10/09/08Biochemistry: Nucleic Acid Struct II

Nucleic AcidStructure II

Andy HowardIntroductory Biochemistry

9 October 2008

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

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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!

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Summaries of A, B, Z DNA

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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

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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!

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Instances of inter-calators

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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

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Heat denaturation Figure 11.14

Heat 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.)

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GC content vs. melting temp High salt and

no chelators raises the melting temperature

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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

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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

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Steps in denaturation and renaturation

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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

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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.

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Typical c0t curves

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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

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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

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Density as

function of GC

content

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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

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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

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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

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Examples with a tube

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How this works with real DNA

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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

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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

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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

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< 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

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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

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Cruciform DNA example

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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

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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

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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

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Histones: table 11.2

Histone #lys, #arg Mr, kDa Copies per Nucleosome

H1 59, 3 21.2 1 (not in bead)

H2A 13, 13 14.1 2 (in bead)

H2B 20, 8 13.9 2 (in bead)

H3 13, 17 15.1 2 (in bead)

H4 11, 14 11.4 2 (in bead)

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Nucleosome core particle

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Half the core particle

Note that DNA isn’t really circular: it’s a series of straight sections followed by bends

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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

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Chromosome structure: levels Each of the

first 4 levels compacts DNA by a factor of 6-20; those multiply up to > 104

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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!

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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

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).

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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

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Activation of the phosphoramidate

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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

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tRNA structure: overview

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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.

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Yeast phe-tRNA Note

nonstandard bases and cloverleaf structure