核酸特性、结构和功能ppt课件

1、Interference FootprintingDNA + MMS + Saturating ProteinGel Shift14Cleave backbone modified bases“Chromatin is the last refuge of scoundrels”A Mitotic Human ChromosomeDNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 mCompaction ratio = 80001ChromatinDNA CompactionDNA AccessibilityChromatinRemod

2、elingMachinesNIHBasic Unit of Chromatin Structure: Polynucleosome2Compaction ratio = 6Limited Micrococcal Nulcease Digestion of Chromatin Generates a 200 bp Ladder3H1H1H1H1H1H1Micrococcal Nuclease Digestion TimeMononucleosome ChromatosomeCore ParticleLinker DNA (variable)Operational Definititions of

3、 Nucleosome ParticlesChromatin4The 30 nm Chromatin Fiber5Compaction ratio = 50A Mitotic Human ChromosomeDNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 mCompaction ratio = 8000130 nm filamentNuclear Scaffold/MatrixDNA Loops (5-200 kb)SAR/MAR6The Nucleosome146 bp DNA 2X H3, H4, H2A, H2BH4H2BH2

4、AH3Not in Handout Identities = 74/82 (90%), Positives = 79/82 (96%)Query: 22 ILRDNIQGITKPAIRRLARRGGVKRISGLIYEEVRAVLKSFLESVIRDSVTYTEHAKRKT 81 +LRDNIQGITKPAIRRLARRGGVKRISGLIYEE R VLK FLE+VIRD+VTYTEHAKRKTSbjct: 21 VLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKT 80Query: 82 VTSLDVVYALKRQGRT

5、LYGFGG 103 VT+DVVYALKRQGRTLYGFGGSbjct: 81 VTAMDVVYALKRQGRTLYGFGG 102Blast Search with S. cerevisiae Histone H4Query: S. cerevisiaeSubject: H. sapiensNot in HandoutMW Chromosomal Proteins200,00036,00097,00066,00055,00031,00021,00014,000116,000H4H2AH2B/H3Histones are Low MW Chromosomal Proteins7Domain

6、 Structure of Histones123L1L2NCCore Domain8H2BH2AH3H42312312312319L1L2NH3H4104 helix-bundleH3H4H3H4114 helix-bundleH4H2BH2A12H4H2BH2AH3Not in HandoutH4H2BH2AH3Diameter = 110 A13H4H2BH2AH3Width = 45 A14H4H2BH2AH315Axis of dyad symmetryHistone CoreTilt+RollTilt-Roll15ATiltRollH2BH2AL1-L2Minor groove 1

7、- 1- 1 1Minor grooveMinor grooveL1-L2Minor groove16H2BH2ANot in HandoutDNA entryDNA exitN-term helixH2BH2AH3Not in HandoutDNA entryDNA exitN-term helixN-term helixH2BH2AH3Not in HandoutDNA entryDNA exitEnergetic and TopologicalConsequences of DNA WrappingBasepair: 151015HistoneCoreTranslational and

8、Rotational PositioningBasepair: 151015HistoneCoreTranslational and Rotational PositioningBasepair: 151015HistoneCoreTranslational and Rotational PositioningARECOUPLED!19Are Nucleosomes “Positioned?”1. Sequence at Unique Position2. Micrococcal Nuclease Digest3. Restriction Digest4. Southern BlotDiscr

9、ete Band PositioningSmear Random Orientation 17Implications of Translational Positioning18N-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTE- H3N-SGRGKGGKGLGKGGAKRHRKVLRDNIQ - H4N-SGRGKQGGKTRAKSKTRSSRAGL- H2AN-SDPAKSAPAAKKGSKKAVTKTQKKDGKKRRKSRKES- H2BH3 -ERA-CH4 -RTLYGFGG-CH2A -ELNKLLGGVT

10、IAQGGVLPNIQSVLLPKKTESSKSTKSK-CH2B -PGELAKHAVSEGTKAVTKYTSAK-CCore Core Histones Have Variable Length Tails20H4H2BH2AH3H3 N-terminusH3 N-terminus21H4H2BH2AH3H2B N-terminus22H4H2BH2A+_23N-SGRGKGGKGLGKGGAKRHRKVLRDNIQ -H4PAcAcAcAcN-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTE- H3Core PP102

11、8AcAc AcAc AcAcN-SGRGKQGGKTRAKSKTRSSRAGL-H2APAcAcN-SDPAKSAPAAKKGSKKAVTKTQKKDGKKRRKSRKES-H2BPAcAcAcAcAc Ac23ANewly-synthesized histonesMMMMMK79ActiveregionInactiveRegion (heterochromatin, X-inactivation)ActiveregionMitosis(Aurora B)SSN6/TUP1?“Beads on a String”30 nm Filament24Low saltHigh saltHow doe

12、s chromatin become compacted?The “Linker” Histone H1/H5 Largest histone (21 kD), less conserved Binds 165 bp: “Chromatosome” Facilitates chromatin compaction Histone H5 reduces salt requirement for chromatin compaction25Helix III - Major groove bindingLoop(Secondary DNA binding site)Globular domain of Histone H5“winged helix-turn-helix domain”26NCCCCCC26Zhou et al., 2019 Nature 395, 402The Solenoid Model for the 30 nm Fiber26AHistone H1The 30 nm Ch