BISC 220 Notes PDF

BISC 220 Notes Chemistry of macromolecules ​ Isomers- molecules with the same molecular formula but different arrangements of atoms ○​ Structural: exhibit different arrangements of the same number/types of atoms that make up the compound (same chemical formula,...

BISC 220 Notes Chemistry of macromolecules ​ Isomers- molecules with the same molecular formula but different arrangements of atoms ○​ Structural: exhibit different arrangements of the same number/types of atoms that make up the compound (same chemical formula, different bond arrangement) ○​ Geometric: exhibit a different angle/ orientation based on the presence of single or double (covalent) bonds ○​ Enantiomers: “mirror images” of one another. Chemically identical pair of molecules that exist in 2 forms. ​ Particularly useful in pharmacology ​ Treat various diseases such as Parkinson’s (PD) because they closely mirror chemicals made naturally in the body ​ 3D orientation and different angles in the bonds matters ​ L-dopa (levodopa) is a chemical precursor to dopamine synthesis in the brain, used to treat PD ​ Dopamine: neurotransmitter that controls functions including movement (hypokinesis, or difficulty in movement, in PD traced to lack of dopamine production) ​ Administering L dopa could alleviate PD by promoting dopamine production. Unfortunately, the body develops a tolerance to it. ​ Essential elements of life (CHONPS)- observed in major macromolecules ○​ Carbon ○​ Hydrogen ○​ Oxygen ○​ Nitrogen ○​ Phosphorous ○​ Sulfur ​ Intramolecular bonds (bonds within the molecules): Ionic & covalent bonds ○​ Ionic bonds: metal + nonmetal forms unequal bond where electrons are transferred, leads to charges ​ Ex: sodium chloride will dissociate in water into Na+ and Cl-  ○​ Covalent bonds: 2 nonmetals form an equal bond that share electrons to balance their shells ​ Ex: H2O (dihydrogen monoxide- polar covalent), CH4 (methane- a nonpolar covalent molecule, gas from flatulence) ​ Polar bonds are covalent bonds where there is a polarity difference between the nonmetal elements (water is polar, giving rise to many unique properties) ○​ Hydrogen bonds: Hydrogen is the smallest, nimblest element and can thus form H bonds with certain elements ​ Bonds form due to electrostatic forces (like tiny little bursts of static electricity that exist b/w atoms: brief) ​ Keeps water molecules together, holds DNA nucleotide bases together, found in the secondary and tertiary structures of proteins ​ More hydrogen bonds are present in a solid state. As it goes solid -> liquid → gas you have fewer H bonds and more free kinetic energy/ movement of water molecules ​ Properties of water ○​ Polar covalent molecules ○​ 2 Hydrogen molecules simultaneously shares its 1 valence electron with oxygen, but it's more like it gives the e- to O b/c O is more electronegative than H ○​ Considered to be a universal solvent, so most can be dissolved in water (except for nonpolar molecules like oil, a lipid) ○​ Like dissolves like ​ Polar molecules are miscible with other polar molecules but not with nonpolar molecules ○​ High heat capacity, allowing it to store a lot of heat energy ○​ High heat of vaporization, meaning it takes a lot of energy to make water boil b/c of the need to break its H-bonds ○​ High cohesion (molecules stick to each other → surface tension) and adhesion (molecules stick to other substances, like in a plant stem) ○​ Less dense as a solid than as a liquid due to lattice structure ​ pH ○​ The concentration of free floating H ions (AKA protons, b/c H has lost its e-) influences pH of a solution ○​ Acidic 7 ○​ Inverse logarithmic relationship between hydrogen ion concentration and pH ○​ More acidic = more H+ ○​ More basic = fewer H+, more OH- ​ Functional groups ○​ Hydroxyl groups: OH or OH- ○​ Carbonyl groups: C=O ○​ Carboxyl groups: COOH or COO-  ​ Found on each amino acid, the monomer for proteins ○​ Amine groups: NH2 ​ Found in amino acids ○​ Sulfhydryl (thiol) groups: SH ○​ Phosphate groups: PO4- ​ Found in every nucleic acid ​ Monomers & polymers ○​ Monomer: a single chemical unit/building block for a larger molecule ○​ Polymer: molecule consisting of several monomers bound together ​ Carbohydrates (sugars) ○​ Many are known as saccharides ○​ Commonly composed of C, H, and O ○​ Many are ring structures, hexo- or pento- sugar rings ○​ Monosaccharides for ester bonds with each other ○​ Most polysaccharides contain glucose ○​ Types of polysaccharides: contain different types of ester bonds ○​ Biggest glycogen reserves in the human body: liver and skeletal muscles ​ Lipids (fats) ○​ Composed of C and H ○​ Long carbon chains ○​ Tail of carbon and hydrogen atoms (hydrocarbon chain/fatty acid tail) ○​ Not water soluble: Nonpolar, hydrophobic ○​ Most of our fat is stored in adipose tissue in the form of triglyceride ○​ Saturated fatty acids: no double bonds present b/w c and h atoms, so the fatty acid tails have a linear structure and are solid at room temperature because they can be packed closely together ○​ Unsaturates fatty acids: ​ Trans: H atoms opposite. Fatty acid tail continues to be linear, allowing it to be solid. Ultra-processed foods tend to be enriched in trans fats. ​ Cis: H atoms on the same side → bent configuration. This bend results in more space between fatty acid tails, leading to more fluidity and liquidity at room temperature (ex: olive oil, avocado oil, etc.) ​ Proteins  ○​ Contain C, H, O, N, and sometimes S (CHON/CHONS) ○​ Polar ○​ Monomer: amino acids (of which there are 20) ​ Amino acids bind together to form polypeptides ○​ Amino acids are composed of a central atom, a hydrogen atom bonded to it, a carboxyl group (COOH), an amine group, and a unique r-group for each of the 20 amino acids ○​ Structure: ​ Primary: polypeptide chain ​ Secondary: alpha helix and/or beta sheet structures are produced by hydrogen bonds ​ Tertiary: 3D overall fold of the protein based on r-groups, occurs when secondary structure folds in on itself ​ H bonds between coils and sheets ​ Presence of sulfur atoms can create disulfide bridges ​ Quaternary: multi-subunit complex where each subunit is a distinct polypeptide chain. Different proteins require different amounts of folding to be functional (some end at tertiary stage). ○​ Case study in protein structure: hemoglobin ​ Quaternary level of structure, each heme has a tertiary structure ​ Replacing a negatively charged amino acid with a nonpolar acid can change the shape of blood cells (point mutation leads to sickle cell anemia) ○​ Enzymes: catalytic proteins ​ Reduce the activation energy required for reactions to occur, dramatically increasing reaction rate ​ Enzymes can be anabolic (reactions they catalyze build a larger product from many individual substrates) or catabolic (break a larger product into many individual substrates) ○​ Wide variety of functions ​ Nucleic acids ○​ Contain C, H, O, N, and P (CHONP)  ○​ Highly energetic macromolecules, including ATP ​ Shearing off of a phosphate molecule in ATP produces ADP, a process that causes the energy release ○​ Nucleotide: conglomerates of molecules. Contain a nitrogenous base, a phosphate group (PO4-), and a sugar (typically ribose) ​ DNA vs. RNA ○​ DNA is double stranded whereas RNA is single stranded ○​ RNA (ex: mRNA) is transient, moves around a lot more than DNA ○​ Thymine in DNA, uracil in RNA ○​ DNA lacks oxygen on the ribose sugar in its backbone, which creates phosphodiester bonds in its backbone. RNA has this oxygen on its ribose sugar (deoxy = missing an oxygen). ​ Central dogma ○​ Purpose of DNA: to be able to selectively express regions of DNA/genes so that proteins may be produced ○​ DNA → mRNA → polypeptide ○​ Transcription: DNA is unwound by RNA polymerase and is transcribed into mRNA (nucleus in eukaryotes, in cytosol in prokaryotes). ○​ mRNA travels to a ribosome ○​ Translation: mRNA is translated into a polypeptide chain (codons → amino acids) where the chain undergoes additional folding to become a functional protein, and is then trafficked to where it is needed in the cell * understand how electronegativity and polarity affect molecular behavior The cell and organelles ​ Cells ○​ Smallest unit of life on earth ○​ Can be physically connected to one another to form tissues, which form organs and organ systems. (cell → tissue → organ → organ system) ○​ All cells originate from an ancestral cell ○​ Typical size order: Eukaryotic cells > prokaryotic cells > organelles ○​ Flu virus can’t be seen by a light microscope ​ Visualization techniques ○​ Light microscope ​ Ocular lens (eyepiece) ​ Objective lens (typically 3 magnification) ​ Stage ​ Coarse adjustment knobs ​ Fine adjustment knobs ​ Light aperture (diagram) ○​ SEM- scanning electron microscopy  ​ Used to look at the surface topography of something smaller than the prokaryotic cell ○​ TEM- transmission electron microscopy ​ Looking at the interior of something small ○​ Fluorescent microscopy ​ Examine specimens pre-labeled with fluorescent markers under a microscope capable of differentiating the different fluorescent light wavelengths from these markers ​ Allows molecules of interest to be tracked/distribution seen ​ Taxonomy of life ○​ 3 domains: bacteria, archaea, eukarya ​ Prokaryotes include bacteria and archaea ○​ Both: plasma membrane, ribosomes, DNA ​ Prokaryotic cells ​ Lack all membrane-bound organelles ​ Nucleoid region rather than nucleus (cluster of genome) ​ DNA is circular ​ Eukaryotic cells ○​ Nucleus: stores/protects DNA ○​ Endoplasmic reticulum: packages proteins, lipids, and other macromolecules ​ Rough ER: speckled with ribosomes- synthesizes proteins ​ Smooth ER: lack ribosomes- manufacture lipids ○​ Golgi apparatus ​ Receives contents from ER ​ Materials move in one direction ​ Protein travels in vesicle to cis side of GA, passes through GA and is modified, exits through trans side of GA (mailing system) ​ Makes protein functional through enzymes, etc. ○​ Vesicles ​ Packages of cellular materials ○​ Ribosomes ​ Site of protein synthesis (translation) ○​ Mitochondria ​ Site of ATP synthesis (cellular respiration) ​ Inner and outer membrane to maximize surface areas ​ Has circular DNA: evidence of evolutionary theory ○​ Lysosomes ​ Digests used materials in the cell ​ “Stomach” of the cell ​ Lysosomes may also burst when programmed cell death (apoptosis) is triggered. ○​ Peroxisomes ​ Break down toxins into less harmful chemicals ○​ Vacuoles  ​ Stores water in the cell ○​ Cytoplasm ​ Aqueous (water based) solution ​ Maintains internal cellular environment and pads organelles ○​ Plasma membrane ​ Semi-permeable membrane that protects internal cellular environment ​ Semi-permeable = selectively allows molecules to freely enter cell Cytoskeletal elements → ○​ Intermediate Filaments ​ Maintain stability of organelles ​ Provide overall cell structure ​ Hold together the internal components of the cell and prevent excessive movement from causing internal damage in the cell ○​ Microfilaments (actin) ​ Help give cells direction ​ Responsible for whole-cell and internal movement/transport ​ Polymer ○​ Microtubules ​ Create centrioles → centrosome ​ Important for cell division and whole cell/internal movement ○​ 3 unique plant cell organelles: ​ Chloroplasts, which like mitochondria, play an important role in cell energy production/ consumption. ​ Large Central Vacuole, used for long-term water storage. ​ Cell wall, typically made of cellulose, that maintains the cell’s structural integrity. ​ Endosymbiotic theory ○​ Ancestral prokaryotic cells exhibit folding of the plasma membrane that gave ride to internal membrane-bound organelles ○​ Uptake of mitochondria and chloroplast ○​ Evidence: mitochondria and chloroplasts, AKA cyanobacteria, both have internal and external membranes and both have their own genomes/DNA  ​ Suggests they functioned independently once ​ Internal and whole cell motility ○​ GFP (green fluorescent protein) can be attached to actin ○​ Actin polymerization ​ Process of assembling (and disassembling) actin filaments ​ When this polymerization reaction occurs over several actin filaments, it can result an whole-cell movement, as shown on the bottom left. Here, the growing and shrinking actin filaments are tagged with Green Fluorescent Protein (GFP) to allow for easy visualization of the way in which the cell is capable of moving as these filaments grow in the direction of the movement. ○​ ○​ Shown in the middle is an example of a unique cellular structure that is actually more commonly observed in prokaryotic cells than eukaryotic cells: cilia are small, hair-like projections that can allow a cell to exhibit some motility (movement) in its environment, though this movement is limited by the size and quantity of the cilia present. Perhaps a more well-known example of whole-cell movement resulting from a unique cellular structure is the swimming movement that results from the present of a flagellum, a tail-like appendage that as with cilia, is more commonly seen in prokaryotic cells than eukaryotic cells. However, sperm cells notably have a flagellum that allows them to swim towards an unfertilized egg. ○​ ○​ Finally (and perhaps my favorite of all the motility mechanisms), cells need to have a way to transport materials within them, and this task is accomplished by specialized motor proteins such as dynein and kinesin (the latter is shown in the gif on the bottom right of this slide). This gif is actually a very accurate representation of what these proteins look like and how they move: their “feet-like” processes undergo conformational (shape) changes that allow them to move along the intermediate filaments and microtubules of the cytoskeleton, while carrying vesicles or other “cargo”. ○​ Kinesin: a motor protein with foot processes that carries a vesicle in 1 direction ​ Carry neurotransmitter to the synaptic terminal Semipermeable membranes & intracellular transport ​ Endomembrane system ○​ Described the transportation sequence of membrane-bound organelles that participate in exo- and endocytosis of key macromolecules (notably proteins) ​ Endocytosis: captures substance outside of cell, engulfs it ​ Exocytosis: cells shift materials such as waste from inside to an extracellular space outside the cell ○​ The order in which a molecule would be shuttled through the endomembrane system:  1.​ Synthesized at a ribosome in the rough ER 2.​ Vesicle transports protein to golgi apparatus 3.​ Vesicle buds off golgi apparatus to transport protein to cell membrane ​ The plasma membrane ○​ A semipermeable (selective) membrane with two chemical components: ​ Outer and inner layers are hydrophilic (polar) due to phosphate groups ​ Middle layer is hydrophobic (nonpolar) due to fatty acids ○​ Most membranes are packed with macromolecules that participate in cell signaling, transport, and identification (in the immune system). ​ Glycoproteins and glycolipids- hybrid macromolecules that contain protein and carbohydrate molecular traces, and play an especially important role in cell signaling and the immune system. ​ Surface proteins- adhered to the intracellular or extracellular side of the cell membrane, depending on their function ​ Integral and channel proteins- span the width of the membrane (all channel proteins are integral proteins, some integral proteins don’t have a pore/allow passage but span the width of the membrane) ​ Protein pumps are usually integral proteins ​ The phospholipid bilayer ○​ Cell membrane is largely made of cholesterol (a steroid liquid) but also a phospholipid bilayer ○​ Phosphate heads face toward the intra and extracellular spaces and are hydrophilic (Water soluble) ○​ Fatty acid (lipid) tails make up the medial layer of the membrane and are hydrophobic  ○​ These conflicting properties of the phospholipid bilayer necessitate the presence of integral proteins and protein channels to allow for polar and charged molecules to cross the cell membrane, as they would not ordinarily be able to do so due to the hydrophobic nature of the internal layer of the membrane. ​ Permeability in the membrane ○​ A semipermeable membrane only allows certain molecules based on their size and polarity/charge to freely cross ​ A more fluid membrane has increased permeability due to the presence of unsaturated (cis) fatty acids, which are kinked/bent and can’t pack as closely together ​ A more viscous membrane has decreased permeability due to presence of saturated fatty acids, which can be packed tightly together ​ Passive transport: diffusion ○​ This form of transport does not require energy on the part of the cell ○​ Typically, molecules move along their concentration gradient (from a region of high concentration to low concentration) ○​ Diffusion is the simplest form ​ Passive transport: osmosis ○​ The diffusion of water molecules across a semipermeable membrane ​ This is not to be confused with the active transport of water molecules via special membrane proteins known as aquaporins. ○​ Osmolarity is affected by the concentration of solutes on either side of the membrane ○​ Changes in water concentration across a membrane can result in changes in osmolarity on either side of the membrane, which can have important health consequences. ○​ Water diffuses in slow rates and low concentrations across the membrane (small polar molecules can diffuse across the membrane passively but only slowly/in low concentrations) ​ Dialysis lab technique ○​ Method for separating molecules by size ○​ A thin, semipermeable dialysis tube (of which the permeability is known) is filled with a solution that typically contains multiple compounds of different sizes. ○​ Measuring the mass of the dialysis tube at the start of the experiment, then comparing this measurement to the mass at the end of the experiment, indicates the net movement of molecules across the membrane. ​ Tonicity ○​ Less water in a hypertonic solution, more water in a hypotonic solution ○​ Molecules don’t ever stop moving ○​ Equilibrium is thus always dynamic ○​ Three possible tonicities of the extracellular environment (i.e. the solution surrounding the cell):  ○​ A hypertonic solution is one in which there is a higher concentration of water inside the cell compared to outside the cell. Thus, water will diffuse out of the cell in order to reach an equilibrium state where the concentration of water on either side of the cell membrane is roughly equal. ​ Ex: cases of dehydration (in which there is not enough water in the extracellular fluid surrounding one’s red blood cells). This can lead to shriveling of the cells and over time, cell dysfunction and death. ○​ An isotonic solution is one in which there are roughly equal concentrations of water on either side of the cell membrane, and thus, an equilibrium state is achieved. It is important to note that once the concentration gradient of water is at equilibrium, that does not mean that water stops moving across the cell membrane. Rather, water will continue to diffuse freely in both directions across the cell membrane. This is considered an “ideal solution” from the standpoint of human health, as this type of solution is one in which the body is fully hydrated and the solute concentration of the blood is also balanced (i.e. not too much salt or sugar in the blood). ○​ A hypotonic solution is one in which there is a lower concentration of water inside the cell compared to outside the cell. Thus, water will diffuse into the cell in order to reach an equilibrium state where the concentration of water on either side of the cell membrane is roughly equal. A real example of this kind of solution is in cases of water poisoning (yes, this is a thing), or when an individual consumes too much water too quickly. This can be extremely dangerous as it can cause the cells to burst or rupture, leading to cell death. There was a high-profile case in the 1990’s of a listening of a radio show partaking in a “water drinking contest” who later died from water poisoning. ​ Functions of membrane proteins ○​ Form intercellular connections to physically adhere cells to one another (this is especially important when considering the formation of different body tissues) ○​ Participate in enzymatic activity, which typically overlaps with signal transduction ○​ Assist in forms of intercellular transport, such as active or passive transport ○​ Play a role in cell-to-cell recognition ○​ Participate directly (or indirectly) in signal transduction ​ Passive transport ○​ These are transport mechanisms that do not require energy and allow molecules to move along their concentration gradient. ○​ Simple Diffusion: Process of a molecule moving from an area of high concentration to an area of low concentration (“along its concentration gradient”) without the assistance of any protein channels. ○​ Facilitated Diffusion: Essentially the same process as diffusion, however, as the name suggests, this is “facilitated” by the presence of specialized protein channels. Intracellular signaling & gene expression ​ Gated channels ○​ Often ion channels (and other protein channels) will exhibit a gating mechanism in order to prevent excessive movement of ions into and/ or out of cells. ○​ Voltage-gated Ion Channels undergo a conformational (shape) change in response to a change in the charge difference across the membrane, also known as the membrane potential ○​ Ligand-gated Ion Channels require a specific molecule, or ligand to bind to its designated active site on the channel in order for the channel to undergo a conformational (shape) change (i.e. change from the “closed” to “open” state). ○​ Tension-gated Ion Channels are observed in specific sensory systems (such as the auditory, vestibular and somatosensory systems), and are channels that undergo a conformational (shape) change in response to a physical perturbation (mechanical stimulus) that essentially “forces” the channel to change shape ​ Mechanical stress and piezo channels ​ When a mechanical force is applied to the cell membrane, it springs open due to being a tension-gated channel, which causes an increase of voltage in the cell ○​ This is the reason we feel touch (mechanically sensitive channels undergoing polarization and depolarization) ​ Types of active transport ○​ Active transportation required energy to move molecules across their concentration gradient ○​ Protein Pump-mediated Transport often utilizes energy in the form of ATP to “force” molecules to move against their concentration gradient. ​ Ex: the Sodium-Potassium Pump, which uses ATP to pump Sodium and Potassium against their respective concentration gradients in order to maintain or reset the internal voltage of a cell. ○​ Cotransporters (AKA secondary active transporters) harness the energy gained from transporting one molecule along its concentration gradient to force the other molecule to move against its concentration gradient. ​ In the case of symporters, both molecules move in the same direction (into or out of the cell), and the energy that’s gained from one molecule moving along its concentration gradient is used to “force” the other molecule to move against its concentration gradient. ​ In the case of antiporters, each molecule is moving in opposite directions, but the form of energy used in this type of cotransporter (i.e. the energy of one molecule moving along its concentration gradient) is consistent with the energy utilized in symporters. ​ Bulk transport ○​ Exocytosis: Process of transporting materials out of the cell via via vesicle fusion with the cell membrane  ○​ Endocytosis: Process of transporting materials into the cell via vesicle fusion with the cell membrane ​ Pinocytosis: Intake of many small, dissolved molecules ​ Phagocytosis: Intake of one or more large molecules ​ Receptor-mediated endocytosis: clathrin-mediated endocytosis ○​ Relies on recognition of the membrane protein clathrin by adaptor proteins that coat the outer surface of the vesicle as it is formed. The clathrin proteins are eventually stripped from the outer surface of the vesicle to allow it to fuse within the endomembrane system ​ Cell junctions: adherence and signaling ○​ Adherens junctions- recruit microfilaments (actin), common in epithelial tissue. Rely on the binding of actin filaments between neighboring cells, using these actin tethers to adhere the cells to each other. No materials can be exchanged between the two cells via these junctions. ○​ Tight junctions- Recruit microfilaments (actin) and fuse two cell membranes together, common in epithelial tissue. Recruit actin filaments to form the physical connection between neighboring cells), however, they are also characterized by physical connections between the cell membranes of the two connected cells. ○​ Gap junctions- Allow for more molecules to pass between cells, important for electrical coupling. They allow for the passage of small molecules (often ions) directly between the two connected cells. Exchange of ions via gap junctions allows for close electrical coupling over time, meaning that a very rapid electrical current can be generated and signal between the two connected cells. This occurs in a small percentage of synapses in the nervous system, known as electrical synapses. ○​ Desmosomes- Similar to adherens junctions, recruit intermediate filaments, common in epithelial and some muscle tissue. They recruit intermediate filaments rather than microfilaments such as actin to form an even stronger physical connection between the neighboring cells. They are commonly observed in epithelial tissue as well as certain muscle tissue, such as cardiac muscle tissue. ​ Chemical signaling mechanisms important to the endocrine system ○​ Autocrine signaling occurs when a cell signals to itself. The cell releases a chemical signal that then binds to its corresponding receptor on that same cell. This chemical signaling mechanism can be especially useful in the context of short-distance negative feedback loops, in which a cell must regulate its own function in order to maintain homeostasis. ○​ Paracrine signaling occurs when neighboring cells release chemical signals that then diffuse and/ or migrate through small capillaries via red blood cells to communicate with one another. This form of chemical signaling typically takes place within a tissue or gland. ○​ Endocrine signaling occurs when cells signal to far away targets. The slowest of these three chemical signaling mechanisms, and occurs when chemicals are released by endocrine cells which then migrate to  their targets via blood vessels. Typically occurs between tissues/ glands. ​ Far travel through cardiovascular system ​ Chemical signaling mechanisms important to the nervous system ○​ Neuron-to-Neuron (Neural) Signaling occurs when two (or more) neurons are connected together via synapses and are capable of relaying electrical and chemical signals in a single direction to one another. One neuron is designated as the presynaptic neuron and is responsible for releasing chemicals called neurotransmitters into the synapse to communicate with the postsynaptic neuron, which receives these chemical signals and produces an electrical response. ○​ Neuron-to-Muscle (Neuromuscular) Signaling occurs when a single neuron communicates with one (or more) muscle cells to induce muscle contraction or muscle relaxation. The type of synapse that connects these two cells is typically referred to as the neuromuscular junction. The presynaptic cell (neuron) released chemicals into the synapse which bind to their corresponding receptors on the postsynaptic cell (muscle cell), causing a cascade of intracellular events in the muscle cell. ○​ Neuron-to-Gland (Neuroendocrine) Signaling occurs when a single neuron (or several neurons) communicate with cells in a gland, such as the pituitary gland, to trigger changes in downstream hormone production/ signaling. The presynaptic cell (neuron) releases chemicals into the synapse that bind to their corresponding receptors on the postsynaptic cell (endocrine cell), causing a cascade of intracellular events in the endocrine cell. ​ Intracellular signaling cascades ○​ GPCR- 7 transmembrane domains, extracellular side has a domain that can bond a ligand, on the intracellular side, a receptor communes with a G protein ○​ Typical cascade: An external stimulus (usually a ligand) binds to a membrane-bound protein known as a receptor. Each receptor binds to a specific ligand, a behavior that is common across all major classes of proteins (similar to the way in which antibodies, which are specialized proteins, only bind to specific antigens). The binding of the ligand to the receptor triggers a cascade of molecular changes within the cell, ultimately resulting in a final “response” from the cell (typically a change in gene expression, which leads to a subsequent change in protein expression)  ○​ G-protein Coupled Receptor (GPCR)-mediated cascade: begins with the binding of a ligand to a special, seven channel transmembrane receptor known as a G-protein Coupled Receptor (GPCR). The binding of the ligand causes a change in the conformation (shape) of the GPCR, which triggers the activation of a G-protein within the cell. The activation of this G-protein causes a series of activational changes in the messenger proteins in the cascade. Once again, this cascade will lead to an “ultimate” cell response, which is almost always a change in gene expression. ​ Molecular “players” & common cellular responses ○​ Membrane proteins- integral proteins with one or more transmembrane domains, capable of “receiving” a (chemical) signal from the extracellular side of the membrane and then “relaying” this signal into the intracellular side of the membrane to begin the signaling cascade. Ex: GPCRs, and Receptor Tyrosine Kinases (which typically consist of two separate protein domains that dimerize, or bind together to form one functional membrane protein receptor). ○​ Secondary messengers- chemicals (often proteins or enzymes) found within the cell that aid in relaying the chemical signal detected by the receptor. Often undergo phosphorylation (adding a phosphate group) or dephosphorylation (removing a phosphate group) of the secondary messenger ○​ Cellular Responses: commonly a change in gene expression, or to be more specific, the upregulation or downregulation of transcription of a particular gene or set of genes. This is caused by direct modifications made to transcription factors, which are a special class of protein localized to the nucleus and responsible for regulating transcription. ​ Ionotropic versus metabotropic signaling ○​ Ionotrophic: ions move across the cell membrane via active or passive transport, using a dedicated ion (protein) channel or pump embedded in the membrane. Because these signaling mechanisms only involve the movement of ions from one location to another (i.e. no secondary messengers relaying a signaling within a cell), they are much faster than metabotropic signaling mechanisms. ○​ Metabotrophic: These types of signaling mechanisms involve secondary messengers within the cell that relay the initial “message” to the target (usually  the nucleus, where changes in gene expression occur). Because these signaling pathways involve several “relay steps” within the cell, they are typically slower as compared to their other signaling counterparts. ​ G-protein coupled receptors ○​ GCPRs are a large class of metabotropic receptor proteins ○​ Contain 7 transmembrane domains ○​ N-terminus faces extracellular side, c-terminus faces intracellular side ○​ GCPRs are associated with G-proteins found on the intracellular side of the cell membrane ​ Inactive G-proteins bind Guanosine Diphosphate (GDP) ​ Active G-proteins bind Guanosine Triphosphate (GTP) ○​ In the inactive state, these G-proteins bind a nucleic acid called Guanosine Diphosphate (GDP). When a GPCR binds its specific ligand (chemical signal), this G-protein becomes activated, resulting in the formation of Guanosine Triphosphate (GTP), which provides the necessary energy to begin the “relay race” of the intracellular cascade. After the activation of this G-protein, secondary messengers are then activated in turn to relay the signal within the cell. ​ GPCR case study on dopaminergic signaling in the basal ganglia ○​ Ex: Neurons in the basal ganglia express two GPCRs capable of binding the neurotransmitter dopamine: ​ D1-type Receptors trigger an intracellular cascade and stimulate a neural circuit to promote movement ​ D2-type Receptors trigger an intracellular cascade and stimulate a neural circuit to prevent movement ​ Receptor Tyrosine Kinases (RTKs) ○​ Metabotropic membrane receptor ○​ Kinases phosphorylate molecules within the cell (attach phosphate groups ot molecules to make them more energetic) ○​ The majority of RTKs have two transmembrane domains which dimerize (become associated with each other) ○​ Consist almost exclusively of hydrophobic (nonpolar) amino acids. They also have an extracellular N-terminus and an intracellular C-terminus. RTKs are especially common receptors in the endocrine system, and several have been implicated in various types of cancers. ​ RTK Case study ○​ Insulin mediates the regulation of blood sugar ○​ Insulin commonly signals through RTKs ○​ At the pancreas, liver, and brain, cells expressing this RTK can bind insulin, which causes increased expression and trafficking of a protein channel known as GLUT4 to the cell membrane. GLUT4 assists in the uptake of excess glucose from the bloodstream, and also assists in the process of forming glycogen from glucose monomers. ​ Gene expression  ○​ The process of regulating when certain genes (discrete regions of DNA) are transcribed into (m)RNA. ○​ Because cells must maintain intracellular homeostasis, they do not “want” to constitutively (continuously) produce certain quantities of proteins, and must be able to switch gene expression (which precedes protein synthesis) “on” and “off”. ○​ Certain types of proteins must bind to particular regions of the DNA that exist ahead of, or upstream of the genes being transcribed, in order to promote or prevent transcription from occurring. ​ Eukaryotic regulation of gene expression ○​ Transcription factors are proteins that regulate gene expression. They can either: ​ Promote the gene expression (transcription) when bound to the promoter region of the DNA ​ Prevent gene expression (transcription), when bound to the repressor region of the DNA ​ More than one transcription factor is usually required for regulating gene expression, and they form what is known as a transcription factor complex (a protein complex). ​ Prokaryotic regulation of gene expression- (don’t have transcription factors) the following components make up an operon: ○​ A regulatory gene (which codes for a protein/ proteins that perform a similar function to transcription factors). These proteins can bind to either the promoter or operator (repressor) regions of the DNA upstream of the structural genes and regulate gene expression. ○​ A promoter and operator (repressor) region of the DNA ○​ Structural genes (genes to be regulated) ○​ These regions together make up an operon. ​ Common operons ○​ Lac operon- regulates the expression of genes that code for enzymes which digest lactose to produce energy for the cell. ​ When lactose is low, the lac repressor will bind to the operator (repressor) region and prevent gene expression. ​ When lactose is high, a structural isomer allolactose will bind to the lac repressor, preventing it from binding to the operator (repressor) region and thus allowing gene expression. ○​ Trp operon- regulates the expression of genes that code for enzymes necessary to synthesize the amino acid tryptophan. ​ When tryptophan is low, the trp repressor dissociates from the operator (repressor) region of the DNA, allowing for transcription to occur. ​ When tryptophan is high, the trp repressor is tightly bound to the operator (repressor) region of the DNA, preventing transcription. DNA replication ​ Semiconservative ○​ In the early 1950’s, Matt Meselson and Frank Stahl were seeking to identify the underlying mechanism for DNA replication. ○​ At the time, there were three reigning theories for how DNA replication workedr: ​ Conservative ​ Semiconservative ​ Dispersive ○​ To confirm which model was accurate, Meselson and Stahl incorporated light nitrogen (N14) into the first generation of DNA in cultured cells, then surrounded this DNA with nitrogenous bases consisting of heavy nitrogen (N15). ​ DNA is double stranded and antiparallel, w/ leading and lagging strands ○​ The DNA strands run antiparallel ○​ The leading strand is replicated by DNA Pol III and is replicated continuously ○​ The lagging strand is replicated by DNA Pol I and is replicated discontinuously, in okazaki fragments ○​ The “leading” strand in a DNA double helix is replicated/ transcribed from the 5’ to 3’ end. ○​ The “lagging” strand in a DNA double helix is replicated/ transcribed from the 3’ to 5’ end. ​ Key enzymes and protein in DNA replication ○​ Helicase- unwinds DNA double helix, forming a replication fork ○​ Single-stranded binding proteins (SSBs)- help to hold replication fork open throughout replication process ○​ Topoisomerase- relieves tension created upstream of the replication fork ○​ Primase- add RNA primers to begin replication ○​ DNA Polymerase III- adds complementary nitrogenous bases to the leading strand, and only works in the 5’ → 3’ direction on the 5’ to 3’ strand. ​ Also removes rna bases ○​ DNA Polymerase I- replicates the lagging strand of the DNA double helix. Because this lagging strand is replicated in the 3’ → 5’ direction, DNA Polymerase I works in short “bursts”, creating separate segments, or fragments of complementary DNA known as Okazaki Fragments. Replicated 5’ to 3’ on the 3’ to 5’. ○​ DNA Polymerase II- primarily responsible for detecting mutations, and can move in the 3’ to 5’ direction unlike the other enzymes, backtracking to an incorrectly matched nucleotide and replacing it with the correct nucleotide. ○​ Ligase- ligates (glues together) the Okazaki fragments produced by DNA Polymerase I during replication of the lagging strand. Ligase also plays an important role, along with DNA Polymerase II, in DNA proofreading and repair (after replication). ​ Phases of DNA replication in eukaryotic and prokaryotic cells  ○​ Eukaryotic DNA is linear, whereas prokaryotic DNA is circular- this necessitates the having double the number of enzymes assisting in prokaryotic DNA replication ○​ Three phases to DNA replication: ​ Initiation- primarily involves unwinding the DNA double helix to create the replication fork (or replication bubble in prokaryotic cells) and adding RNA primers. ​ Elongation (replication)- building the complementary DNA strands from the leading and lagging strand templates (through the actions of DNA polymerases) ​ Termination- occurs once a specific termination sequence has been reached in the DNA that signals the release of the DNA polymerases. Additionally, ligase is recruited to anneal the Okazaki Fragments on the lagging strand together. ​ DNA proofreading ○​ All cells perform a post-replication “check”, known as proofreading, to confirm that no such mutations have been introduced. ○​ DNA Polymerase II detects mismatched nitrogenous bases in the newly-replicated and template DNA strands, then uses its exonuclease subunit to remove the inappropriate nitrogenous base. This allows DNA Pol II to then add the correct nitrogenous base. ○​ In cases of more extreme mutations (i.e. those that result in a frameshift in how the DNA strand is read), ligase may need to be recruited to stitch together fragmented strands of the newly-replicated DNA molecule. ​ Types of mutations ○​ Substitution mutations- occur at a single nucleotide (and can result in Single Nucleotide Polymorphisms, or SNPs), generally the most innocuous as they do not cause a significant shift in the reading frame of the DNA molecule ○​ Frameshift Mutations- result in at least one nucleotide shift in the reading frame of the DNA strand, which can have devastating effects on all the downstream genes from the site of the mutation. There are two subtypes of frameshift mutations: ​ Insertion Mutations occur when a new nucleotide is inappropriately inserted into the replicated DNA strand ​ Deletion Mutations occur when a nucleotide has been inappropriately removed from the replicated DNA strand Transcription ​ First portion of the Central Dogma, transcription is the process of gene expression. ​ In eukaryotic cells, this takes place in the nucleus (as well as post-transcriptional modifications). ​ In prokaryotic cells, this takes place in the cytosol.  ​ DNA exists as a double helix in both cell types, but is linear in eukaryotic cells and circular in prokaryotic cells. ​ 3 phases ○​ Initiation- recruits helicase, SSBs, and topoisomerase to open the DNA double helix to form the transcription bubble in which transcription will begin. Transcription will proceed in the 5 ‘ → 3’ direction. However, unlike replication, only one DNA strand is necessary to serve as the template for synthesizing the mRNA transcript and thus, only the leading strand of the DNA double helix is utilized, mitigating the need for ligase fusing. ○​ Elongation- occurs once all the necessary transcription factors (in the case of eukaryotic transcription) have bound to the promoter region of the DNA, allowing RNA Polymerase to bind and begin synthesizing the RNA transcript. ○​ Termination- occurs once the termination sequence of the DNA has been reached, triggering the dissociation of RNA Polymerase from the DNA and the release of the pre-mRNA transcript. As we will see on the next two slides (and this is the case in eukaryotic cells only), several modifications must be made to the mRNA transcript before it can be used for translation. ​ RNA splicing ○​ Prior to splicing, strand of RNA is known as pre-RNA ○​ Before the pre-mRNA transcript has been adorned with the 3’ poly-A tail and 5’ Guanosine (G)-cap, the non-coding regions of the transcript, known as the introns, are excised by an enzyme complex known as the spliceosome. ○​ The remaining sections of the now mature mRNA transcript are known as exons, and code for proteins necessary for normal cellular functioning. Translation ​ Second portion of the Central Dogma, translation is the process of protein synthesis. ​ In both eukaryotic and prokaryotic cells, this process takes place at the ribosome. ​ However, in the case of eukaryotic cells, there are two locations where ribosomes are found: ​ Floating freely in the cytosol ○​ Ex: The enzyme caspase plays an essential role in the eukaryotic apoptotic (programmed cell death) pathway, and typically exists at relatively constant levels within most eukaryotic cells. Given the function and location of caspase within the cell, where would you expect translation of caspase to occur and why? Given that caspase is an enzyme that remains within the cell (i.e. does not become embedded in the plasma membrane or exit the cell via exocytosis), you would expect translation of caspase to occur at a free-floating (or cytosolic) ribosome. This is because there is no need to package the enzyme into a vesicle via the endomembrane system for transport to the cell membrane or out of the cell entirely. ​ Embedded in the (rough) endoplasmic reticulum ​ Molecular and genetic methods Cell respiration Glycolysis & anaerobic respiration The Krebs cycle Oxidative phosphorylation ----------------------------- Exam 1 -----------------------------