Structure of nucleic acids II Biochemistry 302 January 20, 2006
Intrinsic structural flexibility of RNA antiparallel A-form Fig. 4.19 High Temp Denaturants In vivo conditions Base stacking w/o base pairing/h-bonds In vivo conditions Intra-strand base pairing
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 tertiary structure necessary for function. Fig. 4.27 Theoretical cloverleaf or cruciform structure Fig. 4.20 Yeast trna Phe 76 bases
Ribose conformation (sugar pucker) differs in double-stranded RNA helices 2 3 H DNA is C2 endo 3 2 OH RNA is C3 endo 2 OH restricts C3 to endo
DNA structures dictated by sequence: hairpins & cruciforms inverted repeat w/ twofold symmetry with potential to form hairpin or cruciform Intrastrand bp single strand only symmetric sequence in each strand but cannot form hairpin or cruciform Palindrome = segments of complementary strands that are the reverse of one another Stability of extended DNA vs cruciform? Intrastrand bp both stands involved
Triple helical or H-DNA (regions of high Pur/Pyr asymmetry) Note how the third strand runs in a parallel direction to its complement. C + = protonated C N7, O 6, N 6 of purines known as Hoogsteen positions 1 2 3 6 5 4 H-DNA formation produces a sharp bend (mutagenic, hotspot for DSBs) 6 5 1 4 2 3 1 6 7 5 4 2 3 8 9 Fig. 4.30 Fig. 4.29 RNA can also form triple helices: polyu:polya:polyu This atom should be a purple nitrogen.
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 +, Mg 2+ ). Random coil has a entropy. helix random coil ΔG = ΔH TΔS So, ΔS > 0 & ΔH elrep < 0 favors transition to random coil but ΔH total > 0 because of H- bonding and van der Waals interactions between bps Lehninger Principles of Biochemistry, 4th ed., Ch 8
Concept of DNA thermal stability (practical perspective, DNA melting ) anneal Note the sharp T m transition point which is indicative of a highly cooperative transition. Fig. 4.31 Hypochromism: Pur and Pyr rings of stacked bases absorb light less efficiently than unstacked bases or free nucleotides.
T m depends on base-pair composition AT-rich regions melt (i.e. denature) more easily than GC-rich regions. Why? At T m, ΔG denat = 0 so 0 = ΔH T m ΔS and T m = 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 10 6 for E. coli genome (circular, DS) 6.5 x 10 7 bp for 1 fruit fly chromosome (linear, DS) 3.2 x 10 9 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) ΔG sc, free energy stored in supercoiling is proportional to superhelical density, ΔG sc = Kσ 2 (σ = ΔL/L 0 ) where ΔL = # turns removed or added relative to # in relaxed DNA. When DNA is relaxed..σ = 0 so ΔG sc = 0. Decreasing σ (local unwinding) reduces stored energy ΔG sc. Imposing superhelical stress on DNA may thus promote Localized melting (AT-rich DNA) Formation of short stretches of Z-DNA (alternating CG n tract) Cruciform extension (palindromic sequences) H-DNA formation (asymmetric poly Pur/Pyr tract)
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 protein Solenoidal supercoiling (greater compaction)
Metazoans have major size and biological issues to contend with. Super-sized genome Fit 2 meters worth of DNA (~6 x 10 9 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 Octamer acts like a helical ramp K. Luger et al. (1997) Nature 389:251 Fig. 28.10 146 bp of DNA/octamer; 1.7 left-hand superhelical turns
Other features of nucleosome structure (2 nd 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. beads-on-a-string (3 rd level of organization)
30 nm fiber ~100-fold DNA compaction (need 10 4 fold) Heterochromatin (condensed) Euchromatin (open) MARs (sites of topoisomerase action and gene activation) Fig. 28.12