Lecture 16 Nucleic acid polymers

Transcription

1 Lecture 16 Nucleic acid polymers Key learning goals: Understand 1 and 2 structure of DNA and RNA chains, and how 3 structures arise Understand why DNA is a good genetic storage medium but RNA is much more interesting Understand driving forces that control double helix formation Learn the dimensional (size) properties of B-DNA Understand base-pairing Understand the major factors that control DNA and RNA hybridization Understand what helicase enzymes do.

2 olymerization allows synthesis of periodic polymers Fatty acid elongation Chitin synthesis Cellulose synthesis Glycogen synthesis oly-ubiquitylation

3 Templated polymerization produces aperiodic polymers with defined sequences DNA polymerase DNA RNA polymerase reverse transcriptase RNA RNA-dependent RNA polymerase ribosome protein

4 DNA and RNA: aperiodic polymers that can form double-stranded duplexes Hybridization is the pairing of two DNA or RNA strands to form non-covalent duplexes. Formation of hybrid duplexes allows templated polymerization The backbone is identical in each repeating unit The bases vary from one repeating unit to the next National Institute of General Medical Sciences

5 Essential nomenclature anhydride bonds phosphoester bond γ β α glycosidic bond AM ribose dam deoxyribose

6 A DNA Duplex The backbones in a duplex run in opposite directions: they are antiparallel to one another phosphodiester (, ) glycosidic bonds (1 ) Modified from MCAT45.com (this is not an endorsement; I just liked this picture)

7 A DNA Duplex phosphodiester (, ) pka of this group is ~1.0 It is always ionized & negatively charged Later, we will see that counterions are required to balance these charges glycosidic bonds (1 ) Modified from MCAT45.com (this is not an endorsement; I just liked this picture)

8 olymer nomenclature The structure of a DNA or RNA chain can be specified in simplified notation. Nucleosides: A G C T base sugar A pentanucleotide: C phosphate phosphodiester link A C T G A nucleotide: C deoxycytidine-monophosphate dcm The same pentanucleotide, in even shorter form: -pcpapcptpg- and the most common representation of all: -CACTG- Note: By convention, DNA and RNA sequences are most often written -[ ]-, so the direction is not always indicated: CACTG

9 More nomenclature cytosine C cytidine (C) C glycosidic bond cytidine--monophosphate (CM) cytidine--diphosphate (CD) phosphoester bond C phosphoanhydride bond C

10 olymerization (DNA) n residues + dnt (RNA) n residues + NT (DNA) n+1 residues + i (RNA) n+1 residues + i C T G + C C T G C + All biological DNA and RNA polymerization reactions occur in direction! In contrast: synthetic polymerization reactions (generally done in organic solvents) are usually performed in direction.

11 *Q: Why did nature choose phosphorus? C A C T G R RA hosphates are reasonably good leaving groups, and their hydrolysis has a Goldilocks G (~50 kj mol -1 ) not too big, not too small. hosphoesters are stable in water ( 1 / 2 life = 30 million 25 C, ph 7). * A: Westheimer, Why nature chose phosphates. Science 235:1173

12 *Q: Why did nature choose phosphates? The importance of being ionized Histidine biosynthesis! Every single intermediate is phosphorylated until the charged amino and carboxylic acid groups appear. This is a common feature of many metabolic pathways. * A: Westheimer, Why nature chose phosphates. Science 235:1173

13 *Q: How about some arsenic? GFAJ-1 (from Wolfe-Simon et al.)

14 *Q: How about some arsenic? However... Chemical precedents suggest that arsenodiester linkages in the putative arsenic-containing DNA of GFAJ-1 would undergo very rapid hydrolytic cleavage in water at 25 C with an estimated half-life of 0.06 s. In contrast, the phosphodiester linkages of native DNA undergo spontaneous hydrolysis with a half-life of approximately 30,000,000 y at 25 C. Overcoming such dramatic kinetic instability in its genetic material would present serious challenges to Halomonadacea GFAJ

15 *A: No thanks, I ve already eaten. 27 JULY 2012 SCIENCE 337: This is a great example of how science is self-correcting!

16 Exercise: In a human cell, how many times does the DNA backbone spontaneously break each day? On average, each phosphodiester linkage is hydrolyzed once per 30,000,000 y Our genome has 3 billion base pairs per cell Note: RNA is not as stable as DNA (we ll come back to this point).

17 RNA backbones break through base-catalyzed hydrolysis at least 100 times more often than DNA 2 roduct can have i on 2 or position

18 DNA and RNA hybridization

19 DNA and RNA hybridization (this is extremely important) Denatured (ssdna) T m = melting point Native (dsdna) Factors that control T m : chain length solvent conditions (ionic strength, ph) number of mismatched base pairs base composition (G:C to A:T ratio)

20 Hybridization: base pairing provides specificity

21 Watson-Crick base pairing Remember: the H-bonds between Watson-Crick base pairs are important for pairing specificity, but H- bonds do not provide the major energetic contribution for duplex formation! The C5 methyl that distinguishes T from U does not participate in base pairing.

22 Hybridization for fun and profit Atomic Force Microscopy ted by parallel d (black) runs rst designed, with strands ck triangles to shape, blue with coloured lines, and major/minor grooves by large/small angles between them.. Arrows Rosenmund in c point (2006) to nicks Nature sealed doi: /nature04586 to create green strands in d. Yellow diamonds in c and d indicate a position at which staples may be cut and resealed to bridge the seam. e, A finished design after merges and rearrangements along the seam. Most staples are 32-mers spanning three helices. Insets show a dumbbell hairpin (d) and a 4-T loop (e), modifications

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24 Three dimensional structure of DNA in water water sugar phosphoric acid nitrogenous bases

25 Why DNA prefers to be duplexed hydrophilic hydrophobic hydrophilic phosphodiester pka ~1.0 6 Å Calladine et al., Understanding DNA, 3rd ed.

26 Base stacking A skewed ladder might allow stacking of the bases, to hide them from water X X However: the geometry of the glycosidic bond does not accomodate this structure. Calladine et al., Understanding DNA, 3rd ed.

27 The base and sugar are relatively rigid rings X 5! 4! 3! endo! 1! N! C-2 exo

28 Base stacking can occur only when the backbones spiral into a double helix. X X skewed ladder double helix

29 B-form DNA 10 base pairs per helical turn 3.4 Å i i 20 Å = 2 nm

30 Dimensions of the DNA double helix (B-form) Right-handed double helix Know these: Diameter = 20 Å Rise per base pair = 3.4 Å 10 base pairs per 360 helical turn and you can derive these: Helical twist per base pair θ = 36 Rise per helical turn = 34 Å (=3.4 nm)

31 DNA intercalating agents insert between bases, and thereby unwind DNA Intercalating agents are usually mutagenic. They do not covalently modify DNA but by chaning the conformation of DNA, they can force mis-pairing, resulting in base insertions or deletions (indels) during replication.

32 Base pairs can move a bit in the double helix All base-pairs assume a propeller twist configuration to varying degrees depending on the base pair (15º to 25 º for AT stacks, and 5 º to 15 º for GC stacks) glycosidic bond The propeller twist minimizes exposure of water between the base pairs. The hydrogen bond network is not disrupted.

33 Other DNA conformations A B Z A-DNA is scrunched, with a fatter helix. When RNA forms a double helix, it looks more like the DNA A-form. Z-DNA forms a left-handed double helix.

34 Optical properties of nt s, ssdna, dsdna A 260 nm da dg du dc nucleotides ssdna dsdna The conjugated!-electron systems of the purine & pyrimidine bases absorb strongly in the UV band. The excited states of two interacting molecules can be described as linear combinations of the excited states of each. In certain geometries, some of the absorption strength in the near-uv moves to bands at higher energies. This is the hyperchromic effect.

35 The ratio of A:T to G:C base pairs influences T m ? % Denatured ? % GC Temperature / o C 110? A 260 nm 1. As DNA melts, what happens to the UV absorbance? 2. As the fraction of A:T base pairs rises, what happens to T m? 3. What might we expect the base compostion of an arctic fish to look like vs. a tropical fish? 4. How can we best explain the effects of base compostion on T m?

36 DNA and RNA hybridization base stacking energies depend on the sequence!

37 Helicases are motors that hydrolyze AT to melt DNA and RNA Helicases hydrolyze the terminal (γ) phosphate on AT: AT > AD + i E. coli alone has twelve DNA helicases and more RNA helicases; DnaB unwinds DNA for replication. Helicases do not break the backbone!!