Structure of nucleic acids IIBiochemistry 302January 20, 2006Intrinsic structural flexibility of RNAHigh TempDenaturantsIn vivo conditionsBase stacking w/o base pairing/H-bonds In vivo conditionsIntra-strand base pairingFig. 4.19A-formantiparallelStructure 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 tRNAPhe76 basesFig. 4.20Theoretical cloverleaf or cruciform structureFig. 4.27Ribose conformation (sugar pucker) differs in double-stranded RNA helicesDNA is C2′ endoRNA is C3′ endo2′ OH restricts C3′ to endo3′2′H3′2′OHDNA structures dictated by sequence: hairpins & cruciformsPalindrome = 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 cruciforminverted repeat w/ twofold symmetry with potential to form hairpin or cruciformLehninger Principles of Biochemistry, 4th ed., Ch 24Intrastrand bp single strand onlyIntrastrand bp both stands involvedTriple helical or H-DNA (regions of high Pur/Pyr asymmetry)RNA can also form triple helices: polyU:polyA:polyUC+= protonated C123456625134987546123This atom should be a purple nitrogen.Fig. 4.30Fig. 4.29N7, O6, N6of purines known as HoogsteenpositionsH-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 bpsLehninger Principles of Biochemistry, 4th ed., Ch 8Concept 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. 4.31annealTmdepends 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-DNAFig. 4.32Structural 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 106for E. coli genome (circular, DS)– ∼6.5 x 107bp for 1 fruit fly chromosome (linear, DS)– ∼3.2 x 109bp for 23 human chromosomes (linear, DS)• B-form except where sequence dictates otherwise• Relaxed or supercoiledE. coli cell (2 μm) chromosome (1.7 mm)Lehninger Principles of Biochemistry, 4th ed., Ch 24Tertiary 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)Lehninger Principles of Biochemistry, 4th ed., Ch 24Utility 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 CGntract)– Cruciform extension (palindromic sequences)– H-DNA formation (asymmetric poly Pur/Pyr tract) Lehninger Principles of Biochemistry, 4th ed., Ch 24Characteristics 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 2Plasmid DNA treated with type I topoisomerase for different timesLehninger Principles of Biochemistry, 4th ed., Ch 24Tertiary structure of DNA in vitro and in vivo: importance of compactionPlectonemic (“twisted thread”) supercoilingSolenoidal supercoiling (greater compaction)⇌proteinLehninger Principles of Biochemistry, 4th ed., Ch 24Metazoans have major size and biological issues to contend with….• Super-sized genome– Fit 2 meters worth of DNA (~6 x 109bp) 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 Lehninger Principles of Biochemistry, 4th ed., Ch 24The 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)Lehninger Principles of Biochemistry, 4th ed., Ch 24Structure of the nucleosome core particle (histone octamer plus DNA)H3H4H2AH2B146 bp of DNA/octamer; 1.7 left-hand superhelical turnsOctameracts like a