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CHAPTER 3: Chromosomes, chromatin, and the nucleosome

CHAPTER 3: Chromosomes, chromatin, and the nucleosome. 3.1 Prokaryotic chromosome ( 原核 染色体 ). 3.1.1 The E. coli chromosome. A single closed-circular DNA, 4.6 X 10 6 bp

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CHAPTER 3: Chromosomes, chromatin, and the nucleosome

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  1. CHAPTER 3: Chromosomes, chromatin, and the nucleosome

  2. 3.1 Prokaryotic chromosome (原核 染色体)

  3. 3.1.1 The E. coli chromosome • A single closed-circular DNA,4.6 X 106bp • The DNA packaged into a region known as Nucleoid (类核) that contains high concentration of DNA (up to 30-50 mg/ml) as well as all proteins associated with DNA. • Continuous replication (no cell cycle) • Attach to a part of cell membrane

  4. Nucleoid (类核,拟核) – Bacterial chromosome 细菌染色体

  5. 3.1.2 DNA domains/loops • Observed under electron microscope • 50-100 domains or loops per E. coli chromosome, with 50-100 kb/loop • The ends of loops are constrained (束缚) by proteins

  6. Domain/ Loop Basic protein Member binding proteins? Supercoiled DNA

  7. Supercoiled domain Non-supercoiled domain Protein-membrane core or scaffold

  8. 3.1.3 Supercoiling of the genome • E. coli chromosome as a whole is negatively supercoiled • Individual domains may be supercoiled independently (topological independent) because the protein-membrane scaffold may prevent DNA rotation.

  9. 3.1.4 DNA-binding proteins • Histone-like proteins essential for DNA packaging to stabilize and constrain the supercoiling. • HU: a small basicdimeric (碱性双体) protein, non-specific binding to DNA, most abundant. • H-NS (protein H1): neutral monomeric (中性单体), partially non-specific binding • Site-specific DNA binding proteins important for organization of DNA domains (RNA polymerases, IHF etc).

  10. Supplementary 1: • Organelle genomes: • circular, multiple copies • ctDNA: chloroplast (叶绿体) DNA, 140kb in plants, and <200kb in lower eukaryotes. Similar to cyanobacteria (兰细菌) • mtDNA: mitochondrial (线粒体) DNA, 16.5 kb in mammals, 80 kb in yeast, >100kb in plant. Similar to -purple bacteria, Rickettsia) mtDNA

  11. Supplementary 2: Range of genome size found in different organism phyla.

  12. 3.2 Chromatin Structure (染色质结构)

  13. Solving the packing problem:chromatin The length of a chromosomal DNA can be up to several centimeters (cm), but the the diameter of nucleus is about 1-10 mm. (104-fold of condensation) The DNA concentration in nucleus is about 200 mg/ml 3.2.1 Chromatin (染色质) A highly organized complex of DNA and protein (nucleoprotein complex), which makes up the eukaryotic chromosomes (染色体). >50% of the mass is protein

  14. Chromatin structure enables the chromosomes toalter their compactnessas the cell progress the cell cycle. Mitosis Interphase Chromatin (diffused) Chromosome (condensed)

  15. 3.2.2 Histones (组蛋白) and Nucleosomes (核小体) • The major protein components of chromatin • Four families of core histone: H2A, H2B, H3 and H4. An additional non-core histone H1 • Small, 10 kDa for core histones and 23 kDa for H1. • Basic (rich in lysine and arginine) and tightly binds to DNA

  16. Histone octamer (组蛋白八聚体) Top view Nucleosome core Side view

  17. DNA Histone octamer Nucleosome core 146 bp, 1.8 superhelical turn Histone H1 Chromatosome 166 bp, 2 superhelical turn

  18. 3.2.3 The role of H1 • Stabilizes the point at which DNA enters and leaves the nucleosome core. • C- tail of H1: stabilizes the DNA between the nucleosome cores. • 23 kDa, located outside of nucleosome core, binds to DNA more loosely • Less conserved in its sequence

  19. 3.2.4 Linker DNA The additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bp

  20. “Beads on a string” structure Nucleosome repeat: Core + linker DNA 200 bp Histone H1 Nucleosome Linker DNA <10 to > 100 bp average 55 bp

  21. 3.2.5 The 30 nm fiber - Steps to make it

  22. Step 1:form a Nucleosome (核小体) DNA + Histone octamer (组蛋白八聚体) Nucleosome core (核小体核心 146bp) + H1  Chromatosome (染色小体 166bp) + linker DNA Nucleosome (核小体) (~200 bp of DNA) Nucleosome (核小体) is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins.

  23. Step 2: “Beads on a string” structure Nucleosome repeat: Core + linker DNA 200 bp Histone H1 Nucleosome Linker DNA <10 to > 100 bp average 55 bp

  24. Ø10-11 nm

  25. The 30 nm fiber 30 nm fiber (30nm in diameter) Solenoid (螺线管） Step 3: 30 nm fiber • Higher ordered • Left-handed helix • Six nucloesomes per turn

  26. 30nm fiber

  27. Step 4:looped domain structure Highest level of chromatin organization 30 nm fiber 300 nm Nuclear matrix (核基质), protein complex

  28. Supplementary 1: Nucleosomes and micrococcal nuclease treatment

  29. Supplementary 2: Steps from DNA to chromosome

  30. 染色体的包装（chromosome packing） ◆核小体(nucleosome) ◆螺线管(solenoid) ◆超螺线管(supersolenoid) ◆染色体(chromosome) 从核小体开始到染色体, DNA总共压缩: 压缩7倍 压缩6倍 压缩40倍 压缩5倍＝10000 DNA →核小体→螺线管→超螺线管→染色单体

  31. 染色体的包装 chromosome packing 1400nm 染色体（每个染色单体含10圈） 染色体 5 螺旋圈 700nm 30个座/每圈 玫瑰花结 300nm 6环超螺旋/座 突环 150nm,75000bp/突环 40 30nm 6 核小体/环 螺线管 6 11 nm/珠 核小体 7

  32. 3.3 Eukaryotic chromosome (真核染色体)

  33. Supplementary : Cell cycle (细胞周期)

  34. Cell cycle Interphase 间期: G1 + S + G2 (G0) M phase (mitosis 有丝分裂):

  35. D3 Eukaryotic Chromosomal Structure Cell cycle M phase includes: prophase (前期), metaphase (中期), anaphase (后期), telophase (末期)

  36. Mitotic chromosome

  37. 3.3.1 Mitotic chromosome (有丝分裂期的染色体) more condensed than chromatin, and most highly condensed at mitosis

  38. Telomere Sister chromatid 姊妹染色单体 Centromere 中心粒 Loops of 30nm fiber Mitotic chromosome at metaphase Nuclear matrix 核基质 Chromatid 染色单体

  39. The centromere (中心粒) • The region where two chromatids (姊妹染色体）are joined • The sites of attachment to the mitotic spindle (纺锤体) via kinetochore (动原体) • Centromere DNA: Yeast: AT-rich (88bp)

  40. D3 Eukaryotic Chromosomal Structure Mitotic chromosome Mitotic spindle

  41. Mitotic chromosome - centromere Yeast centromere Mammalian cells: much longer, flanked by satellite DNA (卫星DNA)

  42. The Telomere (端粒) • Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosome • Contains up to hundreds copies of a short repeated sequence(5’-TTAGGG-3’ in human) • Synthesized by the enzyme telomerase(aribonucleoprotein) independent of normal DNA replication. • The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation

  43. D3 Eukaryotic Chromosomal Structure Telomere & Telomerase Repeat sequence: Tetrahymena- TTGGGG; human- TTAGGG

  44. Telomere: structure A loop structure forms at the end of chromosomal DNA

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