哈佛分子生物学讲义--核酸特性、结构和功能.ppt

1、“Chromatin is the last refuge of scoundrels”,A Mitotic Human Chromosome,DNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 m Compaction ratio = 8000,1,Chromatin,DNA Compaction,DNA Accessibility,NIH,Basic Unit of Chromatin Structure: Polynucleosome,2,Compaction ratio = 6,Limited Micrococcal Nulce

2、ase Digestion of Chromatin Generates a 200 bp Ladder,3,4,The 30 nm Chromatin Fiber,5,Compaction ratio = 50,A Mitotic Human Chromosome,DNA length: 4.6 x 107 bp = 1.5 cm Chromosome length: 2 m Compaction ratio = 8000,1,30 nm filament,Nuclear Scaffold/Matrix,DNA Loops (5-200 kb),SAR/MAR,6,The Nucleosom

3、e 146 bp DNA 2X H3, H4, H2A, H2B,H4,H2B,H2A,H3,Not in Handout,Identities = 74/82 (90%), Positives = 79/82 (96%) Query: 22 ILRDNIQGITKPAIRRLARRGGVKRISGLIYEEVRAVLKSFLESVIRDSVTYTEHAKRKT 81 +LRDNIQGITKPAIRRLARRGGVKRISGLIYEE R VLK FLE+VIRD+VTYTEHAKRKT Sbjct: 21 VLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLE

4、NVIRDAVTYTEHAKRKT 80 Query: 82 VTSLDVVYALKRQGRTLYGFGG 103 VT+DVVYALKRQGRTLYGFGG Sbjct: 81 VTAMDVVYALKRQGRTLYGFGG 102,Blast Search with S. cerevisiae Histone H4,Query: S. cerevisiae Subject: H. sapiens,Not in Handout,MW Chromosomal Proteins,200,000,36,000,97,000,66,000,55,000,31,000,21,000,14,000,116

5、,000,H4,H2A,H2B/H3,Histones are Low MW Chromosomal Proteins,7,Domain Structure of Histones,1,2,3,L1,L2,N,C,Core Domain,8,H2B,H2A,H3,H4,a2,a3,a1,a2,a3,a1,a2,a3,a1,a2,a3,a1,9,L1,L2,aN,H3,H4,10,4 helix-bundle,H3,H4,H3,H4,11,4 helix-bundle,H4,H2B,H2A,12,H4,H2B,H2A,H3,Not in Handout,H4,H2B,H2A,H3,Diamete

6、r = 110 A,13,H4,H2B,H2A,H3,Width = 45 A,14,H4,H2B,H2A,H3,15,Histone Core,Tilt,+Roll,Tilt,-Roll,15A,Tilt,Roll,H2B,H2A,L1-L2 Minor groove,1- 1 Minor groove,L1-L2 Minor groove,16,H2B,H2A,Not in Handout,DNA entry,DNA exit,N-term helix,H2B,H2A,H3,Not in Handout,DNA entry,DNA exit,N-term helix,N-term heli

7、x,H2B,H2A,H3,Not in Handout,DNA entry,DNA exit,Energetic and Topological Consequences of DNA Wrapping,Basepair:,1,5,10,15,Histone Core,Translational and Rotational Positioning,Basepair:,1,5,10,15,Histone Core,Translational and Rotational Positioning,Basepair:,1,5,10,15,Histone Core,Translational and

8、 Rotational Positioning,ARE COUPLED!,19,Are Nucleosomes “Positioned?”,1. Sequence at Unique Position,2. Micrococcal Nuclease Digest,3. Restriction Digest,4. Southern Blot,Discrete Band Positioning Smear Random Orientation,17,Implications of Translational Positioning,18,Histones Have Variable Length

9、Tails,20,H4,H2B,H2A,H3,H3 N-terminus,H3 N-terminus,21,H4,H2B,H2A,H3,H2B N-terminus,22,H4,H2B,H2A,+,+,+,_,_,_,23,N-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTE- H3,Core,10,28,N-SDPAKSAPAAKKGSKKAVTKTQKKDGKKRRKSRKES-H2B,23A,M,M,M,M,M,K79,Active region,Inactive Region (heterochromatin, X-

10、inactivation),Active region,Mitosis (Aurora B),?,“Beads on a String”,30 nm Filament,24,Low salt,High salt,How does 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 requ

11、irement for chromatin compaction,25,Helix III - Major groove binding,Loop (Secondary DNA binding site),Globular domain of Histone H5 “winged helix-turn-helix domain”,26,26,Zhou et al., 1998 Nature 395, 402,The Solenoid Model for the 30 nm Fiber,26A,Histone H1,The 30 nm Chromatin Fiber,5,Compaction ratio = 5