Nucleus, chromosomes, DNA structure and function

Details about nuclear, chromosomes, DNA structure and function

1. Nucleus, chromosomes, DNA Dr. Vividha Raunekar

2. Nuclear Envelope: The nuclear envelope (NE) is a double membrane structure that encloses the nucleus in eukaryotic cells, separating the nuclear contents from the cytoplasm. It plays a crucial role in regulating material exchange and maintaining nuclear integrity. Structure of the Nuclear Envelope Double Membrane : Composed of an inner and an outer membrane, each with distinct functions. Perinuclear Space : The space (~20-40 nm wide) between the inner and outer membranes, continuous with the lumen of the endoplasmic reticulum (ER). Outer Membrane : Continuous with the rough ER and may have ribosomes attached, contributing to protein synthesis. Inner Membrane : Contains specific proteins that interact with the nuclear lamina and chromatin to provide structural support. Nuclear Lamina : A fibrous network of intermediate filaments ( lamins ) underlying the inner membrane, essential for nuclear shape, chromatin organization, and structural integrity.

3. Function of the Nuclear Envelope Compartmentalization : Separates nuclear processes (e.g., DNA replication and transcription) from cytoplasmic processes (e.g., translation). Selective Transport : Facilitates regulated exchange of molecules between the nucleus and cytoplasm via nuclear pores. Chromatin Organization : The inner membrane associates with chromatin and nuclear lamina to regulate gene expression. Cell Cycle Regulation : During mitosis in higher eukaryotes, the NE disassembles and reassembles, allowing chromosome segregation.

4. Structure of Nuclear Pores The Nuclear Pore Complex (NPC) is a massive protein assembly that regulates the exchange of materials between the nucleus and cytoplasm. It is embedded in the nuclear envelope , a double membrane that surrounds the nucleus in eukaryotic cells. The NPC is highly selective, allowing passive diffusion of small molecules and controlled transport of larger molecules like proteins, RNAs, and ribosomal subunits.

5. Structural Organization of the Nuclear Pore Complex The NPC has an octagonal symmetry and consists of multiple substructures , each with a specific function: A. Cytoplasmic Ring Located on the cytoplasmic side of the nuclear envelope. Composed of cytoplasmic fibrils , which extend into the cytoplasm. Functions: Acts as a docking site for import receptors (importins) . Helps capture cargo proteins that need to enter the nucleus. B. Nuclear Ring Located on the nucleoplasmic side of the nuclear envelope. Connected to the nuclear basket , a filamentous structure extending into the nucleus. Functions: Assists in nuclear export by interacting with export receptors (exportins) . Plays a role in gene regulation by interacting with chromatin .

6. C. Central Transport Channel (Central Pore) The core region of the NPC, lined with FG-nucleoporins . Forms a hydrogel-like barrier that selectively allows passage of molecules. Functions: Small molecules (<40 kDa ) diffuse freely. Large molecules (e.g., proteins, ribosomal subunits) require active transport via nuclear transport receptors. D. Cytoplasmic Filaments Extend outward from the cytoplasmic ring into the cytoplasm. Function: Capture and guide cargo that needs to enter the nucleus. Interact with Ran GTPase , which regulates nuclear transport. E. Nuclear Basket A cage-like structure attached to the nuclear ring inside the nucleus. Composed of long filaments. Function: Acts as a filter for nuclear export. Interacts with chromatin and transcription factors. Plays a role in gene regulation .

7. Nuclear Pore Complex (NPC) Composition Each NPC is made up of approximately 30 different nucleoporins ( Nups ) , which assemble into a symmetrical octagonal structure . These nucleoporins can be categorized into three major types based on their function and location: Structural Nucleoporins (Scaffold Nups ) Provide the architectural framework of the NPC. Form the outer rings and spokes that hold the complex together. Examples: Nup107, Nup133, Nup85. Membrane-anchoring Nucleoporins (Pore Membrane Nups ) Help anchor the NPC into the nuclear envelope by interacting with the membrane. Examples: Ndc1, Pom121, GP210. FG-Nucleoporins (Phenylalanine-Glycine Repeat Nups ) Line the central channel of the NPC. Contain disordered FG-repeats , which form a selective permeability barrier that blocks non-specific diffusion. Interact with nuclear transport receptors (e.g., importins and exportins) to regulate selective transport. Examples: Nup62, Nup98, Nup153.

8. Function of the Nuclear Pore Complex The NPC is a selective transport gate that regulates the movement of molecules between the nucleus and cytoplasm. Its functions include: A. Passive Diffusion Small molecules (e.g., ions, ATP, water) pass through the NPC freely. B. Selective Transport of Large Molecules Proteins, RNAs, and ribosomal subunits require nuclear transport receptors for passage. Transport is regulated by Ran GTPase , a protein that controls directionality: Import : Cargo is recognized by importins , which bind to FG-nucleoporins and guide it into the nucleus. Export : Cargo binds to exportins , which carry it out of the nucleus. C. Regulation of Gene Expression The nuclear basket interacts with chromatin and transcription factors to regulate gene activity. The NPC is involved in RNA processing and transport. D. Cell Cycle Regulation During mitosis , NPCs disassemble and reassemble to allow nuclear envelope breakdown and reformation. Certain nucleoporins help in spindle assembly and chromosome segregation .

9. Transport Mechanism of the NPC A. Nuclear Import (Into the Nucleus) Cargo Recognition : A protein with a Nuclear Localization Signal (NLS) binds to importin . Transport through the NPC : Importin interacts with FG-nucleoporins , allowing the cargo to move through the central channel . Release into the Nucleus : The protein is released inside the nucleus using Ran GTPase . B. Nuclear Export (Out of the Nucleus) Cargo Recognition : A protein with a Nuclear Export Signal (NES) binds to exportin . Transport through the NPC : Exportin interacts with FG-nucleoporins, allowing cargo to pass through the central channel . Release into the Cytoplasm : Ran GTP hydrolysis causes the cargo to be released into the cytoplasm.

10. Special Features of Nuclear Pores Large Size : NPCs are about 100 nm in diameter and 30-50 nm thick . High Copy Number : A typical eukaryotic nucleus contains 2,000 – 5,000 NPCs . High Transport Efficiency : NPCs can transport up to 1,000 molecules per second . Dynamic Nature : NPCs disassemble and reassemble during cell division.

11. Chromosomes and Chromatin: Overview 1.1 Chromosomes Chromosomes are long, linear DNA molecules that carry genetic information. In eukaryotes, chromosomes are found inside the nucleus and are associated with proteins to form a highly organized structure. Number of Chromosomes : Each species has a characteristic number of chromosomes (e.g., humans have 46 chromosomes in somatic cells, 23 pairs). Structure : Chromatid : Each chromosome consists of one or two chromatids , depending on the phase of the cell cycle. Centromere : The central region where sister chromatids are attached. Telomeres : Repetitive DNA sequences at the ends of chromosomes, which prevent degradation. Arms : p-arm (short arm) q-arm (long arm) Chromosomes are visible under a microscope only during cell division , when chromatin condenses into distinct structures.

12. 1.2 Chromatin: The Functional State of DNA Chromatin is the complex of DNA and proteins found in the nucleus of eukaryotic cells. It exists in two forms: Euchromatin (Lightly Stained, Active) Less condensed and transcriptionally active . Found in regions of high gene expression. Appears as a diffuse, light-staining region in the nucleus. Heterochromatin (Densely Stained, Inactive) Highly compacted and transcriptionally inactive . Two types: Constitutive heterochromatin (e.g., centromeres, telomeres) – always compacted . Facultative heterochromatin (e.g., inactive X chromosome in females) – can switch between compact and loose states . Chromatin allows the dynamic regulation of gene expression by altering the accessibility of DNA to transcription factors and polymerases.

13. 2. DNA Packaging: From Double Helix to Chromosome Eukaryotic DNA (~2 meters long) must be efficiently packaged into the nucleus (which is only ~6 µm in diameter). This is accomplished through a hierarchical organization involving nucleosomes, chromatin fibers, and chromosome loops. Step 1: DNA Double Helix (2 nm diameter) The fundamental structure of DNA is the right-handed double helix , consisting of: Two strands of nucleotides running antiparallel . Base pairing (A-T, G-C) stabilized by hydrogen bonds. Major and minor grooves , which serve as binding sites for proteins.

14. Step 2: Formation of Nucleosomes (10 nm Fiber) – “Beads on a String” The first level of compaction is the nucleosome , the fundamental unit of chromatin structure. 2.1 Nucleosome Structure Each nucleosome consists of: Core histone octamer : Composed of eight histone proteins : H2A, H2B, H3, and H4 (two copies each). Histones are basic (rich in lysine & arginine) , allowing tight binding to negatively charged DNA. 146 bp of DNA wrapped around the histone core (1.7 turns of a left-handed supercoil). Linker DNA (~20-60 bp) connects adjacent nucleosomes. 2.2 Histone H1 and Higher Order Folding Histone H1 (linker histone) binds to the outside of the nucleosome. It stabilizes nucleosomes and promotes higher-order chromatin folding . At this stage, chromatin appears as "beads on a string" in an electron microscope.

15. Step 3: Formation of the 30 nm Fiber (Higher-Order Packing) Nucleosomes further fold into a more compact 30 nm chromatin fiber : This involves interactions between adjacent nucleosomes , stabilized by histone H1 and other factors. Two proposed models: Solenoid model – nucleosomes form a spiral-like helix. Zigzag model – nucleosomes alternate in a zigzag pattern. This level of compaction reduces DNA length by ~50-fold .

16. Step 4: Chromatin Looping (300 nm Fiber) The 30 nm fiber is further organized into chromatin loops , anchored to a scaffold of non-histone proteins . Loops contain active genes and are arranged for efficient transcription . The scaffold consists of SMC proteins (Structural Maintenance of Chromosomes) . This step compacts DNA by ~1,000-fold .

17. Step 5: Metaphase Chromosome (Highly Condensed, 1400 nm Fiber) During mitosis , chromatin undergoes its final level of condensation: Chromatin loops coil into a tightly packed structure (~700 nm per chromatid) . Two sister chromatids align and are connected at the centromere . Condensation is mediated by proteins like condensins and cohesins . At metaphase, chromosomes become visible as highly condensed structures ready for segregation. This final stage reduces DNA length by ~10,000-fold .

18. 3. Epigenetic Regulation of Chromatin Structure The structure of chromatin is dynamically regulated by post-translational modifications of histones. These modifications influence gene expression by altering nucleosome stability. 3.1 Histone Modifications Acetylation (by Histone Acetyltransferases, HATs) Adds acetyl groups (-COCH₃) to lysine residues. Reduces histone-DNA interaction , leading to open chromatin (euchromatin) and active transcription . Deacetylation (by Histone Deacetylases, HDACs) Removes acetyl groups. Leads to chromatin compaction (heterochromatin) and gene silencing . Methylation (by Histone Methyltransferases, HMTs) Can activate or repress genes , depending on the modified residue. Example: H3K9 methylation = heterochromatin formation (gene silencing). Phosphorylation & Ubiquitination Involved in DNA damage repair and cell cycle regulation .

19. Level Structure Approx. Width Compaction Factor 1 DNA double helix 2 nm 1x 2 Nucleosome ("beads on a string") 10 nm ~6x 3 30 nm chromatin fiber 30 nm ~50x 4 Chromatin loops 300 nm ~1,000x 5 Condensed chromosome (mitosis) 1,400 nm ~10,000x Summary of DNA Packaging Hierarchy

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