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Nucleic Acid Structure II. Andy Howard Introductory Biochemistry 9 October 2008. What we’ll discuss. Folding kinetics Supercoils Nucleosomes Chromatin and chromosomes Lab synthesis of genes tRNA & rRNA structure. Getting from B to Z. Can be accomplished without breaking bonds

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nucleic acid structure ii

Nucleic AcidStructure II

Andy HowardIntroductory Biochemistry9 October 2008

Biochemistry: Nucleic Acid Struct II

what we ll discuss
What we’ll discuss
  • Folding kinetics
  • Supercoils
  • Nucleosomes
  • Chromatin and chromosomes
  • Lab synthesis of genes
  • tRNA & rRNA structure

Biochemistry: Nucleic Acid Struct II

getting from b to z
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

summaries of a b z dna
Summaries of A, B, Z DNA

Biochemistry: Nucleic Acid Struct II

dna is dynamic
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

intercalating agents
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

instances of inter calators
Instances of inter-calators

Biochemistry: Nucleic Acid Struct II

denaturing and renaturing dna
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

heat denaturation
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

gc content vs melting temp
GC content vs. melting temp
  • High salt and no chelators raises the melting temperature

Biochemistry: Nucleic Acid Struct II

how else can we melt dna
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

what happens if we separate the strands
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

steps in denaturation and renaturation
Steps in denaturation and renaturation

Biochemistry: Nucleic Acid Struct II

rate depends on complexity
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

second order kinetics
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

typical c 0 t curves
Typical c0t curves

Biochemistry: Nucleic Acid Struct II

hybrid duplexes
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

gc rich dna is denser
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

density as function of gc content
Density as function of GC content

Biochemistry: Nucleic Acid Struct II

tertiary structure of dna
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

dna is wound
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

linking twists and writhe
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

examples with a tube
Examples with a tube

Biochemistry: Nucleic Acid Struct II

how this works with real dna
How this works with real DNA

Biochemistry: Nucleic Acid Struct II

how gyrases work
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

typical gyrase action
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

superhelix density
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

0 and spools
 < 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

cruciform dna
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

cruciform dna example
Cruciform DNA example

Biochemistry: Nucleic Acid Struct II

eukaryotic chromosome structure
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

nucleosome structure
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

histone types
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

histones table 11 2
Histones: table 11.2

Biochemistry: Nucleic Acid Struct II

nucleosome core particle
Nucleosome core particle

Biochemistry: Nucleic Acid Struct II

half the core particle
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

histones continued
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

chromosome structure levels
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

synthesizing nucleic acids
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

solid phase oligonucleotide synthesis
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

slide41

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).

solid phase synthesis
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

activation of the phosphoramidate
Activation of the phosphoramidate

Biochemistry: Nucleic Acid Struct II

secondary and tertiary structure of rna
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

trna structure overview
tRNA structure: overview

Biochemistry: Nucleic Acid Struct II

amino acid linkage to acceptor stem
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

yeast phe trna
Yeast phe-tRNA
  • Note nonstandard bases and cloverleaf structure

Biochemistry: Nucleic Acid Struct II