Structure of nucleic acids II Biochemistry 302 January...

Structure of nucleic acids IIBiochemistry 302

January 20, 2006

Intrinsic structural flexibility of RNA

High Temp

Denaturants

In vivo conditions

Base stacking w/o base pairing/H-bonds

In vivo conditions

Intra-strand base pairing

Fig. 4.19

A-form

antiparallel

Structure of transfer RNA (tRNA) and the concept of self-complementarity

• Self-complementary regions form A-type antiparallel helices or hairpins.

• Triple-base H-bonding & non-canonical bp

• Folding of helices produces a tertiarystructure necessary for function.

Yeast tRNAPhe 76 basesFig. 4.20

Theoretical cloverleaf or cruciform structure

Fig. 4.27

Ribose conformation (sugar pucker) differs in double-stranded RNA helices

DNA is C2′ endo

RNA is C3′ endo2′ OH restricts C3′ to endo

3′

2′ H

3′

2′OH

DNA structures dictated by sequence: hairpins & cruciforms

Palindrome = segments of complementary strands that are the reverse of one another

Stability of extended DNA vs cruciform?

symmetric sequence in each strand but cannot form hairpin or cruciform

inverted repeat w/ twofold symmetry with potential to form hairpin or cruciform

Lehninger Principles of Biochemistry, 4th ed., Ch 24

Intrastrand bp single strand only

Intrastrand bp both stands involved

Triple helical or H-DNA (regions of high Pur/Pyr asymmetry)

RNA can also form triple helices: polyU:polyA:polyU

C+ = protonated C

1 2

3

45

6

6

25

1

34

9

875

4

6

1

2 3

This atom should be a purple nitrogen.

Fig. 4.30 Fig. 4.29

N7, O6, N6 of purines known as Hoogsteenpositions

H-DNA formation produces a sharp bend (mutagenic, hotspot for DSBs)

Note how the third strand runs in a parallel direction to its complement.

Stability of DNA ds structure

• DNA does not fall apart under physiological conditions of pH and ionic strength….

• but some inherent instability is built in. Why?– Phosphate backbones of

opposing DNA strands →electrostatically repulsive (an effect reduced by dissolved counterions Na+, K+, Mg2+).

– Random coil has a ↑ entropy.• helix → random coil

ΔG = ΔH – TΔS– So, ΔS > 0 & ΔHelrep < 0 favors

transition to random coil– but ΔHtotal > 0 because of H-

bonding and van der Waals interactions between bps


Concept of DNA thermal stability(practical perspective, DNA “melting”)

Hypochromism: Pur and Pyr rings of stacked bases absorb light less efficiently than unstacked bases or free nucleotides.

Note the sharp Tmtransition point which is indicative of a highly cooperative transition.

Fig. 4.31

anneal

Tm depends on base-pair composition

• AT-rich regions melt (i.e. denature) more easily than GC-rich regions. Why?

• At Tm, ΔGdenat = 0 so……0 = ΔH – TmΔSand Tm = ΔH/ΔS– ΔS is the same for most

polynucleotides on a per bp basis.

– ΔH is higher for G ≡ C base pairs.

• Thermal stability of hybrids: RNA-RNA >RNA-DNA > DNA-DNA

Fig. 4.32

Structural features of DNA molecules in living organisms

• Single or double-stranded• Linear or circular• Small or large

– 5243 bp for SV40 genome (circular, DS)

– 6407 b for bacteriophage M13 genome (circular, SS)

– ∼4.6 x 106 for E. coli genome (circular, DS)

– ∼6.5 x 107 bp for 1 fruit fly chromosome (linear, DS)

– ∼3.2 x 109 bp for 23 human chromosomes (linear, DS)

• B-form except where sequence dictates otherwise

• Relaxed or supercoiled

E. coli cell (2 μm) chromosome (1.7 mm)


Tertiary structure of DNA (supercoiling of the helix)

• Higher-order folding of regular secondary structural elements

• Supercoiling– Twist of DNA strands

around one another– Extra twists in the helix

itself– Normal state of closed

circular DNA molecules (to relieve “strain” of being underwound)


Utility of superhelical density (σ)ΔGsc,free energy stored in supercoiling is proportional to superhelical density, ΔGsc = Kσ2

(σ = ΔL/L0) where ΔL = # turns removed or added relative to # in relaxed DNA.

• When DNA is relaxed…..σ = 0 so ΔGsc = 0.

• Decreasing σ (local unwinding) reduces stored energy ΔGsc.

• Imposing superhelical stress on DNA may thus promote…– Localized melting (AT-rich DNA)– Formation of short stretches of

Z-DNA (alternating CGn tract)– Cruciform extension

(palindromic sequences)– H-DNA formation (asymmetric

poly Pur/Pyr tract) Lehninger Principles of Biochemistry, 4th ed., Ch 24

Characteristics of naturally occurring circular DNA (e.g. plasmids, mito DNA)

• Underwound (common)– Right-handed superhelical

twist, negative supercoiling – σ = –0.05 to –0.07 (5-7% of

helical turns removed)• Overwound (rare)

– Left-handed superhelical twist, positive supercoiling

– Processive enzyme movement• Exist as topoisomers

– Relaxed– Supercoiled

• Topoisomerases– Cut and reseal DNA– Type I or II change L by

increments of 1 or 2

Plasmid DNA treated with type I topoisomerase for different times


Tertiary structure of DNA in vitro and in vivo: importance of compaction

Plectonemic (“twisted thread”) supercoiling

Solenoidal supercoiling (greater compaction)⇌protein


Metazoans have major size and biological issues to contend with….

• Super-sized genome– Fit 2 meters worth of DNA (~6

x 109 bp) into a nucleus ~8 μm in diameter

– Compaction → solenoid• Exquisite control of Gene

Expression– Maintain and regulate genetic

programs essential to cell growth and differentiation

– Structure must accommodate chromosomal organization where only ∼5-10% of DNA is actually transcribed


The Solution….Use special proteins to form chromatin• Histones

– Small (~11-23 kDa), basic, & highly conserved

– Building blocks of chromatin – Subject to posttranslational

modification– Five types – “core” & “linker”

• Non-histone chromosomal proteins– SMC (structural maintenance of chromosomes) proteins

• Cohesins – link sister chromatids after replication• Condensins – mediate chromosome condensation as

cells enter mitosis– Polymerases and other nuclear enzymes plus gene

regulatory proteins (e.g. transcription & remodeling factors, ∼1000 different proteins)


Structure of the nucleosome core particle (histone octamer plus DNA)

H3

H4

H2A

H2B

146 bp of DNA/octamer; 1.7 left-hand superhelical turns

Octameracts like a helical ramp

Fig. 28.10K. Luger et al. (1997) Nature 389:251

Other features of nucleosome structure (2nd level of organization)

• Histone core-DNA binding is not random (positioned near clusters of A=T bp).

• Nucleosomes separated by ∼20 to 100 bp (but linker distance varies among organisms and cell types.

• Linker DNA is occupied by H1-type histones and other non-histone proteins.

• H1 plays important role in higher order organization (one H1/nucleosome core).

• Gene-specific nucleosomes occupy defined positions.

• Subject to “remodeling”.Lehninger Principles of Biochemistry, 4th ed., Ch 24

“beads-on-a-string”

(3rd level of organization)

30 nm fiber → ~100-fold DNA compaction (need 104 fold)

Fig. 28.12

Heterochromatin (condensed)

Euchromatin (open)

MARs (sites of topoisomerase action and gene activation)


Structure of nucleic acids II Biochemistry 302 January...

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