Jovine et al JMB (2000) 301:401 tRNA folding

Fig. 3.52 & 3.53 Detailed Interaction between D- interactions and T- loops

Sharp turns in the tRNA structure (Anticodon, T- loop, D-loop, 9-11 U- turn)

Fig. 3.54 & 3.55 Modified bases • Conserved • Not all the roles are clear yet Ψ

• Often aids in stabilizing m5U long range interactions m1A Mg2+ 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 Base triples

Fig. 3.56 Juncons

Fig. 3.42 & 3.43 “Hammerhead ” ribozyme

Fig. 3.58 & 3.59 Reaction

Hypothetical transition state Tetrahymena group I intron P4-P6 domain (1gid)

• First ribozyme described (Tom Cech - Nobel) Cech et al Cell (1981) 27:487-96 • Self catalyzes removal of intron • Stable tertiary domain of P4-P6

Doudna & Cech Labs: Cate et al Science (1996) 273:1678-85 Structure (from the text)

Fig. 3.62 & 3.63 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) Pseudoknot structure

A pseudoknot structure contains more than one stem-loops where at least one is intercalated into another.

Fig. 3.46 & 3.47 Biotin bound pseudoknot aptamer (1f27) Central

3’ Major

Tetraloops • Helices are capped by a limited pool of 4 residue 5’ sequences • General rule for stable

3’ Minor helix capping

Woese et al PNAS (1990) 87:8467 U RNA tetraloop C U motif G • Hyperabundunt 4 nucleotide terminal loops 5’ • 3 Classes 3’ – UNCG A – GNRA A – CUYG G – 256 possible but 16 A dominate!

5’

Tuerk et al PNAS (1988) 85:1364 Woese et al PNAS (1990) 87:8467 1j5e 3’ 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 Motifs from P4-P6

A-platform Ribose zipper

Fig. 3.64 & 3.65 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 1gid Steitz & Moore Labs: Nissen et al PNAS (2001) 98:4899-903 A-minor interacons from 23S rRNA

Fig. 3.49 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 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 connues to be shown in biology class today. It has since spawn a series of similar funny aempts at vulgarizing protein synthesis. Narrated by Paul Berg, 1980 Nobel prize for Chemistry. hps://www.youtube.com/watch?v=u9dhO0iCLww