Structure of nucleic acids IIBiochemistry 302Name that polynucleotideStructures of single-stranded nucleic acids (RNA)Structure of transfer RNA (tRNA) and the concept of self-complementarity(fold back to form a bp antiparallel helix)The importance of water in RNA base pair (U:A) openingConformational possibilities of DNA and RNA in vitro and in vivoZ-DNA (A. Rich and coworkers report crystal structure of CGCGCG in 1979)Purines are always syn in Z-DNA (normally anti in B-DNA)Sequences predicted to form cruciforms are often palindromicSequences forming hairpins/cruciforms tend to be in gene regulatory regionsTriple helical or H-DNA(regions of high Pu/Py asymmetry in which one strand loops back)Tertiary structure of DNACharacteristics of naturally occurring circular DNA (e.g. plasmids, mito DNA)DNA structure is flexible and can be described in quantitative termsConcept of superhelical density (measure of the “intensity” of supercoiling)Concept of stored superhelical energy and its use to drive structural changesConcept of DNA “stability”(thermodynamic perspective)Concept of DNA stability(practical perspective, DNA “melting”)Tm depends on base-pair compositionStructural features of DNA molecules in living organismsPhysical organization of DNA in cells….the importance of proteinMetazoans have two major problems that prokaryotes don’t have….The Solution….Complex DNA with special proteins to form chromatinStructure of the nucleosome core particle (histone octamer plus DNA)Other features of nucleosome structureBut the nucleosome only gets you several-fold DNA compaction….Structure of nucleic acids IIBiochemistry 302Bob KelmJanuary 23, 2004Name that polynucleotide= carbon = nitrogen = oxygen = phosphorusStructures of single-stranded nucleic acids (RNA)Fig. 4.19High TempDenaturantsIn vivo conditionsBase stacking w/o base pairing/H-bonds In vivo conditionsIntrastrand base pairingStructure of transfer RNA (tRNA) and the concept of self-complementarity(fold back to form a bp antiparallel helix)Yeast tRNAPhe76 basesFig. 4.20Theoretical cloverleaf or cruciform structureFig. 4.27• Self-complementary regions form A-type hairpin.• Triple-base H-bonding• Additional folding of helices produces a defined tertiarystructure necessary for function.The importance of water in RNA base pair (U:A) openingGUA*New 2′OH(n)-O4′(n+1) H-bond**Y. Pan and A. D. MacKerell Jr. (2003) NAR 31:7131Conformational possibilities of DNA and RNA in vitro and in vivo• Common– B-form (DNA in vivo)– A-form (RNA in vivo, DNA too but only in vitro)– Random coil (no defined secondary structure)– RNA hairpins and cruciforms (descriptive)• Rare – Left-handed or Z-DNA (CGnrepeats, favored configuration when Cs are methylated on carbon 5)– DNA hairpins and cruciforms– Triple helices and H-DNAZ-DNA (A. Rich and coworkers report crystal structure of CGCGCG in 1979)B-DNA Z-DNAZigzag pattern of phosphatesReverse sense of helixAlternating Pu/Pybases in alternat-ing syn/antiorientationAll basesin anti orientation with respect to the deoxyribose ringFig. 4.26Fig. 4.11Purines are always syn in Z-DNA (normally anti in B-DNA)http://www.bioc.aecom.yu.edu/labs/brenlab/courses/Brenowitz/GB_MB2003_Lecture02-v01.pdfSequences predicted to form cruciforms are often palindromicFig. 4.28Palindrome = segments of complementary strands that are the exact reverse of one another Unpaired ends, stability of extended vs cruciform?Sequences forming hairpins/cruciforms tend to be in gene regulatory regionsModel of mouse VSM α-actin gene 5′ MCAT enhancer regionTriple helical or H-DNA(regions of high Pu/Py asymmetry in which one strand loops back)C+= protonated C1234566123This should be a purple N.Fig. 4.30Fig. 4.2962513498754RNA can also form triple helices: polyU:polyA:polyUTertiary structure of DNA• Higher-order folding (bending) of regular secondary structural elements• Supercoiling– Twist of DNA strands around one another– Extra twists in the helix itself– Normal state of closed circular DNAsCharacteristics of naturally occurring circular DNA (e.g. plasmids, mito DNA)• Handedness– Left superhelical twist, negativesupercoiling (most common)– Rightsuperhelical twist, positivesupercoiling• Exist as topoisomers– Relaxed– Supercoiled• Topoisomerases– Cut and reseal DNA– Some (e.g. E.coli DNA gyrase) require ATPFig. 4.18Mitochondrial DNA 16.5 kbDNA structure is flexible and can be described in quantitative termsLinking number (L) =# times strands of closed DNA are interlinkedWrithe (W) =# superhelical turns required to restore original Twist Unnatural twist or underwoundL = T + WTopoisomeraseschange L.Left (-)Fig. 4.24Twist (T) = # bp/10.5 bp/turnConcept of superhelical density (measure of the “intensity” of supercoiling)• Strain imposed by putting extra twists (T) and turns (W) in the DNA helix, ∆L = ∆T + ∆W.• Strain, ∆L, is positive or negative depending on whether the extra twists or superhelical turns are right-handed (+) or left-handed (–).• Superhelical density, σ = ∆L/L0 where L0is the linking number for DNA in the relaxed state.• Naturally occurring DNA molecules have superhelix densities of –0.06.• Real DNAs minimize strain by writhing into superhelical turns rather than untwisting.Concept of stored superhelical energy and its use to drive structural changes• Energy stored in supercoiling is proportional to superhelical density, ∆Gsc= Kσ2 (σ = ∆L/L0)• When DNA is relaxed σ = 0 so ∆Gsc = 0.• Decreasing σ reduces stored energy ∆Gsc.• Superhelical stress on the DNA molecule may thus promote…– Localized melting (AT-rich DNA)– Z-DNA formation (alternating CGntract)– Cruciform extension (palindromic sequences)– H-DNA formation (asymmetric poly Pur/Pyr tract)Concept of DNA “stability”(thermodynamic perspective)• B-DNA does not fall apart under physiological conditions of pH and ionic strength.• Some inherent instability must be built into the system. Why?– Phosphate backbone of opposing strands are electrostatically repulsive (an effect reduced by counterions like Na+, K+, Mg2+).– Random coil has a higher entropy.• helix → random coil ∆G = ∆H – T∆S– So, ∆S > 0 and ∆Helrep< 0 favors transition to random coil– But, ∆Htotal> 0 because of H-bonding and van der Waals interactions between base pairsConcept of DNA stability(practical perspective, DNA “melting”)Hypochromism: Pu and Py rings