11 nucleus - Objectives 1. To know about how nucleus is...

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Unformatted text preview: Objectives 1. To know about how nucleus is organized in a eukaryotic cell 2. To understand the molecular mechanisms by which nucleus regulates the movement of molecules in and out of the region 3. To discuss the complications and advantages of having partially enclosed region for holding the genetic materials 1 Nucleus • The site within a eukaryotic cell where: 1. Chromosomes are localized and replicated 2. DNA is transcribed 3. Repository and control centre of most of the cell’s genetic information 2 A. Nuclear envelope • Trilamellar appearance; 2 membranes separated by a perinuclear space: 1. Outer nuclear membrane • • Continuous with endoplasmic reticulum (perinuclear space is continuous with ER lumen) (perinuclear Studded on its outer surface with ribosomes 2. Inner nuclear membrane • Rests on a network of supporting fibers (nuclear lamina) 3 euchromatin (eu); heterochromatin (he); nucleous (nu); nuclear pore (NP) 4 3. Nuclear pores • Small cylindrical channels; fusion of inner and outer membranes; direct continuity between cytosol and nucleoplasm Lined with an intricate protein structure (nuclear pore complex NPC; Molecular Mass ~125 million): Cytoplasmic side Nucleoplasmic side • 5 6 - Octagonal arrangement of at least 50 polypeptides (nucleoporins); protrudes on both cytoplasmic and nucleoplasmic sides - Proteins extend into perinuclear space to anchor the pore complex - Transporter moves macromolecules across (9 (9 nm → 26 nm); fibers extend from the rings into cytosol (cytoplasmic filaments) and nucleoplasm (forming a (forming basket) 7 Transportation • IN: Enzymes / proteins for chromosome replication and DNA transcription OUT: RNA molecules / partially assembled ribosomes for protein synthesis To protect newly synthesized RNAs from being acted upon by cytoplasmic organelles / enzymes before it has been fully processed • - 8 A. Passive Transport • 8 separate aqueous diffusion channels (periphery of the pore complex between the (periphery spokes) / 1 channel (center of transporter) (center Freely permeable to small molecules (< (< 5,000 daltons) Impermeable to large molecules (> 60,000 daltons) 9 - B. Active Transport • • Large proteins and RNAs (mRNAs, tRNAs, and rRNAs) Requires energy and transport receptors (karyopherins) to escort the proteins IN • Amino acid sequences enabling the protein to be recognized and transported into nucleus by NPC (nuclear localization signals NLS): 10 - There are about 10,000,000 ribosomes in a HeLa cell; the nucleus must import ~560,000 ribosomal proteins and export ~14,000 ribosomal subunits EVERY MINUTE ! ! ! Proteins with NLS moving through NPC Gold particles coated with NLS proteins moving through NPC 11 - NLS consists of stretch(es) of positively charged amino acids (e.g., (e.g., pro-lys-lys-lys-arg-lys-val-) - Precise location of NLS within the protein is not important phenylalanine- and glycine-rich (FG-repeats) adaptor protein lysine- and arginine-rich 12 1. The karyopherins (importins α and β) bind to the NLS of the protein Docking of the importins-NLS complex onto a cytoplasmic filament of NPC The filament bends to bring the complex onto its specific binding sites of the NPC; conformational change of the transporter opens its aqueous channel Binding of RanGTP (monomeric GTPase) induces dissociation of the importins and discharge of the protein (RanGTP remains bound to the importinβ) Recycle of the importins Importin 2. 3. 4. 5. 13 1. Transportation RanGDP RanGAP GTPase-activating Protein GTPase(located solely in the cytosol) RanGTP 3. Recycle 2. Discharge Cellular Apoptosis Susceptibility protein (CAS) - Exportin for α 14 Guanine Exchange Factor (located solely in the nucleus) OUT • Less is known • RNAs are associated with proteins (ribonucleoproteins) for exports (e.g., mRNAs are (e.g., exported as heterogeneous nuclear ribonucleoproteins hnRNPs) - Amino acid sequences targeting the complexes for export through NPC (nuclear export signals NES) • Exportins bind to NES; mediate the complexes to transport out through NPC 15 (An exportin) 16 • There are some proteins which have both NLS and NES (e.g., gene regulatory proteins) (e.g., The steady state of localization is determined by the relative rates of their import into and export out of the nucleus: 1. Regulation of NLS and NES by phosphorylating the adjacent amino acids phosphorylating 2. Binding to inhibitory cytosolic proteins that inhibitory either anchor them in the cytosol (with (with cytoskeleton/specific organelles) or mask their NLS 17 1. Calcineurin dephosphorylates NF-AT and exposes NLS 2. Calcineurin may also block NES Nuclear Factor of Activated T Cells 18 B. Nucleoplasm • The interior space of nucleus: 1. Nuclear matrix • • A protein network maintaining the shape of nucleus To provide an organizing scaffold for chromatin fibers: Organizing DNA for orderly replication and transcription Guiding the newly formed mRNA to nuclear pores; transportation to cytoplasm 19 2. Nuclear lamina • A thin dense meshwork of 10-nm intermediate filaments, composed of lamins and oriented roughly perpendicular to one another, lining the inner surface of inner nuclear membrane: To support nuclear envelope As attachment sites for chromatin Nucleus Cytoplasm Fibers of nuclear matrix Nuclear lamina - 20 (by the cyclin-dependent kinase) cyclinkinase) reassembly of nuclear pore complexes; rebind of lamins and inner nuclear membrane proteins onto the chromosomes 21 3. Nucleolus • The ribosome factory • A membrane-free organelle consisting of DNA that is being transcribed into rRNA (fibrils); rRNA molecules being packed with proteins imported from cytoplasm (granules) Fibrils Granules 22 • Stretches of DNA carrying multiple copies of rRNA genes (nucleolus organizer region NOR) reside on a number of chromosomes (e.g., 10 in (e.g., human) Size of nucleolus is correlated with its level of activity: Cell approaches division → chromatin condenses (as compact (as chromosomes) → shrinkage → nucleoli disappear Mitosis is ending → chromatin uncoils → NORs loop out again → rRNA synthesis resumes → near the tip of 10 chromosomes → 10 NORs → 10 tiny nucleoli → nucleoli enlarge → fuse into a single large nucleolus 23 • - - Nucleolar Fusion After mitosis, each of the 10 human chromosomes carrying a cluster of rRNA genes begins to form a tiny nucleolus Single large nucleolus in an interphase cell 24 ...
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This note was uploaded on 09/26/2010 for the course BIO BIO2110 taught by Professor Kwam during the Spring '10 term at 한동대학교.

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