BMS1025 - Cell Biology - the nucleus 2023.pdf

BMS1025 – Cell Biology • Dr Penny Lympany 28AY04 April Chloe Terrazas ; Cellular Biology: organelles, structure, function Content In these sessions we will be looking at Nucleus – DNA packaging and chromatin Ribosomes and protein synthesis Endoplasmic reticulum This Photo by Unknown Author is...

BMS1025 – Cell Biology • Dr Penny Lympany 28AY04 April Chloe Terrazas ; Cellular Biology: organelles, structure, function Content In these sessions we will be looking at Nucleus – DNA packaging and chromatin Ribosomes and protein synthesis Endoplasmic reticulum This Photo by Unknown Author is licensed under CC BY Why do we need to know? Links to other areas of the course – Biochemistry Pharmacology Structures of genes and other chromosomal structures will be covered in molecular biology in year 1 and 2 Learning outcomes • Identify features of the nucleus • Describe the structures of the • Endoplasmic Reticulum/Ribosomes • Describe how DNA is packaged • Describe how proteins are synthesised More information and reading lists For definitions – check the Glossary at the end of this presentation For access to the Reading List for this module, go to the Course Materials page and click on reading list You can also use BibliU for your Reading List Source: Alberts et al. Molecular Biology of the Cell 7th Ed The Nucleus Repository of genetic information and the cell’s control centre The nucleus is a membrane bound organelle in eukaryotic cells containing DNA Discovery Antony van Leeuwenhoek (1632– 1723) - probably the first to observe nucleus in the blood cells of birds and amphibians. Felice Fontana (1730–1805) discoverer of nucleus by observing epidermal cells of eel Robert Brow, Scottish Botanist (1773–1858) observed the nucleus in plant cells and was the first to call these structures ‘nuclei’. What is the nucleus? This Photo by Unknown Author is licensed under CC BY Nuclear lamins Source: Alberts et al. Molecular Biology of the Cell 7th Ed Key facts – • contains DNA arranged in chromosomes • surrounded by the nuclear envelope, a double nuclear membrane (outer and inner), which separates the nucleus from the cytoplasm • nuclear membrane is supported by a meshwork of intermediate filaments (nuclear lamins) • outer membrane is continuous with the rough endoplasmic reticulum • nuclear envelope contains pores which control the movement of substances in and out of the nucleus. • RNA is selectively transported into the cytoplasm, • proteins are selectively transported into the nucleus. Why is the nucleus important? • Separates fragile chromosomes from cell contents – crucial for proper function of cell • DNA replication, transcription and RNA processing all in the nucleus • Separates RNA transcription in the nucleus from translation machinery in the cytoplasm • Nuclear envelope allows gene expression to be regulated • mRNA undergoes posttranscriptional processing before moving from nucleus to cytoplasm • control of gene expression at the level of transcription e.g. expression of some eukaryotic genes controlled by regulated transport of transcription factors from cytoplasm to nucleus The Nucleus – more information Most cells have a single nucleus, some have none (i.e. red blood cells), and some have several (i.e. skeletal muscle) Nuclear lamins Nucleolus • one or more nucleoli are found inside the nucleus • most prominent in cells that are synthesising large amounts of protein • sites at which ribosomes are assembled and ribosomal RNA is transcribed Histology Guide © Faculty of Biological Sciences, University of Leeds The nuclear envelope • Encloses DNA • 2 concentric membranespenetrated by nuclear pore complexes • Inner membrane contains proteins that act as anchoring sites for chromatin and for the nuclear lamina • outer membrane – continuous with ER and studded with ribosomes • Perinuclear space • Proteins made are transported into perinuclear space Source: Alberts 7th Edn Fig. 12-54 Nuclear envelope during mitosis When nucleus disassembles during mitosis, lamina depolymerises & NPCs disperse in cytosol During process some NPC proteins bound to nuclear import receptors – important in reassembly of NPCs at end of mitosis Nuclear envelope membrane proteins disperse throughout ER membrane Later in mitosis, nuclear envelope reassembles close to surface of chromosomes Nuclear pores Each nuclear pore complex (NPC) ~30 proteins (nucleoporins) Eightfold rotational symmetry A – 8 fold symmetry, proteins making up central portion of NPC orientated symmetrically – nuclear and cytosolic sides look identical. B – SEM of nuclear side of nuclear envelope C – EM of side view of 2 NPCs D – Face on view of NPC Source: Fig 12-55 Alberts et al. Molecular Biology of the Cell 7th Ed (A, adapted from A. Hoelz et al., Annu. Rev. Biochem. 80:613–643, 2011. B, © 1992 M.W. Goldberg and T.D. Allen. Originally published in J. Cell Biol. https://www.doi.org/10.1083/jcb.119.6.1429. With permission from Rockefeller University Press. C, courtesy of Werner Franke and Ulrich Scheer. D, courtesy of Ron Milligan.) Nuclear pores – cont. • • • • Nuclear envelope of typical mammalian cell – 3-4000 NPCs Each NPC can transport up to 1000 macromolecules per sec and in both directions simultaneously The internal diameter of each NPC is ∼40 nm - large enough to accommodate ribosomal subunits Pore filled with unstructured protein - contains numerous repeats of phenylalanine–glycine (FG) motifs - weak affinity for each other creates a gel-like mesh inside the NPC. • Mesh acts as a sieve - restricts diffusion of large macromolecules but allowing smaller molecules to pass through. • Small molecules (<5000 daltons) rapid diffusion = freely permeable • Many cell proteins (40,000 daltons (∼5 nm diam) too large to diffuse passively through the NPCs nuclear compartment and the cytosol can maintain different protein compositions. • Mature cytosolic ribosomes (~30 nm diam) can’t diffuse through - protein synthesis confined to the cytosol Regulation of transport through NPCs small proteins continually shuttle back and forth between the nucleus and the cytosol • need import or export signal • other proteins contain both nuclear localisation signals (NLS) and nuclear export signals (NES). • relative rates of import and export determine the steady-state localisation of such shuttling proteins • changing rate of import, export, or both change the location of a protein This Photo by Unknown Author is licensed under CC BY-NC-ND Nuclear localisation signals Nuclear localisation signals (NLSs): • responsible for the selectivity of active nuclear import process • Most common signal - 1 or 2 short sequences rich in aas lysine and arginine (+ charge) - precise sequence varying for different proteins • can be located almost anywhere in the aa sequence - form loops or patches on the protein surface. • Many function even when linked as short peptides to the surface of a cytosolic protein - precise location of the signal within the amino acid sequence not important. • as long as one of the protein subunits of a multicomponent complex displays a NLS, entire complex will be imported into the nucleus • NPC transport occurs through a large, constitutively open, mesh-filled pore- fully folded proteins and large multiprotein complexes can be transported in either direction through the nuclear pore. Aa – amino acid Nuclear Import Receptors Source: Fig 12-58 Alberts et al. Molecular Biology of the Cell 7th Ed Nuclear localization signals must be recognized by nuclear transport receptors Most receptors are karyopherins Receptors can use adaptor proteins that form an import receptor/NLS bridge Variety of import receptors and adaptors - cells recognize range of NLS Import receptors FG - phenylalanine and glycine Source: Fig 12-55 Alberts et al. Molecular Biology of the Cell 7th Ed • soluble cytosolic proteins • contain multiple low-affinity binding sites for FG repeats found in the unstructured domains of several nucleoporins. • FG repeats - recruit import receptors and their bound cargo proteins to NPCs • import receptors then bind the FG repeats that form the mesh inside the nuclear pore to disrupt interactions between the repeats. • Receptor–cargo complex locally dissolves the gel-like mesh and can diffuse into and within the NPC pore Nuclear Export inactive • The nuclear export of large molecules through NPCs depends on a selective transport system. • Relies on nuclear export signals on the macromolecules to be exported Export receptors bind to both the export signal, either directly or via an adaptor, and to NPC proteins • Import and export transport systems work in similar ways but in opposite directions: the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse export receptors function in the opposite fashion active Source: Fig 12-61 Alberts et al. Molecular Biology of the Cell 7th Ed Ran present in both cytosol and nucleus required for the active transport of macromolecules into and out of the nucleus through nuclear pore complexes Nuclear Lamina Nuclear Lamina – localised nuclear side of inner nuclear membrane Meshwork of interconnected protein subunits (nuclear lamins) Lamins – intermediate filament proteins – polymerise into 2D lattice. Gives shape and stability to nuclear envelope Anchored by attachment to NPCs and integral membrane proteins Interacts directly with chromatin Source: Alberts et al. Molecular Biology of the Cell 7th Ed Nuclear lamin structure Lipid like anchor on COOH attaches lamina to INM Source: Fig 16-62 Alberts et al. Molecular Biology of the Cell 7th Ed DNA Structure This Photo by Unknown Author is licensed under CC BY DNA – some key points • Deoxyribonucleic acid (DNA) consists of two long polynucleotide chains composed of four types of nucleotide subunits • Each chain is a “DNA strand” • The two strands run antiparallel to each other, and hydrogen bonds between the base portions of the nucleotides hold the two strands together • A nucleotide is composed of a five-carbon sugar to which a phosphate group and a nitrogen-containing base are attached • The nucleotides are covalently linked together in a chain through the sugars and phosphates, to form a “backbone” of alternating sugar–phosphate–sugar– phosphate Nucleotides A nucleotide consists of a nitrogen containing base, a five-carbon sugar (ribose) and a phosphate group The phosphate makes the nucleotide negatively charged. Nucleoside Glycosidic bond DNA structure Alberts et al. Molecular Biology of the Cell 7th Ed In DNA, the deoxyribose attached to a phosphate group (deoxyribonucleic acid), and the base may be either adenine (A), cytosine (C), guanine (G), or thymine (T). Sugars Nucleic Acid Polymers To form nucleic acid polymers, nucleotides are joined together by phosphodiester bonds between the 5' and 3' carbon atoms of adjacent sugar rings. The linear sequence of nucleotides in a nucleic acid chain is abbreviated using a one-letter code, such as AGCTT, starting with the 5' end of the chain. Base Nucleoside Abbreviation Adenine Adenosine A Guanine Guanosine G Cytosine Cytidine C Thymine/Uracil Thymine/Uracil T/U Creating the phosphodiester bond Nucleotide 1 The 5' group of a nucleotide triphosphate is held close to the free 3' hydroxyl group of a nucleotide chain. Nucleotide 2 The 3' hydroxyl group forms a bond to the phosphorus atom of the free nucleotide closest to the 5' oxygen atom. Meanwhile, the bond between the first phosphorus atom and the oxygen atom linking it to the next phosphate group breaks. A new phosphodiester bond now joins the two nucleotides. A pyrophosphate group has been liberated. The pyrophosphate group is hydrolyzed (split by the addition of water), releasing a great deal of energy and driving the reaction forward to completion. The double helix Nucleotides - covalently linked Strands are held together by hydrogen bonds between base pairs All bases the inside of the double helix, sugar–phosphate backbones on the outside The strands run anti-parallel Source: Fig 4-3 Alberts et al. Molecular Biology of the Cell 7th Ed Base pairing A bulkier two-ring base (a purine) is paired with a single-ring base (a pyrimidine) This base-pairing is the most energetically favourable arrangement Each base pair is of similar width - sugar–phosphate backbones a constant distance apart along the DNA molecule. (A) Two hydrogen bonds form between A and T, whereas three form between G and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel (A) A short section of the double helix viewed from its side. Four base pairs are illustrated; note that they lie perpendicular to the axis of the helix. Source: Fig 4-5 Alberts et al. Molecular Biology of the Cell 7th Ed The Alpha Helix • To maximise the efficiency of base pairing, the double stranded DNA molecule winds into a right-handed double helix • One complete turn per 10.4 bp • This creates two grooves, the major and minor groove Source: Fig 4-6 Alberts et al. Molecular Biology of the Cell 7th Ed DNA packaging The chromosome • Eukaryotic DNA - 22 pairs of homologous chromosomes and 1 pair of nonhomologous • Exception of the gametes (eggs and sperm) and some highly specialized cell types, each human cell nucleus contains two copies of each chromosome. • Each chromosome - a single long linear DNA molecule along with the proteins that fold the fine DNA thread into a more compact structure. • Chromosomes associated with other proteins (as well as numerous RNA molecules) required for the processes of gene expression, DNA replication, and DNA repair. • The complex of DNA and tightly bound protein is called chromatin Karyotype centromeres A – chromosomes “painted” and visualised as they were obtained from a lysed cell B – lined up in numerical order – showing the Karyotype Chromosomes 1–22 numbered in approximate order of size. The red knobs (chromosomes 13, 14, 15, 21, and 22) indicate the positions of genes that code for the large ribosomal RNAs and form the nucleolus (Adapted from U. Francke, Cytogenet. Cell Genet. 31:24–32, 1981.) Centromeres and telomeres Centromere • Not always in centre of chromosome • Keep chromosomes properly aligned during cell division • Centromere - attachment site for the two halves of each replicated chromosome (sister chromatids) Arms Telomeres • Repetitive stretches of DNA located at the ends of linear chromosomes • Protect the ends of chromosomes to keep them from unravelling – think shoelaces • In many types of cells, telomeres lose a bit of their DNA every time a cell divides. When all of the telomere DNA is gone, the cell cannot replicate and dies • WBC and other frequently dividing cells have an enzyme (Telomerase) that prevents their chromosomes from losing their telomeres – cells live longer • Telomerase adds TTAGGG repeats to the ends of chromosomes • Role in cancer - chromosomes of malignant cells usually do not lose their telomeres – fuels the uncontrolled growth DNA – size is important • Most important function of DNA is to form genes – (specify all the RNA molecules and proteins) - including information about when, in what types of cells, and in what quantity each RNA molecule and protein is to be made. • If the double helices of all 46 chromosomes in a human cell could be laid end to end, they would reach approximately 2 meters • BUT the nucleus is ~6 μm in diameter. • This is equivalent to packing 40 km (24 miles) of extremely fine thread into a tennis ball. • DNA packaging is accomplished by specialized proteins that bind to the DNA and fold it, generating a series of organized coils and loops that prevent the DNA from becoming an unmanageable tangle. • Although the DNA is very tightly compacted, it remains accessible to the enzymes in the cell that replicate it, repair it, and use its genes to produce RNA molecules and proteins. How do we package DNA into an individual chromosome? Human genome DNA length – 3.1 x 109 nucleotide pairs Number of genes coding for proteins - ~20,000 Largest gene coding for proteins – 2.5 x 106 nucleotide pairs Source: Fig 4-16 Alberts et al. Molecular Biology of the Cell 7th Ed If drawn with a 1 mm space between each nucleotide pair, the human genome would extend for nearly 3100 km – across the centre of Africa DNA in chromosomes If chromosome 22 was laid out as one long double helix, it would extend to around 1.5 cm But at mitosis, it measures only 2 µm Compaction of 7000x How? – proteins which coil and fold the DNA DNA of interphase chromosomes is still tightly packed but can decondense to allow access to specific sequences for gene expression, DNA repair and replication and then recondense. Chromatin • Chromatin - diffuse mass of DNA found in interphase cells • What is interphase? • period in the eukaryotic cell cycle characterized by • G1 phase - cell undergoes growth, • S phase - cell makes a copy of its DNA • G2 phase - cell continues to grow, and prepares for cell division • Heterochromatin - chromatin regions that are condensed during interphase and transcriptionally inactive • Euchromatin - chromatin regions that are decondensed and DNA sequences are being transcribed into RNA • Heterochromatin stains more densely than euchromatin Histology Guide © Faculty of Biological Sciences, University of Leeds Nucleosomes Proteins binding to DNA to form eukaryotic chromosomes divided into 2 classes – Histones non-histone Complex of both classes of protein with nuclear DNA of eukaryotic cells = chromatin Histones Histones – responsible for most basic level of chromosome packing – a protein DNA complex called the nucleosome A – EM of interphase nuclei showing chromatin in form of a fibre B – unfolded chromatin showing a DNA string with beads (nucleosome core particle – DNA wound round a histone core Source: Fig 4-21 Alberts et al. Molecular Biology of the Cell 7th Ed Nucleosomes - cont • Each individual nucleosome core particle consists of a complex of eight histone proteins—two molecules each of histones H2A, H2B, H3, and H4— and double-stranded DNA that is 147 nucleotide pairs long. • This histone octamer forms a protein core around which the double-stranded DNA is wound. • 147 nt pairs wraps 1.7 times around the histone core • Each nucleosome separated from next by linker DNA (few nucleotide pairs up to about 80) • Nucleosomes repeat at intervals of approx. 200 nucleotide pairs. Source: Fig 4-22 Alberts et al. Molecular Biology of the Cell 7th Ed Structure of nucleosome core particle Source: Fig 4-23 Alberts et al. Molecular Biology of the Cell 7th Ed Nucleosomes packed into a chromatin fibre Source: Fig 4-28 Alberts et al. Molecular Biology of the Cell 7th Ed Chromatin in a living cell probably rarely adopts the beads on a string form Nucleosomes packed on top of each other – DNA more condensed Nucleosome to nucleosome linkages formed by histone tails, notably H4 tail Looped domains 30 nm fibre pulled into loops – “looped domains” DNA packaging - overview What we have covered Nucleus Nuclear envelope Nuclear lamina DNA DNA packaging That’s all folks This Photo by Unknown Author is licensed under CC BY-NC Remember – for more information Alberts B et al. Molecular Biology of the Cell or your favourite Molecular Cell Biology text book The module discussion board Email – [email protected]