Cell Biology Textbook PDF

The cell is the basic structural and functional unit of all living organisms. Cells are very diverse: filamentous fungi, bacterial, blood cells, radiolarian, protozoan, gametes (eggs and sperm), intestinal, xylem (plant), retinal neuron ( Figure_01_01.jpg ). But, first, A brief history of Cell Biology ( Figure_01_02.jpg ) Genetics, Biological Chemistry and Cytology combined to form a science far greater than the sum of the parts. Cellular Biology is the basis of all Biology and Applied Biologies (from Agriculture & Environmental Sciences to Nutrition & Medicine). Cell Theory (-mid 1800s): 1) All organisms consist of one or more cells 2) The cell is the basic unit of all living organisms 3) All cells come from pre-existing cells Before Cell Theory: Believed that life could arise spontaneously and from rotting organic matter as illustrated by "The Recipe for the Production of Mice" by the Chemist: J.B. van Helmont. "If you press a piece of underwear soiled with sweat together with some wheat in an open mouth jar, after about 21 days the odour changes and the ferment, coming out of the underwear and penetrating through the husks of wheat, changes the wheat into mice." 5 Cell Behaviours Cell Behaviour is the key to all of biology. 1. Cell-cell communication 2. Cell shape changes 3. Cell movement 4. Cell proliferation 5. Cell death (apoptosis) Resolution: The ability to see adjacent objects as distinct from one another Microscopy has been essential in understanding Cell Biology Resolving Power: Light Microscopy (LM): 200 nm - 350 nm There are different types of light microscopy (LM): Bright field (stained & unstained), fluorescence, phase contrast, differential interference contrast, confocal ( Figure_01_01T.jpg ) Resolving Power: Transmission Electron Microscopy (TEM): 0.1 - 0.2 nm There are different types of electron microscopy (EM) Scanning electron microscopy (SEM), Transmission electron microscopy (TEM) ( Figure_01_04.jpg ) Model Organisms ( Figure_01_01B.jpg ) Out of all the organisms out there, we have selected a few to study extensively E. coli, S. cerevisiae, Drosophila melanogaster, C. elegans, Mus musculus, Arabidopsis Hierarchical Nature of Cellular Structure ( Figure_02_14.jpg ) Level 1: small organic molecules Level 2: macromolecules Level 3: supramolecular structures Level 4: organelles & sub-cellular structures Level 5: the cell The Chemistry of the Cell Originally organic chemistry was biological chemistry. Now separate due to the synthesis of non-biological compounds. Now biological chemistry (biochemistry) is part of modern cell biology. Carbon Carbon has a valence of four: outer electron orbital has four out of eight electrons required to be stable. Can form four chemical bonds: stable molecules ( Figure_02_01.jpg ). Can easily form molecules with hydrogen, oxygen and nitrogen. Can form double and triple bonds. As the carbon atom has a tetrahedral structure, when four different atoms or groups are bonded to produce stereoisomers or mirror-image forms ( Figure_02_07.jpg ). Water Water is the universal solvent in biology. Water molecules are polar: unequal distribution of electrons or charge. Water molecules are "sticky" or cohesive: form hydrogen bonds ( Figure_02_08.jpg ). Results in high surface tension, high boiling point, heat of vaporization and other properties important to biology. Water is an excellent solvent as can be seen with sodium chloride ( Figure_02_10.jpg ). hydrophilic versus hydrophobic. Selectively Permeable Membranes To define and control the content of cells, a membranes act as a barrier between inside and outside of the cell. Membrane lipids and proteins are not simply hydrophilic or hydrophobic but have regions of both: amphipathic. Amphipathic phospholipids are prime examples ( Figure_02_11.jpg ). The lipid bilayer is (3 to 4 nm X 2) 7 to 8 nm thick and forms the basic structure of the membrane ( Figure_02_12.jpg ). Membrane proteins are embedded and help with various duties ( Figure_02_13B.jpg ). Importantly, the membranes are semi-permeable. Synthesis by Polymerization Many biological molecules are linear polymers of simple monomers ( Figure_02_15.jpg ). Proteins (amino acids), DNA & RNA (nucleotides), starch, glycogen and cellulose (simple sugars). Lipids are complex polymers. Self-Assembly Many biological compounds, especially proteins, undergo a process of “self-assembly”. Denatured proteins can return to their native form via renaturation: spontaneous protein/peptide folding ( Figure_02_18.jpg ). Molecular chaperones: proteins that bind temporarily to new or unfolded proteins to allow the eventual proper folding. Weak interactions such as non-covalent bonds are important in the formation of macromolecules. The Macromolecules of the Cell Most biological macromolecules are made from only about 30 small molecules. Proteins Polymers of amino acids. About 60 different amino acids are commonly found in a cell. Only 20 amino acids are used in protein synthesis ( Figure_03_02.jpg ) Please note that amino acids are represented by both 1 letter and 3 letter codes/abbreviations ( Figure_03_02T.jpg ). After the proteins/peptides are made, the amino acid sub-units can be modified. Amino acids have a central alpha-carbon, an amino group, a carboxyl group, and an R group. R groups are different for each amino acid: hydrophobic versus hydrophilic. Proteins and peptides are linear polymers of amino acids. Made in an "amino (N) to carboxyl (C)" direction. Linearly, the N-terminal end is the beginning and the C-terminal end is the end! Polymerization is through formation of peptide bonds ( Figure_03_03.jpg ). Stabilized by a) disulfide bonds, b) hydrogen bonds, c) ionic bonds and d) Van der Waals/hydrophobic interactions ( Figure_03_05.jpg ). Protein structures depend upon the amino acid sequence and the many interactions among the amino acids. Four levels of organization of protein structure: primary, secondary, tertiary and quaternary protein structures build upon the previous level ( Figure_03_06.jpg ). Nucleic Acids Polymers of nucleotides. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) differ in the 5 prime carbon sugar part of the nucleotide (deoxyribose versus ribose). Primary Roles of DNA and RNA: DNA is the store of "genetic information" and RNA is involved in using the information ( Figure_03_14.jpg ). A nucleotide is made up of a nitrogenous base (purines or pyrimidines), a 5 prime carbon sugar and a phosphate group ( Figure_03_15.jpg ). Nucleic acids are linear polymers of nucleotides ( Figure_03_17.jpg ). Made in an "5 prime to 3 prime" direction. Linearly, the "5 prime" is the beginning and the "3 prime" end is the end! Nucleic acids are joined by "3 prime", "5 prime" phosphodiester bridges. DNA is a double-stranded helix ( Figure_03_19.jpg ). DNA has hydrogen bonds between A-T pairs and G-C pairs of opposite strands ( Figure_03_18.jpg ). Polysaccharides Polymers of simple sugars such as glucose ( Figure_03_21.jpg ). Polysaccharides are for storage, such as starch & glycogen ( Figure_03_24.jpg ) and structure such as cellulose( Figure_03_25.jpg ). Lipids Polymers of various monomers including fatty acids ( Figure_03_05T.jpg ). Heterogeneous group of hydrophobic macromolecules ( Figure_03_27.jpg ). Serve in energy storage, membrane structure and specific biological functions. Prokaryotes and eukaryotes are very different ( Figure_04_01T.jpg ) Prokaryotes: bacteria ( Figure_04_03.jpg ) plasma membrane circular DNA ribosomes (70S) binary fission Eukaryotes: the Animal Cell ( Figure_04_05A.jpg ) and the Plant Cell ( Figure_04_06A.jpg ) plasma membrane and internal membrane-bound compartments / organelles DNA plus proteins linear chromosomes histones larger ribosomes (80S) mitosis & meiosis cytoskeleton endocytosis & exocytosis Evolution of the Eukaryotic Cell and the origin of chloroplasts and mitochondria Uniquely, Mitochondria and chloroplasts are "semi-autonomous organelles" Two theories: 1) Autogenous theory: The eukaryotic cell evolved directly from a single prokaryote ancestor through compartmentalization of functions arising from invaginations of the prokaryotic plasma membrane. The endoplasmic reticulum (ER), Golgi, and the nuclear membrane, and of organelles enclosed by a single membrane According to this theory, mitochondria and chloroplasts have evolved within by compartmentalizing plasmids within vesicles from the plasma membrane. 2) Serial Endosymbiotic Theory (SET) of the Evolution of Eukaryotic cells ( Figure_11_11A.jpg ) Prokaryotic cell engulfs aerobic bacteria Rather than digesting them, the bacteria remain, as symbionts, benefiting the host cell by removing harmful O2 and helping in the production of ATP. As interdependence between the aerobic bacterium and the host cell grows, the bacterium becomes the mitochondrion. Some of these cells also engulf and keep blue-green algal cells which become chloroplasts. What evidence do we have for SET and the origin of mitochondria and chloroplasts from free-living bacteria? 1) size 2) appearance 3) double membrane 4) binary fission 5) common enzymes for respiration, photosynthesis 6) Genetic material: single circular DNA molecule no histone proteins; few proteins continuous S phase similarity in gene sequences for example: rDNA, tDNA 7) Similarity in ribosomes of organelles and prokaryotes (compared to cytoplasm of eukaryotes): 70S ribosomes in organelles and prokaryotes (vs 80S in cytoplasm) sensitivity to antibiotics start codons for the start of protein synthesis Cell Structures and Organelles 1) Cell membranes cell boundary: plasma membrane [PM] ( Figure_04_09.jpg ) internal compartments selectively permeable lipids and proteins ~8 nm 2) Extracellular boundaries: 1) plant cell walls ( Figure_04_25.jpg ) 2) fungal cell walls 3) animal extracellular matrix (ECM) reinforcement, protection and communication 3) Ribosomes ( Figure_04_22.jpg ) site of protein synthesis, translation (mRNA to protein) Free or associated with membranes (Rough endoplasmic reticulum: rER) eukaryotes: 30 nm diameter (and in the mitochondria and cytoplasm) 4) Nucleus ( Figure_04_10.jpg ) compartmentalizes genetic material site of DNA synthesis (S) site of RNA synthesis (transcription) double membrane, nuclear pores, chromatin, nucleolus 5) Endomembrane System a series of internal membranes endoplasmic reticulum (ER) ( Figure_04_15.jpg ), Golgi apparatus ( Figure_04_16.jpg ) & vesicles structurally and functionally related for synthesis and secretion of proteins, carbohydrates and lipids ( Figure_04_17.jpg ) 6. Lysosomes ( Figure_04_18.jpg ) recycling hydrolases 7. Peroxisomes ( Figure_04_19.jpg ) variety of processes generate hydrogen peroxide as a by-product catalase Semi-autonomous organelles: DNA - circular molecule, ribosomes, fission Energy Flow 8. Chloroplasts ( Figure_04_14.jpg ) Photosynthesis energy of sunlight to fuel molecules 9. Mitochondria ( Figure_04_11.jpg ) fuel molecules to ATP ACR (heat) 10. Cytoskeleton ( Figure_04_24.jpg ) 3D interconnected array of protein-based filaments microtubules, microfilaments, intermediate filaments cell shape, movement, cell signalling, endocytosis, mitosis Bioenergetics: The Flow of Energy in the Cell The capacity to obtain, store and use energy, one of the obvious features of most living organisms. The Importance of Energy Energy is required for 6 processes ( Figure_05_02.jpg ). 1. Synthetic work 2. Mechanical work 3. Concentration work 4. Electrical work 5. Heat generation 6. Bioluminescence Phototrophs (light-feeders) or chemotrophs (chemical feeders) Energy constantly flows through the biosphere: oxidation and reduction ( Figure_05_05.jpg ). Matter is recycled: carbon, oxygen, nitrogen and water. Bioenergetics First Law of Thermodynamics: Energy is Conserved Second Law of Thermodynamics: Reactions have Directionality Entropy and free energy Exergonic versus endergonic reactions Life and the Steady State Living cells are very close to maintaining a steady state: an open system with inputs of large amounts of energy. Enzymes: The Catalyst of Life Brief Notes (Introductory Biochemistry is a co-requirement) Enzymes act to catalyze reactions that may not occur, to any noticeable extent, without it! Activation Energy and the Metastable State Catalysis can overcome an activation energy barrier and increase the number of involved molecules can be increased without temperature change ( Figure_06_01.jpg ). A catalyst increases the rate of a reaction by lowering the activation energy requirement. A catalyst acts by forming transient, reversible complexes with substrates. A catalyst changes the rate at which equilibrium is achieved. Enzymes as Biological Catalysts Most enzymes are proteins. Enzymes have active sites where the substrate binds and where the catalytic event occurs such as lysozyme ( Figure_06_02.jpg). Enzymes display a very high degree of substrate specificity and can distinguish among closely-related isomers. Enzymes are dependent upon temperature, pH and molecules that act as inhibitors and activators. Major classes of enzymes are oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases ( Figure_06_01T.jpg ). Substrate binding often leads to conformational change of the enzyme such as hexokinase ( Figure_06_05.jpg ). Substrate binding can change the structure of the active site of the enzyme such as carboxypeptidase A ( Figure_06_06.jpg ). Substrate activation includes common mechanisms: bond distortion, proton transfer, electron transfer. The catalytic cycle of an enzyme maintains the structure of the enzyme while altering substrates ( Figure_06_07.jpg ). Enzyme Kinetics Most enzymes display Michaelis-Menten Kinetics (Think about monkeys shelling peanuts). Enzymes can be inhibited: either competitively or non-competitively ( Figure_06_14.jpg ). Enzyme Regulation Feedback regulation: Allosteric enzymes by downstream products ( Figure_06_15.jpg ). Allosteric enzymes can be multi-subunit proteins with catalytic and regulatory subunits (inhibition or activation) ( Figure_06_16.jpg ). Enzymes can be controlled by simple modification (i.e. phosphorylation or dephosphorylation). Some enzymes are activated by proteolytic cleavage: inactive precursor zymogens that become active in the target tissue ( Figure_06_18.jpg ). RNA Molecules as (part of) Enzymes: Ribozymes Ribozymes are RNA containing biological catalysts. Functions of Cell Membranes (Figure_07_02.jpg) 1) Define boundaries (permeability barrier) 2) Sites of specific functions 3) Regulation of solute transport 4) Signal detection and transmission 5) Cell to cell communication Structure of Cell Membranes 1) Lipids as a bilayer 2) Proteins may transverse the lipid bilayer via alpha helices; integral proteins Models of Membrane Structure "The Fluid Mosaic Model" (Singer & Nicholson: 1972) ( Figure_07_03.jpg) Proteins are retained the lipid bilayer and proteins may also be discontinuously embedded in the membrane: integral proteins Integral proteins have one or more series of hydrophobic sequences that span the lipid bilayer (Unwin & Henderson 1975) i.e. bacteriorhodopsin: a single peptide chain folded back and forth across the lipid bilayer 7 times! The Fluid Mosaic Model is universally accepted model for cell membrane structure. ( Figure_07_05.jpg ) "Mosaic of proteins in a fluid lipid bilayer" 1) Lipids are fluid and this fluidity can be regulated. 2) Proteins form a "mosaic", perform a variety of functions, and protein fluidity may be restricted. 3) Both are asymmetrically distributed in membranes. Membrane Lipids and the Fluid Part of the Model Membrane Lipids: Classes Three classes of membrane lipids ( Figure_07_06.jpg ) 1) phospholipids - most prominent component 2) glycolipids - addition of carbohydrate/sugar group Lipids are amphipathic molecules that have hydrophobic or fatty-acids tails and a hydophillic or polar head The fatty acids tails can be of differing length and (saturation or) number of double bonds (12 - 20 carbon atoms long) ( Figure_07_08.jpg ) 3) sterols - rings in their structure hydrophobic e.g. cholesterol, ergosterol, and phytosterols (these absent from prokaryotes & inner membrane of mitochondria and chloroplasts). Membrane Lipids: Behaviour ( Figure_07_10.jpg ) Asymmetry Little or no transverse diffusion (flip-flopping) from one lipid layer to the other e.g. glycolipids - outside e.g. phosphatidlyethanolamine ? inside Note that enzymes known as flippases exist that under special conditions do this. Fluidity 1) rotational movement 2) lateral diffusion within the lipid layer demonstrated experimentally by fluorescence recovery after photobleaching (a few microns per second or less) ( Figure_07_11.jpg ) Functioning of the lipid bilayer and thus the membrane is affected by temperature. Transition temperature (Tm): temperature at which a membrane changes between the fluid and gelled state The Tm is effected by 1) the length of the fatty acids ( Figure_07_13.jpg ) 2) the number of double bonds ( Figure_07_14.jpg ) & 3) the proportion of sterols (e.g. cholesterol) ( Figure_07_15.jpg ) Tm increases as the length of the fatty acid increases Tm decreases as the number of double bonds increases Sterols (e.g. cholesterol) moderate in both directions! Acts as a buffer up to 50% in mammalian cells Less fluid at higher temps More fluid at lower temps Organisms regulate membrane fluidity primarily by changing lipid composition cooler - shorter fatty acid chains cooler - increase the number of double bonds Poikilotherms & homeoviscous adaptation e.g. Micrococcus - enzyme cuts two carbons off the 18 carbon fatty acids to produce 16 carbon fatty acids in cool temperatures E. coli - desaturase enzyme increases number of double bonds in cool temperatures cold-hardy plants: desaturase enzyme activity triggered at lower temperatures warm blooded hibernating mammals: increase numbers of double bonds Membrane Proteins and the Mosaic Part of the Model Membrane Proteins Freeze fracture microscopy shows us that proteins are embedded or "floating" in and on the lipid bilayer of cell membranes ( Figure_07_17.jpg ) Correlates well with the known protein content of cell membranes e.g. erythrocyte (red blood cell: RBC) membrane vs the inner mitochondrial membrane Proteins, like lipids, show membrane asymmetry (freeze fracture microscopy) many are glycosylated (2 - 60 sugar units!) = glycoproteins The glycocalyx of animal cells for recognition in antibody binding and tissue formation thus membranes are often functionally asymmetric Membrane Proteins: Main Classes ( Figure_07_19.jpg ) 1) integral membrane proteins : monotopic or transmembrane transmembrane proteins cannot be readily removed, require detergents 2) peripheral membrane proteins : weak electrostatic forces 3) lipid anchored proteins: covalently bound Transmembrane proteins have one or more transmembrane segments cannot be readily removed require detergent to isolate ( Figure_07_20.jpg ) Membrane Proteins: mobility 1) some move freely 2) others are anchored: e.g. on the inside to the cytoskeleton (scale?) e.g. on the outside to the ECM e.g. integrin - cell attachment and signalling Or via lipid rafts Some move freely as shown by fluorescent antibody tagging and cell fusion ( Figure_07_26.jpg ) or by putting the cell in an electrical field Membrane Proteins: Methods Application of molecular techniques to the study of membrane proteins X-ray crystallography vs hydrophobicity plots Difficult for hydrophobic proteins but 1988 Nobel Prize for resolution of alpha-helical domains. ( Figure_07_21.jpg ) Proteins and SDS-polyacrylamide gel electrophoresis Membrane proteins and Molecular Biology ( Figure_07_A02.jpg ) Proteins can be separated by SDS-polyacrylamide gel electrophoresis = SDS PAGE Electrophoresis of Sodium dodecyl sulphate (SDS) coated proteins: Coats with a negative charge which disrupts membranes and denatures proteins May use the gel to identify specific molecules by immunoblot (Western) * Transport Across Membranes Cellular transport processes are quite diverse and employ a number of mechanisms ( Figure_08_01.jpg ). The hydrophobic inner portion of the lipid bilayer allows some substances to pass through it. However, it is an absolutely important barrier to a majority of other substances. This permeability must be overcome to allow some important small molecules and ions in and out of the cell. How do substances cross membranes? ( Figure_08_03.jpg) 1) Simple diffusion (via lipids): no cellular energy expended. Down the gradient toward equilibrium. 2) Facilitated diffusion - protein mediated (carrier and/or channel proteins): no cellular energy expended. Down the gradient toward equilibrium. 3) Active Transport - protein mediated: cellular energy expended. Up the gradient. Simple Diffusion Occurs via lipid interaction: 1) lipophilic/hydrophobic substances, 2) simple non-polar molecules e.g. di-oxygen, carbon dioxide, carbon monoxide, 3) some drugs (nicotine, heroin), anesthetics. The cell membranes are not (and cannot be) selective in simple diffusion. Facilitated Diffusion Occurs via proteins: mostly lipophobic/hydrophilic. Dependent upon the proteins present in the cell membrane. This is a very specific process. The cell membranes are very selective in facilitated diffusion of certain solutes. 1) carrier proteins (transporters, permeases) 2) channels (ions, small molecules, water [aquaporins] etc.) Glucose Transporter Protein: the red blood cell (RBC) glucose transporter ( Figure_08_08.jpg ) is an integral membrane protein with 12 transmembrane segments. Glucose in blood plasma is about 3.6 - 5.0 mM but inside RBC, it is about 0.5 - 1.0 mM (a concentration gradient) because there is a glucose selective transporter in the RBC membrane. Facilitated diffusion occurs 50,000 X as fast as in a lipid bilayer alone. In this case, very selective for glucose and a small group of similar sugars. Antiporter carrier protein: RBC anion exchange protein The chloride and bicarbonate exchanger with obligatory reciprocal exchange. Very selective and is used to remove carbon dioxide from the tissues to the lungs. Channel proteins : hydrophilic transmembrane channels across the membrane e.g. Ion channels are specific for one of the following: calcium ions, potassium ions, sodium ions, chloride ions These can move 1 million ions per second! Regulated by changes in voltage, ligand-binding, and mechanical forces (i.e. stretching). Active Transport: Direct and Indirect ( Figure_08_11.jpg ) How do substances move across membranes against the concentration gradient? Active Transport occurs via proteins and requires energy! Active Transport is endergonic; and produces a thermodynamically unfavourable difference in concentrations. Active Transport enables a cell to maintain a constant intracellular non-equilibrium concentration of specific ions. e.g. Sodium Potassium Pump: ( Figure_08_13.jpg) Direct active transport maintains electrochemical ion gradients in mammalian cells. potassium ions at a ratio of 35 inside: 1 outside (35:1). sodium ions at a ratio of 0.08 inside: 1 outside (0.08:1). Three sodium ions are moved out; 2 potassium ions are moved in for each ATP molecule hydrolyzed This forms a electrochemical gradient P-type: phosphorylation event. Model of Mechanism of Na+/K+Pump ( Figure_08_14.jpg ) Functions of the Na+/K+ pump/ATPase: maintains osmotic equilibrium maintains voltage difference across the membrane. conduction of electrical impulses by nerve and muscle cells driving force for cotransport / indirect active transport There are many different types pumps/ATPases: e.g. Na+ /K+ pump of animal cells, e.g. H+ pump of fungi and plants, e.g. H+ pump of the gastric epithelium; e.g. Ca++ pump of the sarcoplasmic reticulum Prokaryotes can pump antibiotics out of the cell: Antibiotic resistance Indirect Active Transport co-transport: symport or antiport This moves one solute down its gradient which is coupled with one solute moving up its gradient. e.g. Na+ in animals, H+ in plants and fungi which drives a second solute up its gradient such as an monosaccharide or amino acid. Sodium/Glucose Symporter ( Figure_08_15.jpg ) driven by sodium gradient in epithelium of the intestine. Bacteriorhodopsin Proton Pump of Halobacteria ( Figure_08_16.jpg ) Photon of light drives the Halobacterium (purple bacteria) proton pump. Energy Metabolism: Energy Flow, Glycolysis & Fermentation; Aerobic Respiration; & Photosynthesis 1) Energy Flow, Glycolysis & Fermentation The flow of energy in ecosystems is unidirectional (for a review see Chapter 5). Its source is the sun and we, here in the biosphere, are the transient custodians of an almost infinitesimally small portion of that energy; it is ultimately lost from the ecosystem as heat. In the biosphere, energy is coupled to matter. Although it enters and leaves unaccompanied, while in the biosphere, energy exists primarily in the form of chemical energy and thus is coupled to matter. That matter is recycled while the energy cannot be; energy flows uni-directionally, not cyclically. Energy originates from thermonuclear reactions in our sun. The flow of energy in living organisms begins with the photo-synthesizers which use light energy to drive electrons energetically up-hill into new chemical bonds. (P) This energy is then released in both plants and animals etc. in "downhill" reactions. (R) This flux of energy from solar to heat, drives the molecular machinery of all life processes. Requirements of cells: Energy and Organic building blocks Phototrophs gain these from photosynthesis / stored organic molecules Chemotrophs gain these from the foods they eat / organic molecules (Both burn them for energy to do work) ATP: The Universal Energy Coupler ( Figure_09_01.jpg ) ATP: adenosine triphosphate Two energy rich phosphate bonds ATP hydrolysis is highly exergonic responsible, directly or indirectly for almost all of the work done by living cells synthesis, active transport, movement ( Figure_09_04.jpg ) Glycolysis ( Figure_09_07.jpg ) and Fermentation ( Figure_09_08.jpg ) Can be used to conserve and release energy under aerobic and anaerobic conditions (respectively). Biological oxidations of food molecules usually involve the removal of electrons and protons Highly exergonic Eg. Aerobic cellular respiration Production of ATP Glycolysis in the cytoplasm: partial oxidation of glucose to pyruvate ( Figure_09_06.jpg ) produces a total 2 ATP (uses 2 ATP to produce 4 ATP = 2ATP) Oxidation of glucose is highly exergonic (alternative substrates, other carbohydrates, fatty acids and proteins, can be used as fuel as well) Fermentation The fate of pyruvate depends on whether oxygen is present. If none, then fermentation: lactic acid or alcohol 7% efficiency only, but well conserved in ATP = Anaerobic glucose catabolism] Gluconeogenesis ( Figure_09_12.jpg ) Glucose can be made from three carbon (3C) and four carbon (4C) precursors by gluconeogenesis. -> 2) Energy Metabolism: Aerobic Respiration ( Figure_10_01.jpg ) The role of mitochondria in aerobic cellular respiration is crucial. Mitochondrial structure and function with a range of zero (in red blood cells) to thousands (some liver cells) in number. 1) Glycolysis (in the cytoplasm) 2) Pyruvate Oxidation (in the mitochondrial matrix) 3) The Citric Acid (or TCA) cycle (in the mitochondrial matrix) 4) Electron transfer chain (ETC) and proton pumping (in the mitochondrial inner membrane) 5) ATP synthesis (in the mitochondrial inner membrane) How is the energy in the organic molecules made available for cellular work? Biological oxidations of food molecules usually involve the removal of electrons and protons Highly exergonic e.g. Aerobic cellular respiration Production of ATP The Citric Acid Cycle ( Figure_10_07.jpg) For details: see ( Figure_10_09.jpg ) The Citric Acid cycle in the mitochondrial matrix: (pyruvate to) Acetyl CoA to CO2 in a ten-step reaction sequence (TCA or Kreb's cycle) Products: CO2, 2 ATP, NADH, FADH2 Electron Transport (Figure_10_19.jpg ) Oxidative phosphorylation in the inner mitochondrial membrane electron transport chain- from coenzymes to oxygen 1) electrons flow from coenzymes, NADH and FADH2 2) are passed along the ETC complexes to oxygen 3) forming water. 4) This is coupled to the accumulation of protons in the mitochondrial cristae (the cristae are the highly convoluted folds of the inner mitochondrial membrane). 5) An electrochemical proton gradient ( Figure_10_26.jpg ) 6) This H+ gradient is used to drive ATP synthase ( Figure_10_23a.jpg ) Mitochondrial respiratory complexes: integral proteins of the inner mitochondrial membrane Complex I: pumps 4 protons from the matrix to the intermembrane space and passes electrons from NADH to Coenzyme Q. Complex II: (not pictured) does NOT pump protons but does pass some electrons to the quinone pool. Complex III: pumps 4 protons (2 indirectly) from the matrix to the intermembrane space and passes electrons from Coenzyme Q to cytochrome c. Complex IV: pumps 2 protons from the matrix to the intermembrane space and passes electrons from cytochrome c to oxygen to help form water. These complexes move freely in the membrane as Coenzyme Q and cytochrome c shuttle electrons. Notably, this unique inner membrane contains lots of unsaturated phospholipids and no cholesterol. Finally, the ATP synthase complex generates ATP on the matrix side of the membrane due to protons passing through the complex (1 ATP per 3 protons). * 3) Energy Metabolism: Photosynthesis ( Figure_11_01.jpg ) How is solar energy trapped and converted to chemical energy? The activities of the chloroplasts are central to the process of photosynthesis. Light capturing reactions = Energy Transduction reactions in the chloroplast thylakoid membranes ( Figure_11_03.jpg ). Energy is converted to chemical energy of ATP and NADPH in chloroplasts, the eukaryotic photosynthetic organelle, is comprised of 3 membrane systems:1) outer membrane; 2) inner membrane; and 3) thylakoids. The stroma, the "chloroplasm", contains internal structures including organelle chromosome (DNA), ribosomes and the thylakoids. The thylakoids:stacks of sack-like grana thylakoids interconnected by tube-like stroma thylakoids. Essentially all photosynthetic components are on/in the thylakoid membranes. Photosynthetic energy transduction: Light Harvesting ( Figure_11_06.jpg ) First stage of photosynthetic energy transduction: energy transfer to chlorophyll. Photons carry energy (a quantum). Chlorophyll, a pigment receives the energy by increasing the energy of an electron from the ground state (lower energy orbital) to the excited state (higher energy orbital). Chlorophyll is a complex lipid molecule with a porphyrin ring structure, a long hydrocarbon tail and a central Magnesium ion. The tail embeds chlorophyll in the lipid bilayer of thylakoid. Transfer of the energy to another molecule is called photochemical reduction. Photosynthetic energy transduction: NADPH Synthesis ( Figure_11_08.jpg) Second stage of photosynthetic energy transduction: from chlorophyll to the coenzyme NADPH ( Figure_11_09.jpg ) Photosystem II: removes electrons from water to release oxygen and photon excites electron in chlorophyll P680 Cytochrome complex: passes electrons from PSII to plastocyanin. Photosystem I: accepts electrons from plastocyanin and photon excites electrons in chlorophyll P700 which then pass through 3 iron-sulfur centres containing proteins to ferredoxin. Ferredoxin-NADP reductase (FNR) transfers electrons from ferredoxin to NADP. The major energy complexes are arranged in the Thylakoid Membrane. Photosynthetic energy transduction: ATP Synthesis The ATP synthase complex uses the proton gradient to generate ATP. Photosynthetic Carbon Assimilation: Calvin Cycle ( Figure_11_12.jpg ) For details of the Calvin Cycle (see Figure_11_13.jpg ). Carbon fixing (dark) reactions occur in the chloroplast stroma. During the Calvin Cycle, the energy from the products of the "light reactions" ATP and NADPH, is transferred to carbohydrate as carbon dioxide is reduced, to form Glyceraldehyde-3-phosphate which leads to the synthesis of sucrose and starch ( Figure_11_16.jpg ). Sucrose is made in the cytosol (cytoplasm). Starch is made in the chloroplast stroma. The "food"/energy source for all organisms. The Endomembrane System ( Figure_12_01 ) Endomembrane system of eukaryotic cells consists of the endoplasmic reticulum (smooth and rough), the Golgi Complex, endosomes and lysosomes. Endoplasmic Reticulum A continuous network of flattened sacs, tubules and vesicles. ER cisternae (the membrane of the sacs) and ER lumen (the enclosed material). Although 50-90% of the cell's membranes are in the ER, it is largely not observed through light microscopy. Rough Endoplasmic Reticulum (RER): ribosomes on cytoplasmic side for the biosynthesis and processing of protein. Smooth Endoplasmic Reticulum (SER): no ribosomes on cytoplasmic side and is involved in steroid biosynthesis, calcium storage, detoxification and carbohydrate metabolism ( Figure_12_03b ). Central role in membrane biosynthesis. Golgi Complex ( Figure_12_04a ) Membrane and protein processing and trafficking. The Golgi is stacked into a number of flattened cisternae. Surrounded by transport vesicles: 1) from the ER to the Golgi; 2) between stacks of the Golgi; and 3) from the Golgi to a number of destinations. Two "faces" of the Golgi (with an active middle): the cis-Golgi network (newly synthesized lipids and proteins) and the trans-Golgi network (transport away from the Golgi). The medial cisternae (the middle) is where more of the processing occurs. The two models of Movement through the Golgi ( Figure_12_05 ): Stationary cisternae model and the cisternal maturation model. Stationary cisternae model: Shuttle vesicles carry material from the ER to Golgi while ER & Golgi remain in place. Cisternal maturation model: the Golgi is made up of transient compartments with the step-by-step transformation of the cisternae as they age and mature. ER and Golgi in Protein Glycosylation ( Figure_12_06 ) and Trafficking ( Figure_12_08 ) Many proteins are translated by ribosomes on the ER, then moved into the lumen of the ER to be processed. Proteins are initially glycosylated in the ER then further in the Golgi ( Figure_12_07 ). Vesicles: Vesicles carry proteins and lipid through the endomembrane system and the compartments depend upon retention and retrieval mechanisms. Retrieval and retention "tags" help sort. For example, a mannose-6-phosphate “tag” helps target lysosomal enzymes to the lysosome ( Figure_12_09 ). Exocytosis ( Figure_12_12a ) and Endocytosis ( Figure_12_13 ) The process of exocytosis releases proteins to the cell exterior. Exocytosis depends upon interaction of vesicles with ( Figure_12_19 ) 1) membrane tethering proteins; 2) a RAB GTPase activates interaction between V-SNARE and T-SNARE; 3) V-SNARE and T-SNARE drive fusion of membranes; and 4) binding NSF and SNAPs release “SNARE” complex The process of endocytosis allows the uptake of materials from the cell exterior. Phagocytosis is the ingestion of large particles, often by specialized cell: phagocytes ( Figure_12_14a ). Receptor-mediated endocytosis depends upon specific receptors to specific ligands at sites identified as coated pits ( Figure_12_15 ) Lysosomes are central to the digestion of a wide range of ingested materials ( Figure_12_21 ). Peroxisomes and Protein Import ( Figure_12_24 ) Peroxisomes come from 1) division of old peroxisomes or 2) by fusion of vesicles from the ER. Lipids and membranes can be added to existing peroxisomes and protein for peroxisome matrix enzymes and co-factors. Division of peroxisomes and fusion of vesicles contribute. Cytoskeletal Systems ( Figure_13_T1 ) The major structural elements of the cytoskeleton are microtubules, microfilaments and intermediate filaments. The range of techniques are used to understand the cytoskeleton include fluorescence microscopy, digital video microscopy and electron microscopy ( Figure_13_T2 ). Microtubules ( Figure_13_03 ) Microtubules (MTs) are largest element of the cytoskeleton. Cytoplasmic microtubules: 1) maintain axons; 2) maintain shape; 3) orient cellulose microfibrils (in plants); 4) mitotic and meiotic spindles for chromosome movements; and 5) vesicle movement. Microtubule structure Axonemal microtubules: highly organized, stable microtubules in specific movement associated subcellular structures (cilia, flagella and attachment basal bodies). The central shaft (axoneme) consists of a highly ordered bundle of axonemal MTs (see sperm tail axoneme). Microtubule structure is made up of heterodimers of alpha-tubulin and beta-tubulin (form an alpha-beta-heterodimer). The alpha-tubulin and beta-tubulin molecules are 4-5 nm in diameter and 55 kDa, have almost identical shapes but only share 40% amino acid sequence identity. A microtubule is a hollow cylinder of 13 protofilaments around a lumen with an outer diameter (25 nm), inner diameter (15 nm) and dimer width (8 nm). Singlet microtubules are 13 protofilaments; doublets are: 13 plus 10 (or 11) [in cilica or flagella] and triplets are 13 plus 10 (or 11) plus 10 (or 11) [in basal bodies or centrioles]. Treadmilling ( Figure_13_04 ) Microtubules form by addition of tubulin dimers at the ends ( Figure_13_07a ). New microtubules form nucleation centres (oligomers) and grow by addition of subunits on either end: elongation. Critical concentration is when disassembly and assembly is exactly balanced: The plus end: rapidly growing end; the minus end: the slower growing end. Microtubule-organizing centre (MTOC) serves as a site for initiation and as an anchor. A centrosome (animal cells) has two centrioles surrounded by pericentriolar material ( Figure_13_11 ). The microtubule polarity can vary with cell function ( Figure_13_10 ). Microtubule-associated protein (MAPs) act as stabilizing and bundling proteins (Figure_13_12 ). Microfilaments ( Figure_13_13 ) Microfilaments (MFs) are smallest element of the cytoskeleton. Muscle contraction (with myosin filaments); and cell migration (lamellipodia & filopodia; amoeboid movement; and cytoplasmic streaming) ( Figure_13_14 ). Monomers of G-actin [gobular actin] have a 7 nm diameter and polymerize into microfilaments of F-actin. Actin MFs take various roles in crawling cells (such as macrophages): 1) contractile bundle (in stress fibres); 2) gel (cell cortex); and 3) parallel bundle (in filopodium). Actin-binding proteins regulate actin organization ( Figure_13_16 ). Ordered actin arrays can be found in microvilli structures that are prominent in intestinal mucosal cells ( Figure_13_17 ). Actin MFs can be linked to membranes by proteins such as spectrin and ankyrin ( Figure_13_19a ). Networks of actin can be formed by polymerization ( Figure_13_20 ). The rho family of proteins regulate actin dependent protrusions ( Figure_13_21 ). Intermediate Filaments ( Figure_13_T4 ) Intermediate filaments (IFs) are 8-12 nm in diameter. Ifs are tissue specific and come in 6 classes. Keratin, a well-known protein, is essential for structures from animal skin (hair, claws & fingernails, horns & beaks, turtle shells, feathers, scales and outermost layer of skin. Intermediate Filaments are usually assembled coils of homodimers into protofilaments ( Figure_13_23 ), except keratin are made up of obligate heterodimers of type I and type II polypeptides. Cellular Movement: Motility & Contractility In addition to scaffold functions, the cytoskeleton plays a role in cellular motility. Proteins interact with the cytoskeleton ( Figure_14_T1 ): specialized motor proteins (mechanoenzymes). Two main systems: 1) specialized motor proteins & microtubules; and 2) actin microfilaments and myosin motor molecules. Intracellular microtubule-based movement Kinesins and dyneins are motor proteins that couple ATP hydrolysis with the ability to walk along the microtubules ( Figure_14_01 ). Kinesins ( Figure_14_03 ) move by hydrolyzing ATP to move forward and releases the nucleotide to release then move. Most move towards the plus end of the microtubules ( Figure_14_04 ). Dynein/dynactin complexes link to cargo vesicles and move towards the minus end of microtubules ( Figure_14_05 ). The endomembrane system moves on microtubules and motor proteins. Microtubule-based motility Cilia and flagella are driven by microtubules ( Figure_14_08a ). Flagellum have a core axoneme and depend on protofilaments and dynein for movement (and linked by nexin) ( Figure_14_09 ). Actin-based cell movement The myosins act as actin-based motor proteins ( Figure_14_10 ). Skeletal muscle cells are made up of thick and thin filaments and are arranged into sarcomeres. Sarcomeres are made up of structural proteins ( Figure_14_T2 ) and support proteins ( Figure_14_11 ). Muscle contraction ( Figure_14_22 ) Nerve impulse (as an action potential) travels down the neuron. Action potential reaches the neuromuscular junction: a synapsis between the neuron and muscle cell ( Figure_14_21 ). Depolarization of neuronal termini result in release of acetylcholine. Acetylcholine bind acetylcholine receptors on muscle cell surface. Initiates depolarization of muscle cell. Releases calcium ions. This leads to activation ( Figure_14_25b ) of myosin light-chain kinase (MLCK) and phosphorylation of myosin II. Filament-based movement in muscle Muscle contraction is a four-step cycle ( Figure_14_19 ): 1) myosin binds loosely to actin filament; 2) the power stroke, a trigger of the conformational change associated with move of thick versus thin filament 3) binding of ATP leads to change in myosin and weakening of bond to actin (no ATP leads to rigour or stiffness). 4) ATP hydrolysis returns myosin to high energy state, ready for the next round of movement. Actin-based motility in non-muscle cells ( Figure_14_27 ) Actin microfilaments (MFs) drive cell migration through cycles of 1) protrusion; 2) attachment; 3) translocation; and 4) detachment. Extracellular Structures The cell does not end at the cell membrane ( Figure_15_01 ). Many animal cells are intrinsically linked to other cells and to the extracellular matrix or ECM ( Figure_15_T2 ). Bone and cartilage are mostly ECM plus a very few cells ( Figure_15_11 ). Connective tissue, that surrounds glands and blood vessels, is a gelatinous matrix containing many fibroblast cells. The ECM contains three classes of molecules: 1) structural proteins (collagens and elastins); 2) protein-polysachharide complexes to embed the structural proteins (proteoglycans); 3) adhesive glycoproteins to attach cells to matrix (fibronectins and laminins), N-CAMs and cadherins mediate cell-cell recognition and cell-cell adhesion ( Figure_15_03a ). Neural cell adhesion molecules (N-CAMs) are large plasma membrane glycoproteins that all share a similar extracellular domain that contains a binding site involved in cell-cell adhesion. When embryonic tissue is exposed to antibodies that interact with N-CAMs, the cells do not bind to each other and neural tissue is not formed ( Figure_15_05 ). Cadherins are a group of adhesive glycoproteins that are similar to the N-CAMs that require extracellular Calcium ions to function and are required for development. E-cadherin (epithethial tissue), N-cadherin (nervous tissues) and P-cadherin (placental tissue) act to drive the adhesion of cells of particular tissue type. The carbohydrate groups of N-CAMs and cadherins determine the strength and specificity of cell-cell recognition and adhesion. N-CAMs have repeating chains of negatively charged sialic acid which changes during development. Vesicles with N-CAMs having little sialic acid bind tighter than those with large amounts. The loss of sialic acid groups from glycophorin may target old erythrocytes for destruction in the spleen. The enzyme neuraminidase can cleave the terminal sialic acid groups as a mechanism to identify old red blood cells for retirement. During inflammation, leukocytes initiate attachment to the endothelial cell surface through the selectins then stabilize the adhesion through the interaction of an integrin and an ICAM ( Figure_15_07 ). Cell Junctions ( Figure_15_02 ) The cell junction are the structures where long term association between neighbouring cells are established ( Figure_15_08a ). In animals, the three most common kinds of cell junctions are adhesive junctions, tight junctions and gap junctions. Adhesive junctions (desmosomes, hemidesmosomes and adherens junctions) link adjoining cell to each other and to the ECM. Although the various adhesive junction types are similar in structure and function, they contain distinct: 1) intracellular attachment proteins; and 2) transmembrane linker proteins. The intracellular attachment proteins form a thick layer of fibrous material on the cytoplasmic side of the plasma membrane called a plaque which binds actin microfilaments in adherens junctions and intermediate filaments in desmosomes and hemidesmosomes. The transmembrane linker proteins is anchored to the plaque by the cytoplasmic domain and binds the ECM or to the same proteins on other cells. Desmosomes form strong points of adhesion between cells in a tissue such that two adjoining cells are separated by a thin space of 25-35 nm, the desmosome core, in which cadherin molecules mediate cell-cell adhesion ( Figure_15_06b ). The plaques on the inner surfaces of cells joined by desmosomes have a mixture of intracellular attachment proteins (desmoplakins and plakoglobin) which interact with the tonofilament intermediate filaments ( Figure_15_06c ). Hemidesmosomes connect a cell, through a plaque, to the basal lamina (ECM) by integrins (Figure_15_20). As in desmosomes, hemidesmosomes interact with tonofilament intermediate filaments. Adherens junctions resemble desmosomes except two adjoining cells are separated by a thin space of 20-25 nm and connect to actin microfilaments in the cytoplasm. Some of the transmembrane glycoproteins are cadherins. Adherens junctions called focal contacts can join a cell to the ECM, primarily through fibronectin receptors. Tight junctions leave no space between plasma membranes of adjacent cells to prevent the movement of molecules across cell layers. The structure of tight junctions consists of fused ridges of tightly packed transmembrane junctional proteins. Tight junctions block lateral movement of lipids and membrane proteins to keep a cell polarized ( Figure_15_09 ). In intestinal epithelial cells transport of glucose from the intestinal lumen through the cell to the blood stream requires the uptake of glucose through apical surface sodium/glucose symport proteins and export by glucose transport proteins on the basalateral surface and tight junctions prevent the lateral movement of these transport proteins. Gap junctions separate cells by 2-3 nm and allow direct electrical and chemical communication ( Figure_15_10 ). Connexons are tightly packed 7 nm wide hollow cylinders in two adjacent cell membranes that form a 3 nm thin hydrophilic channel that allows the passage small molecules and ions. * Collagens & Elastins Collagens the most abundant proteins in the ECM (25-30% of total protein in vertebrates), are secreted by cells, such as fibroblasts ( Figure_15_12 ). Collagens are responsible for the strength of the ECM and form high tensile strength fibres and are prominent in tendons and ligaments. Collagens occur in a triple helix of three polypeptide chains and are high in glycine, hydroxylysine and hydroxyproline. Collagen fibres are bundles of collagen fibrils which are, in turn, bundles of collagen molecules which consist of three alpha chains of collagen polypeptides ( Figure_15_13 ). Procollagen forms many types of tissue-specific collagens. Many tissues require flexibility and strength (lung tissues, arteries, skin and intestines) constantly change shape ( Figure_15_T3). The elastins impart elasticity and flexibility to the ECM and can stretch several times their length ( Figure_15_14 ). Elastins are rich in glycine and proline and are crosslinked by covalent bonds between lysines. The crosslinks allow elastin fibres to recoil back to original shape after extension. During aging, collagens become more crosslinked and elastins are lost resulting in bones, joints and skin losing flexibility. Proteoglycans A matrix of proteoglycans (many glycosaminoglycans attached to each protein) embed collagen and elastin fibres ( Figure_15_15 ). Glycosaminoglycans (GAGs), the major carbohydrate part of proteoglycans, consist of repeating disaccharide subunits. One of the two sugars in the disaccharide is often an amino sugar (N-acetylglucosamine or N-acetylgalactosamine; usually with an attached sulfate group) and the other is a sugar or sugar acid (galactose or glucuronate). GAGs, which are hydrophilic due to the negative sulfate and carboxyl groups, attract water and cations to form the hydrated gelatinous matrix. Chondroitin sulfate, keratan sulfate and hyaluronate are the most common GAGs. Most GAGs in the ECM are bound to proteins to form proteoglycans (mucoproteins). Numerous GAGs (1-200 per molecule, average length of 800 monosaccharide units) are attached to a core protein and different kinds of proteoglycans can be made by varying the combination of core proteins and GAGs. These large proteoglycans (MW of~ 1 million) can be individual or attached to long hyaluronate molecules to form complexes (as in cartilage). Proteoglycans trap water (up to 50 times their weight) to act as extracellular sponges resistant to physical forces in cartilage and joints. Proteoglycans can be embedded in the plasma membrane or covalently linked to membrane phospholipids or bound to receptor proteins. Proteoglycans and collagen may bind to receptor proteins (often integrins) which are reinforced by adhesive glycoproteins, such as fibronectins and laminins, to anchor cells to the ECM. Fibronectins and Laminins Fibronectins, a family of closely related glycoproteins, are soluble in body fluids (blood), insoluble in the ECM and partially soluble at the cell surface ( Figure_15_16 ). The fibronectins bind cells to the matrix and guide cellular movement. The RGD (arginine-glycine-aspartate) sequence binds to the integrin fibronectin receptor. The fibronectins bind cells to the ECM by bridging cell-surface receptors to the ECM. The intracellular cytoskeleton will align with the extracellular fibronectin to determine cell shape. In many kinds of cancer, cells unable to make fibronectins loose shape and detach from the ECM to become malignant. During cell movement (as during embryogenesis), pathways of fibronectins guide cells to their destinations. Soluble plasma fibronectin promotes blood clotting by direct binding of fibrin. Fibronectins guide immune cells to wounded areas and thus promote wound healing. Laminins bind cells to the basal lamina ( Figure_15_18 ). Laminins are found mostly in the basal laminae, the ~50 nm thick ECM layer between epithelial cells and connective tissue, and surrounding muscle cells, fat cells, and Schwann cells. The basal lamina serves as a structural support for tissues and as a permeability barrier to regulate movement of both cell and molecules ( Figure_15_17 ). Laminin is a very large protein comprised of three proteins that form a cross. The domains of laminin bind type IV collagen, heparin, heparin sulfate, entactin and laminin receptor proteins in overlying cells to allow bridging between the cells and the ECM. Integrins, N-CAMs & Cadherins Integrins are cell surface receptors that bind the ECM ( Figure_15_19 ). Fibronectins, laminins and other ECM components bind specific receptor glycoproteins on the cells surfaces known as the integrins. The fibronectin receptor is the best characterized integrin. Integrins act to integrate the cytoskeleton and the extracellular matrix. Integrins consist of two large non-covalently bound transmembrane proteins (alpha and beta subunits). A number of both alpha and beta subunits combine to produce a large variety of heterodimeric integrins. On the outer surface, the subunits interact to form a binding site for the adhesive glycoprotein, the RGD sequence of the ECM glycoprotein. Most of the binding specificity depend upon the alpha subunit. On the cytosolic side, the receptor binds components of the cytoskeleton to enable the ECM to communicate through the plasma membrane to the cytoskeleton ( Figure_15_21b ). Cellulose ( Figure_15_22a ) Rosettes are cell wall synthesizing structure ( Figure_15_24 ). Plasmodesmata are the structures, continuous with the endoplasmic reticulum, that pass through the cell membrane and cell walls to connect adjacent cells ( Figure_15_25 ). Genomes & the Nucleus ( Figure_16_1 ) In 1952, Watson and Crick proposed that DNA is a double helix ( Figure_16_7 ) which is known to have alternative forms ( Figure_16_8 ). The Genome The organization of the total sum of genetic information (or genome) of an organism is in the form of double-stranded DNA, except that viruses may have single-stranded DNA, single-stranded RNA or double-stranded RNA genomes. In many viruses and prokaryotes, the genome is a single linear or circular molecule. In eukaryotes, the nuclear genome consists of linear chromosomes (usually as a diploid set) and the mitochondrial and chloroplast (in plants) genomes are small circular DNA molecules ( Figure_16_22b ). The DNA is converted from relaxed to supercoiled by DNA specific enzymes ( Figure_16_10 ). In general, genome size increases with the complexity of the organism (more or less). Through the analysis of DNA renaturation studies, the large sizes of eukaryotic genomes reveal large amounts of repeated DNA ( Figure_16_13 ). Heating sheared DNA in solution produces single-stranded DNA. Lowering the temperature allows the DNA to reanneal (the higher the concentration the faster the process of reannealing) which can be followed by optical density readings (single- versus double-stranded). For a set concentration of DNA, viral genomes (small) anneal faster than bacterial genomes (far larger than viral genomes, smaller than eukaryotes). Eukaryotic genomes undergo a complex pattern of reannealing which reveals a large amount of repeated DNA sequences (fast annealing) and unique, non-repeated DNA (slow annealing) ( Figure_16_23 ). For example, calf DNA has ~40% repeated and ~60% non-repeated DNA. In humans, repeated sequences make up more. ( Figure_16_24 ). Repeated DNA ( Figure_16_T2 ) The repeated DNA is present in two categories, 1) tandemly repeated DNA and 2) interspersed repeated DNA. Tandemly repeated DNA Tandemly repeated DNA (10-15% of mammalian genomes) is made up of rows of many copies of the same sequence. The repeated unit ranges from 1 to 2000 base-pairs (bps) in length. Often the repeat is less than 10 bps and is referred to as simple-sequence repeated DNA or satellite DNA (due to centrifugation "satellite" bands). These may provide special physical properties to some stretches of the chromosome. Centromeres and telomeres are rich in simple-sequence repeated DNA. At a given site, the amount of simple-sequence repeated DNA may vary greatly. In DNA minisatellites, the satellites may vary between 100 and 100,000 bps in length. DNA fingerprinting is used to distinguish individuals by analyzing microsatellites (repeats of 1 to 4 bps) which often differ by 10 to 100 bps. A number of human diseases are caused by having triplet repeat amplification such as in Huntington's Disease where 11-34 repeats of CAG in the Huntington's Disease gene is normal but ~50 to 100 results in the disease. Interspersed repeated DNA Interspersed repeated DNA make up 25 to 40% of most mammalian genomes that are hundreds to thousands of bps long. Many interspersed repeated DNA sequences are transposable elements. Genome Packaging The cell must handle amounts of DNA that are many times longer than the cells they are in. DNA packaging must be very efficient while still allowing for DNA replication and transcription to occur. Prokaryotic Genome Packaging Prokaryotes package DNA in bacterial chromosomes and plasmids. The bacterial chromosome is localized in the nucleod region of the cell (no nucleus) and is looped into negative coils ( Figure_16_14 ). The loops are 50,000 to 100,000 bps in length (similar to eukaryotic chromosomes) which are held in place by RNA and small basic (histone-like) proteins. Plasmids, small negatively supercoiled circular DNA molecules, carry usually non-essential genes (often drug resistance). Eukaryotic Genome Packaging Eukaryotes package DNA in the nucleus into chromatin and chromosomes. Chromatin fibres are 10 to 30 nm in diameter which condense into much more compact (packaged) structure for cell division. The histones, a group of positive charged (lysine and arginine rich) proteins, which stabilize DNA (negatively charged). Chromatin contains equal amounts of H2A, H2B, H3 and H4 and ~ one-half the amount of H1 plus numerous non-histone proteins ( Figure_16_17 ). Histones provide the basis for the nucleosome, the basic unit of chromatin structure, as seen as "beads-on-a-string" structures on electron micrographs. The nucleosome core is comprised of a histone octomer [(H2A-H2B)X2,(H3-H4)X2]. The DNA double helix is wrapped around (~1.7 times) the histone octomer. With nuclease digestion, 146 bps of DNA are tightly associated with the nucleosome but ~200 bps of DNA in total are associated with the nucleosome. The difference is the linker DNA. H1 is associated with the linker DNA between nucleosome cores. Nucleosomes are packed to form chromatin fibres and chromosomes ( Figure_16_18 ). The nucleosome is only the first level of packaging nuclear DNA. The "beads on a string" fibres are 10-nm in diameter. The next level of organization forms the 30-nm chromatin fibre. Looped (active?) domains (50 to 100 kilobases) of the 30-nm fibre are attached to the non-histone chromosomal scaffold. Heterochromatin is more compacted and mostly transcriptionally inactive. The most compact level of chromatin is, of course, the microscopically visible chromosomes (chromatids). The mitochondria and chloroplasts also have a DNA genome (or chromosome). These resemble prokaryotic genomes (likely due to the endosymbiotic origin of these organelles) but are much smaller ( Figure_16_26 ). The mitochondrial genome varies in size among eukaryotes (mammals =16.5 kb & 37 genes, yeast and plants are greater than 5X this). Chloroplasts are ~120 kb and have ~120 genes. The Nucleus (Figure_16_28b) A double-membrane nuclear envelope surrounds the nucleus. The inner and outer nuclear membranes (7-8 nm thick) are surrounded by the periplasmic space of 20-40 nm thickness. The inner nuclear membrane is supported by the nuclear lamina fibres. The outer membrane is continuous with the endoplasmic reticulum and ribosomes often on the cytosolic side. The periplasmic space is continuous with the ER's lumen. Nuclear pores are channels that pass through both nuclear membranes that join the cytosol with the nucleoplasm ( Figure_16_30b ). The inner and outer membranes fuse to form a channel that is lined with the nuclear pore complex (diameter 120 nm). A mammalian nucleus may have 3000-4000 nuclear pores. Nuclear pores are site of molecular entry and exit both the passive transport of small molecules and the active transport of large proteins and RNA ( Figure_16_33 ). Proteins that are required in the nucleus contain nuclear localization sequences (NLS) that target them through the nuclear pores. The NLS-containing proteins bind importin (a cytosolic receptor protein) which binds the protein to the nuclear pore. The nuclear pore transporter then passes the protein into the nucleoplasm. Ribosome Synthesis (Figure_16_31) The nucleolus is the intranuclear site where rRNA is made. Ribosomal proteins are imported into the nucleus and ribosomal subunits are made in the nucleolus. The ribosomal subunits are then exported through the nuclear pores to the cytoplasm. The Cell Cycle ( Figure_17_1 ) The cell cycle begins with the formation of two cells from the division of a parent cell and ends when the daughter cell does so as well. Observable under the microscope, M phase consists of two events, mitosis (division of the nucleus) and cytokinesis (division of the cytoplasm). As replication of the DNA occurs during S-phase, when condensation of the chromatin occurs two copies of each chromosome remain attached at the centromere to form sister chromatids. After the nuclear envelope fragments, the microtubules of the mitotic spindle separate the sister chromatids and move them to opposite ends of the cell. Cytokinesis and reformation of the nuclear membranes occur to complete the cell division. Most of the time, cells are in interphase, where growth occurs and cellular components are made. DNA is manufactured during S phase. To prepare the cell for Sphase (DNA synthesis), G1 phase occurs (the preparation of DNA synthesis machinery, production of histones). In an analogous manner, the cell prepares for mitosis in the G2 phase by producing the machinery required for cell division. The length of time spent in G1 is variable. Although growing mammalian cells often spend 8-10 hours in G1 phase, a cellular decision is made that cause cells to become arrested in G1 and thus enter the G0 state. G2 is usually shorter than G1 and is usually 4-6 hours. DNA Synthesis ( Figure_17_3 ) DNA replication is a semiconservative process that results in a double-stranded molecule that synthesizes to produce two new double stranded molecules such that each original single strand is paired with one newly made single strand. This was demonstrated by equilibrium density centrifugation (see chapter 19 for details). The replication of DNA most often occurs in a bidirectional manner from an origin of replication from which two replication forks move in opposite direction. In prokaryotes, bidirectional DNA synthesis of the circular genome produces a theta structure (theta replication) ( Figure_17_6). In eukaryotes, DNA replication is initiated in multiple sites along a chromosomes called replicons ( Figure_17_7 ). Thousands of replicons each covering 50 to 300 kilobases, form replication bubbles that fuse to, in the end, make two daughter double stranded DNA molecules ( Figure_17_8 ). A number of proteins and protein complexes are involved in DNA synthesis. DNA polymerases catalyze the synthesis of DNA by adding nucleotides, in a 5-prime to 3-prime direction, utilizing a single stranded region of DNA as a template. Four deoxynucleotide triphosphates (dATP, dCTP, dGTP,dTTP) only are incorporated into the growing chain by releasing the terminal two phosphate groups and covalently bonding the remaining phosphate to the 3-prime hydroxyl group of the previous nucleotide residue. Since the 5-prime end does not get added to and the 3-prime end repeatedly does, the DNA strand is said to grow in a 5-prime to 3-prime manner. Replicon ( Figure_17_11 ) At a replicon, one strand of DNA is made in a continuous manner (the leading strand) and the other in a discontinuous manner (the lagging strand). DNA is made in only the 5-prime to 3-prime direction and the replication bubble opens the original double stranded DNA to expose both a 3-prime to 5-prime template (Leading strand template) and it complement. The lagging strand must be synthesized as a series of discontinuous segments of DNA. These small fragments are called Okazaki fragments and they are joined together by an enzyme known as DNA ligase. DNA synthesis is not perfect initially, so a proofreading mechanism is performed by a 3-prime to 5-prime exonuclease activity that is part of DNA polymerase enzyme itself ( Figure_17_12 ). Since DNA polymerase requires a template and a free 3-prime hydroxyl group to add nucleotides on to, RNA primers are required to initiate DNA polymerization. An enzyme, primase which is part of a large complex of proteins called the primosome, synthesizes a small stretch of RNA (the primer) of 3-10 nucleotide in length, which will act as a starting site for the DNA polymerase ( Figure_17_13 ). Okazaki fragments are initially made with RNA 5-prime ends which is digested away by the 3-prime to 5-prime exonuclease activity of the adjacent DNA polymerase enzyme just prior to the ligation of the two fragments. To make the DNA single stranded in the first place to allow DNA synthesis, the DNA must be unwound. Unwinding double-stranded DNA requires helicases, topoisomerases and single-strand binding proteins (SSBPs) ( Figure_17_14 ). The proteins discussed above form a ribosome-sized complex referred to as the replisome ( Figure_17_15 ). Arrangement of the replisome such that the lagging-strand is "looped around" allows the machinery to move in one direction while synthesizing DNA from strands in opposing polarities ( Figure_17_16 ). Telomeres and telomerase As DNA synthesis requires a RNA primer that will eventually be digested away, standard DNA replication would result in linear chromosomes that would shrink with every round of replication. This is resolved in bacteria by the circular genome which does not have an end. In linear chromosomes, a specialized structure the telomere solves the “end of DNA replication problem” ( Figure_17_18 ). Telomeres have highly repeated DNA sequences 5'-TTAGGG-3'. Human chromosomes have between 100 and 1500 copies of this sequence. Telomerase, a special DNA polymerase, can add additional copies of the 5'-TTAGGG-3' to the end of a chromosome ( Figure_17_19 ). The telomerase enzyme is actually a complex containing protein and RNA (a "ribozyme"). The RNA portion has a 5'-CCCTAA-3' region that acts as a template for adding the DNA repeat to the chromosome ends. The telomerase enzyme is found mostly in the germ cells of multicellular organisms. In somatic cells, the absence of telomerase results in shorter chromosomal ends with each division and may be the limiting factor in an organism's life span. DNA repair Under normal conditions, spontaneous hydrolysis of DNA leads to depurination (breaking the glycosidic bond between the deoxyribose and the purine or deamination (the loss of the amino group from A, C or G) ( Figure_17_23 ). This damage can result in the inclusion of an incorrect nucleotide to produce a mutation. Damage to DNA occurs in response to mutagens (either chemical or radiation). Mutagenic chemicals include 1) base analogues (similar in structure to the normal bases and can become incorporated into DNA); 2) base-modifying agents (which can change a base) and 3) intercalating agents (cause insertions and deletions) ( Figure_17_24). Ultraviolet (UV) radiation (sunlight) can cause pyrimidine dimer formation (such as covalently linked thymines) block replication and transcription ( Figure_17_25 ). Ionizing radiation (such as X-rays) knock electrons off of biomolecules to generate highly reactive intermediates that causes all sorts of DNA damage. Excision repair mechanism remove abnormal nucleotides to correct mutations. Base excision repair mechanisms first remove a damaged base then causes excision of the remaining sugar-phosphate unit ( Figure_17_26 ). Pyrimidine dimers and other bulky lesions are removed through nucleotide excision repair (NER) ( Figure_17_27 ). NER causes two nicks which leads to the removal of a stretch of damaged single-stranded DNA (12 in E. coli and 29 in humans). Mismatch repair corrects mutations of non-complementary bases that become included in DNA during replication that are not fixed by proof-reading ( Figure_17_28 ). The original strand is recognized as such by the action of DNA methylases (the old DNA strand is methylated). Mismatch repair endonucleases cut the sugar phosphate backbone (a nick) and an exonuclease removes the incorrect nucleotides from the nicked strand. The Holliday Model of Homologous Recombination (Figure_17_31) The current model of the mechanism of exchange of DNA between two homologous chromosomes explains gene conversion and genetic recombination. 1) A double-stranded DNA molecule undergoes a single-stranded break. 2) The single-stranded DNA invades the complementary region of the double-stranded homologue. 3) DNA repair (DNA synthesis) of the dsDNA using the invading ssDNA as template begins. 4) Reciprocal invasion results in the formation of the "double crossover" or Holliday Junction. 5) Branch migration (movement of the crossover structure) is the result of DNA unwinding and rewinding. 6) Resolution of the Holliday Junction will result in either a cross over event or gene conversion (without a cross over) event. ( Figure_17_32 ) Transcription The Central Dogma of Molecular Biology is an “informational flow chart” ( Figure_18_0 ) DNA synthesis -> RNA synthesis -> protein synthesis (DNA replication) (transcription) (translation) DNA synthesis maintains the genetic information and passes this to the next generation. RNA synthesis (transcription) is a transfer of the information from the DNA where it is stored into RNA which can be transported and interpreted. The language (of nucleic acids) is essentially the same. Messenger RNA (mRNA) moves the information from the DNA to the ribosomes to direct the production of protein ( Figure_18_3 ). Translation represents a change in the language from the nucleotide letters in RNA to the amino acid letters in protein. The various roles of RNA ( Figure_18_1 ) All three major classes of RNA (mRNA, tRNA, & rRNA) are synthesized by transcription of the appropriate genes and are involved in protein synthesis. Compare the differences and note the similarities between prokaryotes and eukaryotes ( Figure_18_2 ). All three major types of RNA are involved in directing the formation of protein. 1) mRNAs carry the message from the DNA to the ribosome. 2) rRNAs are major structural components of the protein-synthesizing ribosome. 3) tRNAs act as adaptor molecules in aligning the amino acids according to the sequence present in the mRNA. The genetic code is a system of purines and pyrimidines used to send messages from the genome to the ribosomes to direct protein synthesis. A message written as a sequence of nucleotides in an mRNA molecule has no obvious meaning, until a set of equivalency rules for the genetic code is used to convert the sequence into the amino acid sequence of a recognizable polypeptide. One strand of the DNA duplex (the template strand) is transcribed into a segment of mRNA shown, according to the same base-pairing rules used in DNA replication, except the base U is used in RNA in place of T. The complementary DNA strand, with a sequence essentially identical to that of the mRNA, is called the coding strand. With a non-overlapping code the reading frame advances three nucleotides at a time, and a mRNA segment is therefore read as three successive triplets, coding for amino acids ( Figure_18_9 ). The genetic code’s "words" are three-letter codons present in the nucleotide sequence of mRNA, as read in the 5 prime to 3 prime direction. Each codon specifies either an amino acid or a stop signal ( Figure_18_10 ) To decode a codon, read down the left edge for the first letter, then across the grid for the second letter, and then down the right edge for the third letter. For example, the codon AUG represents methionine. AUG is also the translational start signal. There are 64 possible codons in mRNA, 61 code for amino acids. TAA, TAG and TGA are the stop codons which do not have a corresponding tRNA. The genetic code is universal (for the most part). Transcription in Prokaryotes ( Figure_18_11 ) Transcription is catalyzed by RNA polymerase which makes RNA using DNA as a template. Transcription of DNA occurs in four main stages: 1) binding of RNA polymerase to DNA at a promoter, 2) initiation of transcription on the template DNA strand, 3) subsequent elongation of the RNA chain, and 4) eventual termination of transcription, accompanied by the release of RNA polymerase and the completed RNA product from the DNA template. RNA polymerase moves along the template strand of the DNA in the 3-prime to 5-prime direction, and the RNA molecule grows in the 5- prime to 3-prime direction. The DNA promoter region in prokaryotes is a stretch of about 40 base pairs adjacent to and including the transcription start- point ( Figure_18_12 ). By convention, the critical DNA sequences are given as they appear on the coding strand (the non-template strand, which corresponds in sequence to the RNA transcript). The essential features of the promoter are the start point (designated +1 and usually an A), the six-nucleotide -10 sequence, and the six- nucleotide -35 sequence. The two key sequences are located approximately 10 nucleotides and 35 nucleotides upstream from the start point. Initiation ( Figure_18_13 ) The numbers of nucleotides separating the consensus sequences from each other and from the start-point are important for promoter function, but the identity of these nucleotides is not. Elongation ( Figure_18_14 ) During elongation, RNA polymerase binds to about 30 base pairs of DNA (each complete turn of the DNA double helix is about 10 base pairs). At any given time, about 18 base pairs of DNA are unwound, and the most recently synthesized RNA is still hydrogen-bonded to the DNA, forming a short RNA-DNA hybrid. This hybrid is probably about 12 base pairs long, but it may be shorter. The total length of growing RNA bound to the enzyme and/or DNA is about 25 nucleotides. Termination ( Figure_18_15 ) Termination of transcription requires a termination sequence that triggers the end of transcription. Two classes exist, rho dependent and rho independent In rho independent termination, a short complementary GC-rich sequence (followed by several U residues) will form a "brake" that will help release the RNA polymerase from the template (at the weakly poly-U stretch). In rho dependent termination, binding of rho to the mRNA releases it from the template. * Transcription in Eukaryotes ( Figure_18_T1 ) Although transcription in eukaryotes is similar to that in prokaryotes, the process appears to be complex. Instead of one RNA polymerase, there are three (RNA Polymerases I, II, and III) involved in eukaryotic transcription. RNA polymerase I (localized to the nucleolus) transcribes the rRNA precursor molecules. RNA polymerase II produces most mRNAs and snRNAs. RNA polymerase III is responbsible for the production of pre-tRNAs, 5SrRNA and other small RNAs. The mitochondria and chloroplasts have their own RNA polymerases. Eukaryotic Promoters ( Figure_18_16 ) Eukaryotic nuclear genes have three classes of promoters which are individual for the three types of RNA polymerases RNA polymerase I: The promoter for RNA polymerase I has two components: 1) a core promoter (surrounding the start-point) and 2) an upstream control element. After the binding of appropriate transcription factors to both sites, RNA polymerase I binds to the core promoter. RNA polymerase II: The typical promoter for RNA polymerase II has a short initiator sequence, consisting mostly of pyrimidines and usually a TATA box about 25 bases upstream from the start-point. This type of promoter (with or without the TATA box) is often called a polymerase II core promoter, because for most genes a variety of upstream control elements also play important roles in the initiation of transcription. RNA polymerase III: The promoters for RNA polymerase III vary in structure but the ones for tRNA genes and 5S rRNA genes are located entirely downstream of the start-point, within the transcribed sequence. In tRNA genes, about 30-60 base-pairs of DNA separate promoter elements; in 5S rRNA genes, about 10-30 base-pairs promoter elements. Transcription Initiation ( Figure_18_17 ) General transcription factors and the polymerase undergo a pattern of sequential binding to initiate transcription of nuclear genes. 1) TFIID binds to the TATA box followed by 2) the binding of TFIIA and TFIIB. 3) The resulting complex is now bound by the polymerase, to which TFIIF has already attached. 4) The initiation complex is completed by the addition of TFIIE, TFIIJ, and TFIIH. 5) After an activation step requiring ATP-dependent phosphorylation of the RNA polymerase molecule, the polymerase can initiate transcription at the start-point. The TATA-binding protein (TBP) ( Figure_18_18 ). TBP is a subunit of the TFIID and plays a role in the activity of both RNA polymerase I and RNA polymerase III transcription. TBP is also essential for transcription of TATA-less genes. TBP differs from most DNA-binding proteins in that it interacts with the minor groove of DNA, rather than the major groove and imparts a sharp bend to the DNA. The TBP has been highly conserved during evolution. When TBP is bound to DNA, other transcription-factor proteins can interact with the convex surface of the TBP saddle. TBP is required for transcription initiation on all types of eukaryotic promoters. Termination in Eukaryotes Termination signals end the transcription of RNA by RNA polymerase I and RNA polymerase III without the activity of hairpin structures as seen in prokaryotes. mRNA is cleaved 10 to 35 base-pairs downstream of a AAUAAA sequence (which acts as a poly-A tail addition signal). Ribosomal RNA ( Figure_18_20 ) Involves cleavage of multiple rRNAs from a common precursor ( Figure_18_T3 ). The eukaryotic transcription unit that includes the genes for the three largest rRNAs occurs in multiple copies and arranged in tandem arrays with non-transcribed spacers separate the units. Each transcription unit includes the genes for the three rRNAs and transcribed spacer regions. The transcription unit is transcribed by RNA polymerase I into a single long transcript (pre-rRNA) with a sedimentation coefficient of about 45S. RNA processing yields mature rRNA molecules. RNA cleavage actually occurs in a series of steps which varies in order with the species and cell type but the final products are always the same three types of rRNA molecules. tRNA ( Figure_18_21 ) Every tRNA gene is transcribed as a precursor that must be processed into a mature tRNA molecule by the removal, addition and chemical modification of nucleotides. Processing for some tRNA involves 1) removal of the leader sequence at the 5 prime end 2) replacement of two nucleotides at the 3 prime end by the sequence CCA (with which all mature tRNA molecules terminate) 3) chemical modification of certain bases and 4) excision of an intron. The mature tRNA is often diagrammed as a flattened cloverleaf which clearly shows the base pairing between self-complementary stretches in the molecule. Processing of mRNA ( Figure_18_25 ) Messenger mRNA in eukaryotes are first made as heterogeneous nuclear mRNA (or pre-mRNA) then processed into mature mRNA through the addition of a 5 prime cap structure, addition of poly-A tails and the splicing out of introns. RNA capping ( Figure_18_22 ) To give the mRNA stability, a 5 prime “cap” (a guanosine nucleotide methylated at the 7th position) is joined to the 1st nucleotide in an unusual "5 prime to 5 prime" linkage [sort of "backwards"]. During the capping process, the first two nucleotides of the message may also become methylated RNA tailing ( Figure_18_23 ) Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3 prime end of the mature mRNA. An AAUAAA sequence located slightly upstream from the proper 3prime end then signals that the RNA chain should be cleaved about 10- 35 nucleotides downstream from the signal site, followed by addition of a “poly-A tail” catalyzed by poly(A) polymerase. RNA splicing ( Figure_18_26 ) Spliceosomes remove introns from pre-mRNA Introns were discovered to exist in eukaryotic mRNA by ( Figure_18_24 ) 1) mixing mature mRNA molecules with the genes (DNA) from which they had been transcribed and 2) examining the hydrogen bonded hybrids under an electron microscope. Hybridization of a eukaryotic mRNA molecule with a gene which has one intron will produce two single-stranded DNA loops where the mRNA has hybridized to the DNA template strand plus an obvious double-stranded DNA loop. The double-stranded DNA loop represents the intron, which contains sequences that do not appear in the final mRNA. Restriction enzyme analysis has revealed the presence of introns. The spliceosome is an RNA-protein complex that splices intron-containing pre-mRNA in the eukaryotic nucleus. The substrate here is a molecule of pre-mRNA with two exons and one intron. Splicing mechanism ( Figure_18_27) In a stepwise fashion, the pre-mRNA assembles with the U1 snRNP, U2 snRNP, and U4/U6 and U5 snRNPs (along with some non-snRNP splicing factors), forming a mature spliceosome. The pre-mRNA is then cleaved at the 5 prime splice site and the newly released 5 prime end is linked to an adenine (A) nucleotide located at the branch-point sequence, creating a looped lariat structure. Next the 3 prime splice site is cleaved and the two ends of the exon are joined together, releasing the intron for subsequent degradation. Alternative results in alternate forms of mRNA and, often, proteins ( Figure_18_28 ). Stability of mRNA and expression Most mRNA molecules have a high turn-over rate as the molecules are rapidly degraded and replaced. tRNA and rRNAs are relatively stable. Bacterial mRNAs have half-lives of a few minutes and eukaryotic mRNA range from hours to days. Transcription allows amplification of the genetic information because many copies of the mRNA can be produced to direct a great deal of protein synthesis.

Cell Biology Textbook PDF

Document Details

Tags

Related

Summary

Full Transcript