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Berkeley MCELLBI 110 - Lecture Notes

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1MCB 110:Biochemistry of the Central Dogma of MBProf. NogalesPart 3. Membranes, protein secretion, trafficking and signalingPart 2.RNA & proteinsynthesis.Prof. ZhouPart 1.DNA replication,repair andgenomics(Prof. Alber)MCB 110:Biochemistry of the Central Dogma of MBPart 2.RNA & proteinsynthesis.Prof. ZhouProf. NogalesPart 3. Membranes, protein secretion, trafficking and signalingPart 1.DNA replication,repair andgenomics(Prof. Alber)2DNA structure summary 11. W & C (1953) modeled average DNA (independent of sequence) as an:anti-parallel, right-handed, double helix with H-bonded base pairs onthe inside and the sugar-phosphate backbone on the outside.2. Each chain runs 5’ to 3’ (by convention).Profound implications: complementary strands suggested mechanismsof replication, heredity and recognition.MissingStructural variation in DNA as a function of sequenceTools to manipulate and analyze DNA (basis for biotechnology, sequencing, genome analysis)DNA schematic (no chemistry)3. Duplex strandsare antiparallel andcomplementary.Backbone outside;H-bonded basesstacked inside.2. DNA strands are directional1. Nucleotide = sugar-phosphate + base4. The strands form a double helix3Nucleic-acid building blocksnucleosidenucleotideglycosidicbondGeometry of DNA bases and base pairs!C G T AH-bonds satisfiedSimilar widthSimilar angle to glycosidic bondsPseudo-symmetry of 180° rotation4Major groove and minor groove definitionsMajor groove Major grooveMinor groove Minor grooveSubtended by the glycosydic bondsOpposite the glycosydic bondsComparison of B DNA and A DNA (formed at different humidity)bp/turnBase tiltMajor grooveMinor grooveP-P distance10smallwideNarrow6.9 Å1120°narrow & deepwide & shallow5.9 ÅMajor groove(winds around)Minor groove(winds around)3.4- 3.6 ÅBps near helix axis Bps off helix axis5Average structure of dsRNA (like A DNA)“side” view“End” view 3’5’5’3’Minor grooveshallow and wideMajor groove deepand narrow(distortions neededfor proteins tocontact bases)Twist/bp ~32.7°~11 bp/turnBases tiltedDNA structure varies with sequence1. “Dickerson dodecamer” crystal structure2. Twist, roll, propeller twist and displacement3. Variation in B-DNA and A-DNAProteins recognize variations in DNA structureDNA stabilityDepends on sequence & conditionsForces that stabilize DNA: H-bonds, “stacking”,and interactions with ions and waterDNA structure and stability6Crystal structure of the “Dickerson dodecamer”Synthesize and purify 12-mer: d(CGCGAATTCGCG) = sequenceCrystallizeShine X-ray beam through crystal from all anglesRecord X-ray scattering patternsCalculate electron density distributionBuild model into e- density and optimize fit to predict the dataDisplay and analyze modelExperiment -- 1981ResultsB-DNA!!The structure was not a straight regular rod.There were sequence-dependent variations(that could be read out by proteins).Two views of the Dickerson dodecamer1. Double helix: Anti-parallel strands, bps “stacked” in the middle2. Not straight (19° bend/12 bp, 112 Å radius of curvature)3. Core GAATTC: B-like with 9.8 bp/turn4. Flanking CGCG more complex, but P-P distance = 6.7 Å (like B)5. Bps not flat. Propeller twist 11° for GC and 17° for AT6. Hydration: water, water everywhere on the outside (not shown).7Nomenclature for helical parametersPropeller twist: dihedral angle of base planes.Displacement: distance fromhelix axis to bp centerSlide: Translation along the C6-C8 lineTwist: relative rotation aroundhelix axisRoll: rotation angle of mean bp plane around C6-C8 lineTilt: rotation of bp plane aroundpseudo-dyad perpendicularto twist and roll axesSlidePropeller twist, roll and slideNo roll or propeller twist20° propeller twistSlide = -1 Å to avoid clash *Or roll = 20 ° and slide = + 2Å topromote cross-chain purine stacking8Slide and helical twistSlide = translation along the long (C6-C8) axis of the base pairRegular DNA variationsB-like A-like9Helical parameters of the dodecamerC1/G24G12/C13Range 4.9-18.6° 32.2-41.4° 8.1-11.2 3.14-3.54 Å Helical parameters of the dodecamerC1/G24G12/C13Range 4.9-18.6° 32.2-41.4° 8.1-11.2 3.14-3.54 Å10Helical parameters of the dodecamerC1/G24G12/C13Range 4.9-18.6° 32.2-41.4° 8.1-11.2 3.14-3.54 Å Base “stacking” maximizes favorable interactionsClashes due topropeller twist canbe alleviatedby positive roll(bottom left) orchanges in helicaltwist (right)N atoms closeN atoms separatedΔ roll Δ helical twist11Different patterns of H-bond donors andacceptors bases in different base pairs (gray)Major groove side (w)Minor groove side (S)Most differences inH-bond donors andacceptors occur inthe major groove!Sequence-specificrecognition usesmajor-groove contacts.Seeman, Rosenberg & Rich (1976),Proc Natl Acad Sci USA 73, 804-8.Lac repressor headpiece binds differently tospecific and nonspecific DNAsNonspecific DNASymmetric operator Natural operatorBent DNAStraight DNA12E. coli lac repressor tetramer binds 2 duplexesHeadpieceHinge helixNH2N-subdomainC-subdomainTetramerization helixLacI tetramerE. coli lac repressor tetramer binds 2 duplexesHeadpieceHinge helixNH2N-subdomainC-subdomainTetramerization helixRepressor tetramerloops DNA13E. coli catabolite activator protein (CAP)Stabilizes kinks in the DNAHuman TATA binding protein binds in theminor groove and stabilizes large bendsTwist along the DNADNAbent14Human TATA binding protein binds in theminor groove and stabilizes large bendsView into the saddle End viewDNATBPTBPDNA bending by E. coli AlkA DNA glycosylase Leu125 insertedinto the DNAduplex!66° bend15Base flipping in DNA repair enzymesHuman AlkylAdenine DNAGlycosylasePhage T4A GlycosylTransferase,AGTWhat causes bases to flip out?16What cause bases to flip out?Thermal fluctuationsFluctuations include denaturationT+NativeDenaturedTm = 50/50 native/denatured17Tm depends on?Tm depends on?DNA LengthBase compositionDNA SequenceSalt concentrationHydrophobic and charged solutesBound proteinsSupercoiling density18Length dependence of DNA stabilityFraction denaturedTemperature °C10 20 30No further increase> ~50 base pairsTm depends on G+C contentWhy?19Tm depends on G+C contentWhy? GC bps contain 3 H-bonds and stack better.Calculated base stacking energiesAT worstGC best20Tm depends on ionic strengthHigh KCl stabilizes duplex DNAWhy?Mg2+ ionsPolyamines: spermidine and spermine + +


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Berkeley MCELLBI 110 - Lecture Notes

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