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Biology 1202B Final: Biology 1202 Final Cycle Outcomes (ALL)

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Western University
Biology 1202B
Patrick Mc Donald

Biology 1202 Cycle Outcomes list Has anyone made a layout of how everything interconnects? We should make one connecting the concepts in test 1, test 2, and the final -aint nobuddie got time fa that Cycle 1 Membrane Transport and Signalling Describe the structure and common components of eukaryotic membranes Phospholipids - the membrane is composed of lipid molecules that form a lipid bilayer - hydrophilic head - polar (like water) - hydrophobic tail - non-polar - phospholipids are amphipathic - meaning they have different regions of polarity - lipid molecules can vibrate, flex back and forth, spin around on their axis, move sideways, and exchange places on their bilayer half - very rare for lipid to exchange places between the two halves - membrane is less than 10nm thick - selectively permeable Proteins - transport, attachment, signal transduction, enzyme activity - much larger than lipids - therefore they move slower than lipids - most proteins move within the membrane, but few anchor cytoskeleton filaments to membrane and do not move - Integral Protein: - embedded in bilayer - interacts with aqueous (interior and exterior of cell) and the core - polar outside & inside - non-polar core - distinct domains of polarity - Peripheral Protein - surface of membrane - no interactions with hydrophobic core - proportions of lipids and proteins vary depending on the membrane type. - membrane asymmetry: components of each lipid bilayer half are different - each half have different functions +- proteins and lipids can have carbohydrates attached to them (glycoproteins and glycolipids) Describe how the “fluid mosaic model” fits what is known about the plasma membrane with respect to membrane structure/characteristics Membrane Fluidity - fluid mosaic model proposes that membranes are not rigid with molecules locked into place, but rather consists of proteins that move around within a mixture of lipid molecule that has the consistency of olive oil - we know this to be true because the membrane has proteins that move around - also, the lipid molecules can move and they can be fluid in different temperatures and with different levels of saturation → mosaic refers to the fact that most membranes contain an assortment of types of proteins (transport, signaling, other processes). These proteins are a lot larger so they move slow. -integral proteins - suspended individually in fluid lipid bilayer -peripheral proteins- are attached to integral; proteins or membrane lipids mostly on the cytoplasmic side of membrane -carbohydrate groups of membrane glycoproteins and glycolipids face the exterior ~Experimental evidence of fluid mosaic model is the FRYE-EDDIN experiment (human and mouse cells grown separately in tissue cultures, each were tagged with dye (humans-red, mouse-green) and the cells were then fused together and within minutes they began to intermix, indicating that they had moved around the membrane. Membrane Asymmetry - Freeze Fracture Technique - a block of cells is rapidly frozen in liquid nitrogen - block is fractured with knife splitting the membrane in half and we can see the asymmetry of the cell - each side of the cell performs different functions Describe the factors affecting membrane fluidity 1. Ratios of different types of lipids -Some types of phospholipids make the membrane more fluid than others -Cells can alter the ratios quickly -Different organelles have diff. ratios of lipids 2. Saturated vs. Unsaturated phospholipids SATURATED: all carbons have single covalent bonds w/ other carbons and hydrogens. Fatty acid tails are very hydrophobic and interact tightly w/ each other to avoid water = saturated= membrane rigid and viscous UNSATURATED: contain 1 or more double C=C bonds Double bonds in unsaturated fatty acid tails put kinks (can't pack tightly together) = unsaturated = membrane is more fluid NOTE: organisms can alter their fatty acid compositions through desaturases - desaturases remove 2 hydrogen atoms from a neighbouring carbon causing a carbon-carbon double bond to occur (making the lipid unsaturated, making the membrane more fluid) - desaturases abundance is mediated by gene level transcription - growth temp. decreases, desaturase transcription increases 3. Amount of cholesterol It inserts itself in between the fatty acid tails of the bilayer. - At high concentrations, cholesterol blocks phospholipids from moving laterally (decreases fluidity) - At low concentrations, prevents phospholipid tails from packing in tightly (increases fluidity) 4. Temperature- - high temperatures increases random molecular motion, which in turn increases fluidity - this can also result in the membrane becoming too fluid and losing its structure - ions diffuse too freely, which leads to an ion imbalance causing rapid cell death - sterols restrain lateral movement of molecules in high temperatures - low temperatures decreases molecular motion, which in turn decreased fluidity - fluidity is lost and a semisolid gel is formed from the phospholipids at their “gelling point” - the more unsaturated molecules there are, the lower the temp. that gelling occurs - sterols disrupt the fatty acids from associating in low temps, slowing the gelling process Predict the effects on the membrane/cell properties with changes in membrane fluidity - the membrane can burst if penetrated or if a cell takes in too much water. - increased fluidity results in decreased regulation of diffusion, more substances can diffuse across the membrane - cell death can occur from ion imbalance if the cell membrane loses it temperature - rapid cell death can occur if diffusion is unregulated and lysis occurs from a hydrophobic solution, Describe the major functions of the eukaryotic plasma membrane - marks the boundary line between life and non life, the structure keeps the content of the cell away from the environment surrounding it the plasma membrane has cholesterol molecules and proteins that allow the membrane to function properly. Cholesterol molecules are primarily responsible for giving the membrane the rigidity it needs to hold the cell’s shape. Proteins help the cell communicate with the external environment - two types of proteins, peripheral (cellular communication) and integral (transport of material) proteins - anchors for proteins and cytoskeleton - cell identification Determine if a particular substance (including gases, ions, small organic molecules, large organic molecules and water) could cross a simple phospholipid bilayer by simple diffusion Substance Able to cross a simple phospholipid bilayer by simple diffusion Small uncharged Molecules (Water & GlycerolEven though water is polar, it is still able to move quite rapidly across the membrane Small non polar molecules (O & CO ) Move very rapidly from one side to another 2 2 Steroid hormones & drugs that tend to be Can readily transit the lipid bilayer amphipathic Charged Molecules (Cl-, Na+, etc,.) The membrane is partially impermeable to charged molecules. Compare and contrast passive and active transport across eukaryotic membrane, providing and/or identifying) example of different transport mechanisms Identify the type of transport that is occurring across a membrane, provided information about the substance, relative concentrations on either side of a selectively permeable membrane, presence/absence of proteins involved in transport and energy requirements Predict the flow of a particular substance (e.g. gas, ion, small or large organic molecule, water) along its gradient (concentration and/or electrochemical) provided information about relative concentrations on either side of a selectively permeable membrane Predict the effects on cellular processes/characteristics if a particular membrane component is defective/damaged List and briefly describe the three major steps present in most signal pathways Most signal pathways involve the following three steps: 1. Reception. The binding of a signal molecule (a ligand) with a specific receptor of target cells is known as reception. Target cells have receptors that are specific for the signal molecule, which distinguishes them from cells that do not respond to the signal molecule. Most receptors are found on the outer plasma membrane, but some are found on internal membranes such as the ER. In addition, other receptors are soluble proteins that are found in the cytoplasm. If a signal molecule were to enter the cytoplasm itself, there is no effect, it is the binding that sends the signal. 2. Transduction. The process whereby signal reception triggers other changes within the cell necessary to cause the cellular response is transduction. Transduction typically involves a cascade of reactions that include several different molecules, referred to as a signalling cascade. The more steps in the signal transduction, the more potential there is for signal amplification. 3. Response. In the third and last stage, the transduced signal causes a specific cellular response. Different signalling pathways lead to different downstream responses. For example, some signal transduction pathways lead to the direct activation of a specific enzyme, while others often trigger changes in gene expression. Cycle 2 Energy and Enzymes Compare and contrast the states of kinetic energy and potential energy ● Kinetic Energy: Energy possessed by an object because it is in motion (rock falling from a cliff) ● Potential Energy: Stored energy, the energy an object has because of its position or chemical structure (ball on the edge of a cliff) Compare and contrast energy exchange in isolated, closed and open systems ● Isolated Systems: One that does not exchange matter or energy with its surroundings, the only true isolated system is the universe itself ● Closed Systems: Can exchange energy, but not matter, with its surroundings, Earth is considered to be a closed system ● Open Systems: Both energy and matter can move freely between the system and the surroundings Distinguish between exergonic and endergonic reactions ● Exergonic reactions are reactions that release free energy (ΔG<0)6 The Free energy from reactants to products decreases ie: Cellular respiration(Opposite of Photosynthesis) ● Endergonic are reactions that require a net input of free energy(ΔG>0) The free energy from reactants to products increases ie: Photosynthesis; it needs photons of light energy from the sun Determine, provided adequate information (eg. ∆G) if a particular reaction will occur spontaneously The reaction occurs spontaneously if deltaG is negative. It is enthalpically driven if the change in enthalpy is negative, and entropically driven if the change in entropy is positive negative ∆H,, and positive ∆S = Spontaneous Compare and contrast catabolic and anabolic pathway reactions Catabolic reactions: A series of chemical reactions that break down larger more complex molecules into smaller ones. (exergonic- release energy) (-deltaG) Anabolic reactions: A series of reaction that results in the synthesis of larger more complex molecules from simpler starting molecules. (endergonic- consume energy) (+deltaG) Describe how cells use ATP to couple reactions ATP breaks down to ADP passes its extra phosphorus to a molecule, increasing its energy. Any enzyme or another molecule that interacts with this phosphorus, finishing the reaction. Describe the role of enzymes in biological reactions Enzymes reduce the activation energy which increases the reaction rate for a variety of reactions . They do not change the spontaneity, enthalpy, or free energy change between the reactions and products. They speed up the rate of reaction by allowing the reaction to reach the threshold quicker, it is going to reach it eventually on its own but it may take a lot longer without the enzyme Explain how enzymes serve as catalysts for biological reactions, listing mechanism that can contribute to catalysis They increase the likelihood of a reaction occurring by making the substrates unstable and want to react, and/or positioning them in a situation where they are most likely to react. They can: -act as an acid or base, donating or removing hydrogen cations from the substance -facilitate a redox reaction to oxidize or reduce the substrate(s) -use their functional groups to stretch or compress the bonds in a substrate, and -position substrates in the best position for them to react List and describe the factors that affect enzyme activity temp, pH, substrate concentration, enzyme concentration Explain how and why non-optimal conditions (eg. temperatures, pH) affect enzyme activity An enzyme has an optimal PH at which it is most active. At PH values above or below the optimum the rate of enzyme activity drops. As temp rises the rate of the chemical reactions typically increases. As the temp rises the rate of catalyzed reaction increases until the temperature reaches a point at which the enzyme begins to denature. The rate drops off steeply as denaturation progresses and becomes complete Explain how reaction rates and enzyme activity can be regulated - Regulation is achieved through inhibition - INHIBITING AN ENZYME WILL REDUCE THE RATE OF REACTION Predict the effect on enzyme activity, given information about a particular enzyme, reaction conditions and other factors Cycle 3 Photosynthesis Explain how the structure of a pigment relates to how the colour is observed The conjugated structure of the C-H bonds (alternating double bonds and single bonds) allows pigments to obtain an ability to absorb different wavelengths. The wavelengths absorbed provide energy for the photosystems and the wavelengths reflected and not absorbed provide colour. -colour is the light the pigment does not absorb! (i.e. a yellow shirt does NOT absorb yellow wavelengths) Describe the potential fates of a photon when it encounters matter and explain how one of the possible fates has key biological significance 1. Reflected off the object a. this is the colour that we see 2. Transmitted through the object 3. Absorbed (biological significance as this is the only way the energy from the photons excites the electrons and results in photosynthesis) Describe, in general terms, how the products of photosynthesis sustain most organisms on Earth Photosynthesis produces sugar and oxygen for organisms to survive off gvt. Photosynthetic organisms are near the bottom of the food chain, and many higher organisms need them as a food source List and describe the two parts/stages of oxygenic photosynthesis in general terms HELP! Is this referring to photorespiration? If so, O2 + RuBP → 2 carbon molecule released as CO2 and 3PGA I think it’s asking to explain the light-dependent reactions and then the light-independent (calvin cycle) Light reactions: capture of light energy by pigment molecule and the utilization of that energy to synthesize both NADPH and ATP. The e- needed to reduce NADP+ come from the oxidation of H2O, which releases O2. Calvin cycle: the e- and protons carried by NADPH and the energy of ATP hydrolysis are used to convert CO2 into carbohydrate. This reduction reaction is referred to as carbon fixation, with electrons and protons being added to CO2 Explain how the membranes/compartments of chloroplast (structure) relate to the processes of photosynthesis (function) Thylakoid membranes are used to form a proton charge and concentration difference, or ‘membrane potential.’ This potential causes protons that were previously pumped into the thylakoid membrane to diffuse out of it through ATP synthase channels. Its diffusion energized the creation of ATP. ATP is a fuel for the Calvin Cycle. Explain why there are different pigments in an antenna complex Pigments are used for energy from the light photon to travel through the antenna complex through a series of redox reactions from pigment to pigment molecule in the antenna complex. Describe the possible fates of an excited-state electron in a pigment molecule 1. The excited electron from the pigment molecule returns to its ground state, releasing its energy either as heat or as an emission of light of a longer wavelength—a process called fluorescence. 2. The energy of the excited electron (but not the electron itself) is transferred to a neighbouring pigment molecule. This transfer of energy excites an electron in the second molecule, while the electron in the first pigment molecule returns to its ground state. Very little energy is lost in this energy transfer (Inductive resonance). 3. The excited electron is transferred from the pigment molecule to a nearby electron-accepting molecule. Relate the absorption spectrum of photosynthetic pigments in a given plant with the action spectrum of photosynthesis in that plant You can determine the wavelength of light absorbed by a pigment such as chlorophyll a by producing an absorption spectrum for that pigment using a spectrophotometer and a pure sample of the pigment. An absorption spectrum is a plot of the absorption of light as a function of wavelength. Photosynthesis depends on the absorption of light by chlorophylls and carotenoids, acting in combination. This is supported by the action spectrum for photosynthesis. (often measured as amount of O2 produced). An action spectrum is the plot of effectiveness of light of particular wavelengths in driving a process. Describe, in general terms, what occurs in light-dependent reactions of photosynthesis (including energy investments, energy yields, oxygen, water and carbon inputs/outputs). Describe the basic structure, components, and function of photosystems, including the roles of PSI and PSII in the light-dependent reactions. Describe how ATP is produced in the light-dependent reactions. Proton movement by the reduction-oxidation of plastoquinone creates a concentration gradient of H+ (a proton-motive force) across the thylakoid membrane. The gradient is dissipated as H+ diffuse back into the stroma through the ATP synthase complex, which drives the synthesis of ATP from ADP and Pi Explain the role of light for electron transfer in photosystems. Light supplies energy from which the electron can be transferred Compare and contrast linear electron transport and cyclic electron transport. - Linear: Produces ATP and NADPH, and involves all protein complexes. - Cyclic: Produces ATP only, and does not involve PII or NADP+ reductase Describe, briefly, the following stages of the Calvin cycle: CO 2 fixation, production of G3P, and regeneration of RuBP. Describe, in general terms, what occurs in the light-independent reactions/Calvin cycle (including energy investments, yields, and carbon inputs/outputs). Predict the outcome in cellular respiration or photosynthesis if a particular key molecule (e.g., enzyme, organelle structure) is damaged/missing/inhibited. Explain how photorespiration is different from cellular respiration.p Photorespiration is different from cellular respiration due to to the fact that in photorespiration, RuBP accepts oxygen instead of carbon dioxide and in respiration, oxygen is produced in oxidative phosphorylation / chemiosmosis Describe the challenges faced by and strategies employed by aquatic photosynthesizers in terms of obtaining enough CO 2. In aquatic photoautotrophs, an ATP-dependent process pumps bicarbonate (HCO3-) into the cell, which is converted to CO2 through the action of the enzyme carbonic anhydrase. This makes the CO2/O2 ratio greater at the site of Rubisco and out-competes the o2 present for the active site of Rubisco Describe how the C3, C4, and CAM pathways of carbon fixation differ, indicating the advantages and disadvantages of each pathway. Cycle 4 Cellular Respiration Explain why catabolism of molecules such as glucose is carried out in a stepwise manner in living organisms. By doing it in steps the organisms are able to efficient storage the energy, whereas if all the energy was released at once, barely any energy would be stored. So that the energy of the molecule can be used to do work List and describe the three parts/stages of cellular respiration Glycolysis: glucose is broken down into two pyruvate molecules, net gain of 2 ATP and 2 NADH, occurs when there is no oxygen and when there is oxygen Pyruvate Oxidation: Carboxyl group removed as CO2, pyruvate oxidized by NADH to become acetate, CoA binds to acetate to produce Acetyl-CoA, 2 NADH gained per glucose Citric Acid Cycle: Acetyl-CoA oxidized into two carbon dioxide molecules. 6 NADH, 2FADH2, 2ATP made per glucose Chemiosmosis: NADH and FADH2 oxidized to form ATP Explain how the membranes/compartments of mitochondria (structure) relate to the processes of cellular respiration (function). matrix is where citric acid cycle occurs inner mitochondrial membrane is where ETC/oxidative phosphorylation occurs protons are pumped into intermembrane space during chemiosmosis Describe, in general terms, what occurs in glycolysis (including energy investments, energy yields, and carbon inputs/outputs). glucose is initially given energy, so it is unstable and splits into two three carbon molecules. This is a coupled endergonic reaction. those two molecules, eventually both G3P, are oxidized and react with ADP to take away their high energy electrons Explain what is meant by “glycolysis is an ancient pathway.” Glycolysis is universal, being found in all three domains of life. 1. Glycolysis doesn’t depend on the presence of oxygen gas. Oxygen gas is a relatively new thing in the development of life on earth. 2. Glycolysis occurs in the cytosol using soluble enzymes and does not require any sophisticated process like ETC. Describe the transition from glycolysis to the citric acid cycle. Pyruvate oxidation ● Carboxyl from pyruvate is removed as CO2 ● The 2 carbons left in pyruvate are oxidized and electrons are transferred to NADH Describe, in general terms, what occurs in the citric acid cycle (including energy investments, energy yields, and carbon inputs/outputs). Acetyl-CoA is oxidized a lot until it’s carbon atoms have the energy of carbon in CO2, which is low compared to other organic molecules citrate is regenerated to form oxaloacetate ATP is made through substrate level phosphorylation Describe what occurs in the respiratory electron transport chain and oxidative phosphorylation. Electrons spontaneously pass down a chain of increasing electronegativity facilitated by enzymes and electron carriers. While this happens, their energy is used to do work - by actively transporting protons, in the form of hydrogen cations, across the inner mitochondrial membrane into the intermembrane space. In oxidative phosphorylation, oxygen acts as a terminal electron acceptor to bond with the cations and form water, which are diffusing through ATP synthase in facilitated diffusion Compare and contrast substrate-level phosphorylation and oxidative phosphorylation. Substrate level: (relatively) direct formation of ATP from ADP and Pi - using an enzyme Oxidative phosphorylation: indirect, there are lots of things going on that ultimately lead to the formation of ATP down the road - Also by means of an ETC oxidizing energy rich molecules NADH = 3 ATP FADH2 = 2 ATP Describe how ATP synthase produces ATP via chemiosmosis. Think of this like a secondary active transport symport, or cotransport, coupled reaction. H+ ions are moving along their concentration gradient. Their passing energy is used to do work, which turns a motor, the smallest known to man, that somehow creates ATP from ADP and phosphate… what? Now that’s some complex stuff! Explain what is meant when it is said that electron transport and oxidative phosphorylation are coupled processes. Neither would occur without the other. They both need each other, and highlight each other’s strengths… kind of like a couple Oxygen is great at absorbing electrons, while the electron transport chain is great at pumping protons against their concentration gradient in a way that can create energy for ATP synthase The generation of ATP by the ATP synthase complex is coupled (linked) to electron transport by the proton gradient established across the inner mitochondrial membrane. Explain the role of oxygen as the terminal electron acceptor in aerobic respiration and the effects on the pathways of aerobic respiration if oxygen is not available. Oxygen is the terminal electron acceptor. It gets the electrons, now all used up, out of the way so the next ones can pass through. Think of it like a patrol that helps clear a traffic jam by moving cars out of the way If it is unavailable, there will be nothing to take the electrons away from complex IV and traffic gets backed up, eventually stopping the process. Describe the efficiency of cellular respiration compared to other types of fuel combustion (considering the estimated energy yield from a molecule of glucose). 38 is the maximum theoretical yield, true in bacteria but in eukaryotic cells the theoretical max is 36 ATP. This is due the energy cost in transporting the NADH generated from glycolysis into mitochondrion (consumes 1 ATP for each NADH molecule x2). Secondly this is due to the fact that electron transport and oxidative phosphorylation are rarely completely coupled to each other. (Inner membrane can be leaky to protons.) Third reason is that the proton-motive force is used for other things besides generating ATP (ex. Moving pyruvate into matrix.) Phosphorylation of ADP to ATP requires 7.3kcal/mol, 36 moles max in eukaryotes. 36 x 7.3 = 263kcal of energy. The energy released by glucose is 686kcal/mol. 263/686 x 100 = 38% therefore 38% of the energy in glucose is converted into ATP. Determine the activity level of phosphofructokinase given the presence/levels of ATP, citrate, and ADP. ● ATP = allosteric inhibitor of the enzyme. Too much ATP in cytosol = binding to phosphofructokinase= inhibition= stopping glycolysis respiration ● ADP which accumulates when ATP is being consumed for metabolism= allosteric activator for the enzyme ● Increased levels of Citrate = demand for ATP is low (this may occur when O2 is low) Overall, the functional state of glycolysis and the citric acid cycle can be activated and inhibited which regulates the first two parts of cellular respiration. Compare and contrast catabolism of carbohydrates, fats, and proteins. They enter at different points. Proteins, once converted into amino acids, can enter as pyruvate, acetyl- CoA, or right into the cycle at some intermediate. Fats are great at turning into G3P (if glycerol), and entering glycolysis, right about at the energy payoff phase… nice. Fatty acids can be oxidized in their own pathway and enter as acetyl-CoA. Carbs are broken down into monosaccharides such as fructose or glucose and enter right into glycolysis. Nucleic acids? We don’t metabolize that stuff… at least we haven’t learned that yet Describe how anabolic reactions relate to cellular respiration pathways/ intermediates. Anabolic - small stuff get together to form big stuff. For example, the calvin cycle( whatt?????) w gets small stuff - CO2, into big stuff - glucose. This requires energy, for the free energy of the reactants are higher than the products, and it is not spontaneous. Catabolic make energy by turning large stuff into small stuff. This stuff is spontaneous if it overcomes the activation energy required for it to be…. like lighting a log on fire (that’s glucose burning). But, in a tightly-controlled highly sophisticated system, like eukaryotic cells, this activation energy is not met, and the process occurs in more intermediates. Organic molecules are oxidized by cellular respiration, which is linked to the generation of ATP, these molecules are also the source of carbon atoms found in a wide range of essential molecules. Intermediates of glycolysis and citric acid cycle can be used to synthesize amino acids, fats, and the pyrimidine/purine bases needed for nucleic acid synthesis. Respiratory intermediates supple carbon backbones for array of hormones, growth factors, prosthetic groups and cofactors that are essential to cell function. Excess acetyl-CoA can be used to synthesize fatty acids needed for a range of cellular processes Explain how metabolism is possible in the absence of oxygen. Fermentation yo! Drink that fungus defence mechanism to create a biochemical toxin to other microbes… alcohol from yeast NADH is reduced by one of the products from glycolysis, so it can continue to do its job Compare and contrast aerobic respiration, fermentation, and anaerobic respiration. Aerobic respiration Fermentation Anaerobic Respiration Differences oxygen required oxygen not required oxygen not required - no citric acid cycle - different final acceptor than oxygen Similarities glycolysis Aerobic Respiration Anaerobic Respiration Fermentation Definition Uses oxygen (occurs Without oxygen; uses Production of energy in most cells) respiratory electron from food (sugars, Complete combustion transport chain but lipids and proteins) in does not use oxygen as the absence of oxygen electron acceptors (occurs in mostly prokaryotes) Incomplete combustion Stages Glycolysis, Citric acid Glycolysis, Citric acid Glycolysis, cycle, ETC cycle, ETC fermentation (does not involve citric acid cycle or ETC) Amount of energy High, 36-38 ATP Lower, between 32 and Lowest, 2 molecules released molecules 36 ATP molecules of ATP Sites of reactions Cytoplasm and Cytoplasm and Cytoplasm mitochondria mitochondria Products CO2, water and ATP CO2, reduced species, CO2 and ethanol or ATP Lactic acid (and ATP) Production of Doesn’t produce Produces ethanol or Produces ethanol or Ethanol or Lactic either lactic acid lactic acid Acid Cycle 5 - DNA Structure, Organization & Replication (chapter 12) Explain why an understanding of the structure of the DNA as the hereditary molecule was important in later related work. - Enabled scientists to understand key processes in cells for the first time in terms of structure and interaction of molecules - Model made it possible to understand how genetic information is stored in the structure of DNA and how DNA replicates - Relates genetic traits of living organisms to a universal molecular code present in the DNA of every cell Describe key points of the experiment carried out by Griffith that demonstrated a “transforming principle” that could change pneumococcal bacteria at the genetic level - Mice were injected with live S cells and other mice were injected with live R cells. The mice with the R cells lived and the ones with the S cell died. This knowledge was used for the control of the experiment - When mice were injected with the heat killed S cells the mice lived - but when mice were injected with the heat killed S cells and liveR cells the mice died die to the fact that in the heat killed S cell there must be a factor that was released causing the R cells to genetically change into the virulent S form. - He concluded and called the process of genetic change transformation and the molecule that caused the transformation the transforming principle. Describe key points of the experiment carried out by Avery, McCarty, and MacLeod identifying DNA as the “transforming principle.” - Designed an experiment to identify the chemical nature of the transforming principle that could change avirulent rough streptococcus to virulent smooth form - Used bacteria instead of mice o Used heat to kill virulent S bacteria o Extracted the macromolecules from the cell by using enzymes that broke down protein, DNA and RNA - Conclusion · Destroying RNA = no effect; there was still a transformation of R bacteria to virulent S bacteria · Destroying DNA = no transformation occurred - Summary: Avery experiment: used bacteria to test the transformation principle, they killed the S cell then used an enzyme to separately breakdown protein, DNA, nucleic acid/RNA o with destroyed DNA, there was no transformation ∴ DNA is genetic material Describe key points of the experiment carried out by Hershey and Chase that supported DNA as the hereditary molecule. 35 - Hershey and Chase: bacteria cells were infected with phages that had radioactive S in their protein or 32P in their DNA (phages invade a cell and use it to reproduce), cells infected by35S didn’t show radioactivity but cells infected with 32P show radioactivity 32 o DNA contains phosphorus ( P) and because DNA is the hereditary molecule, it was contained in the reproduced phages Identify the elements of the scientific method in each of the above experiments (i.e., hypothesis, positive control, negative control). - hypothesis: DNA is genetic material - positive control (genetic change/results expected): tests DNAs effects - negative control (expect no change): tests proteins effect Describe the relevance of Chargaff’s observations of base ratios in terms of what we know about DNA structure. - # of purine = # of pyrimidines - A=T, C=G - each nucleotide corresponds to their specific pair - Different species have different percent of each base pair Calculate the percentage of the other bases present in the strands, provided with the percentage of one particular base in DNA. - The base pairings in the strands are Adenine – Thymine and Cytosine –Guanine o All the base pairings account for 100% of our make-up o We know that ea. pair must have the same % amount o Let’s say there is 15% Thymine, we know that there must also be 15% Adenine 15% + 15% = 30% of the total 100% 100% - 30% = the total percentage of cytosine and guanine 70% = the total percentage of cytosine and guanine 70/2 = the percent of each pair Therefore 35% is the percent of Cytosine and Guanine separately Explain how the 5′ end of DNA differs from the 3′ end. DNA is made 5’-3’ but read 3’-5-’. The 3’ has a hydroxyl attached to it and it is the newest end of the newly made DNA, The 5’ end has a phosphate group and it is the oldest DNA since it was made first. List and describe the different bonds present in intact, double-stranded DNA. There are hydrogen bonds and covalent bonds present in double stranded DNA, the hydrogen bonds hold together the two nucleotide chains whereas the covalent bonds connect the individual strands together - H-bonds are between the bases phosphodiester bond: linkage of nucleotides in polynucleotide chains by a bridging phosphate group between the 5 carbon of one sugar and the 3 carbon of the next sugar Explain why Rosalind Franklin can be considered one of the discoverers of DNA structure, along with Watson and Crick. Franklin used X-ray diffraction to analyze DNA, the result DNA diffraction pattern was an X shape of spots separated at intervals of 0.34 and 3.4 nm, Franklin interpreted this as DNA having a helix structure (similar to spiral staircase) then watson and crick discovered the rest of their findings but unfortunately rosalind died before she could receive the nobel prize Describe the process of DNA replication, including the roles of major enzymes involved in particular steps. - First phase: Initiation o Helicase: separates the DNA; creates a y-shaped structure called the replication fork [uses ATP energy to unwind] o SSBP single strand binding protein; occurs after helicase. Prevents the DNA strands from rewinding together - Does this because polymerase can only make copies of a single strand o Primase: occurs after SSBP; puts down a small RNA primer so that polymerase has something to attach to (adds 3’OH to RNA molecules) o Polymerase: the final step. Lays down DNA o Topoisomerase: keeps the DNA from tangling as it unwinds by cutting the phosphate backbone and then putting it back together once it has detangled. - Second Phase: Elongation o Two things are occurring on the strands § On the leading strand: continuous synthesis à continuously adding nucleotides on to the 3’ OH end § On the lagging strand: discontinuous synthesis à okazaki fragment: short pieces of DNA are added to the 3’ to 5’ end o Then, DNA POLYMERASE III extends the chains of the leading and lagging strand o DNA POLYMERASE I: removes the RNA primers put in from the primase step § This is because it has a 5’ to 3’ exonuclease that allows it to pull out RNA while putting down DNA o Ligase: occurs after polymerase § Closes up the phosphate backbone after the RNA has been removed. - SUMMARY OF STEPS OF DNA REPLICATION: o Helicase to SSB to Primase to DNA Polymerase Predict and describe the effect on DNA replication if one or more of the key enzymes is missing/non- functional. - Missing helicase: the process wouldn’t be able to start because there would be no unwinding of the DNA - Missing Topoisomerase: the two DNA strands would get tangled after they are initially unwound - Missing SSB: DNA would be able to unwind but it would not be able to stay unwound - Missing primase: DNA polymerase won’t know where to start since the RNA primers, its starting signal, won’t be laid down - Missing DNA polymerase: no elongation; RNA would not be removed - Missing ligase: there would be seals left open in the phosphate backbone Describe the structure of telomeres and the role of telomerase in linear chromosome replication. - Telomeres: replications added onto DNA. (Their repeat formula is: 5’ – TTAGGG-3’)- Telomerase is needed to place the telomere repeat sequence to the 3’ end o Occurs after DNA has shortened o It is not used in bacteria because bacteria has a circular chromosome. Only active in embryonic and cancer cells ● Does not prevent chromosomes from shortening during a round of replication Explain why DNA repair mechanisms are necessary. - DNA repair mechanisms are necessary because it corrects base pair mismatches that aren’t noticed by proofreading o Base-pair mismatches are a mistake done by DNA polymerase - Can’t form hydrogen bonds if mismatched bases, distorts structure of DNA helix. If not fixed can cause mutations, differences in DNA sequence that appear and remain in replicated copies. Can alter property of protein encoded by the gene and can alter how organism functions Describe chromatin and the role of histones in eukaryotes. - Chromatin: structural building blocks of a chromosome - Histones are a way to organize DNA in eukaryotic cells; packs DNA into small parts of the cell nucleus o Two major types of proteins: the histone and non-histone proteins (collectively known as chromosomal proteins as they are associated with the structure of DNA and regulation in the nucleus) o Histones: positively charged proteins that can attract the negatively charged DNA (there are 5 types of histones: H1, H2A, H2B, H3, H4) - 10 nanometre fibres: looks like beads on a string (consists of two molecules each of H2A, H2B, H3, H4 [makes a total of 8 proteins]) note: the DNA winds around it twice - 30 nanometre fibres: H1 histone - H1 histone is not a part of the nucleosome - histones (+ve charge): coil DNA (-ve charge) into condensed chromatin (building block for chromosomes) o nucleosomes: DNA wrapped around histone octamer (2 histones) o H1 (histone) binds nucleosomes and linker DNA o tails are sites of chemical modification to remodel chromatin o heterochromatin: densely packed.(unavailable for activation) - Represents genes that have been turned off and placed into a compact storage form. (i.e. barr body) - this shows that Histones are also responsible for regulating gene activity, along with DNA organization. o euchromatin: loosely packed, for transcription (accessible) Cycle 6: Chap. 13 Transcription & Translation Describe the key points of the experiment carried out by Beadle and Tatum. · collected data showing a direct relationship between genes and enzymes. · wild-type Neurospora, grows on a medium consisting of inorganic salts, sucrose, vitamin A · Fungus uses only simple chemicals to synthesize more complex molecules needed for growth/reproduction (amino acids for proteins and nucleotides for DNA and bv RNA) · Exposed wild-type Neurospora, to x-ray (caused mutation) after some would not germinant unless medium had nutrients added to it · hypothesized that each auxotrophic strain had a defect in a gene coding for an enzyme needed to synthesize a nutrient that now had to be added to the MM. · by testing if mutant strain would grow with supplemented nutrient they discovered specific nutrients each mutant needed (therefore which gene defect it had) · Therefore, different arg mutants might have defects in different enzymes and therefore have blocks at different steps in the assembly line. ◦ IE: the argH mutant grows on arginine but not on of the other three compounds; this means that the mutant is blocked at the last step in the pathway, which produces arginine. Explain what is meant by the “one gene–one polypeptide hypothesis” and why this has replaced the “one gene–one protein hypothesis.” · Experiment (above) showed which gene encoded the enzyme carried out in each step (found direct relationship between gene and enzyme) · Protein structure now known to be much more complex (many proteins consist of more than one subunit, each is a separate molecule called polypeptide that is coded by separate gene) · Polypeptides can assemble to create a functional cluster of molecules called proteins thus the new name of one-gene-one-polypeptide Explain how the processes of transcription and translation differ between prokaryotes (i.e., bacteria) and eukaryotes. · Prokaryotes: can transcribe and translate a given gene simultaneously · Eukaryotes: transcribe and process mRNA in nucleus before exporting to cytoplasm for translation on ribosomes · They also have different mechanisms of termination in Transcription but otherwise, they are similar (13.2a) Transcription: - transcription in eukaryotes needs transcription factors for polymerase binding...prokaryotes polymerase binds directly Termination... eukaryotes have no “transcription terminator.” They transcribe beyond the end of the gene. 1. - near the 3’ end of the gene, the DNA is transcribed into pre-mRNA. 2. - this creates a polyadenylation signal in RNA and cleaves it farther down the gene 3. - this signals the RNA polymerase to stop transcription 4. then poly a polymerase adds a chain of 50-250 adenine nucleotides to the 3’ end of the pre- mRNA (poly a tail) prokaryotes terminate in hairpin loop (terminator sequence base pairs with itself in the mRNA) or a protein binds to the terminator sequence on the mRNA Transcription: - Initiation = differs - Elongation = same - Termination = differs Explain how each base in DNA/mRNA can be considered a letter of an “alphabet” and how these are used to make “words” in the genetic code. · Straightforward: consist of 4 letters A T G C (DNA bases) and rna alphabet A U G C · 4 RNA bases but 20 amino acids, nucleotide information that specifies amino acid sequence of polypeptide is called genetic code · Genetic code is 3 letter codon, genetic information in DNA is transcribed into complementary 3 letter RNA codon, these can bind to ribosomes and cause tRNA with linked amino acid to bind to ribosome (13.1) Describe the key points of the experiment carried out by Nirenberg and Leder, establishing the identity of most of the codons. · Made 64 short mRNAs each consisting of different single codon. They added mRNAs one at a time to a mixture in test tube containing ribosomes and all different tRNAs, each linked to its own amino acid. · Idea was that each single codon mRNA would link to tRNA carrying amino acid corresponding to codon (worked for 61 of 64 codons allowing them to be assigned to amino acids definitively) Determine, provided with a genetic code table, the amino acid, initiator codon, or termination codon encoded by a given gene (DNA) sequence. · AUG-start/initiator codon · UAA, UAG, UGA- don't specify amino acids they are stop codons (nonsense or termination codons)act as periods · Only 2 are specified by single codon - Methionine and Tryptophan (all rest are represented by at least 2 possible 6) feature known as degeneracy Write sequences of DNA (template), mRNA, anticodon, and codon in appropriate directions (based on polarity). Relate the degeneracy (or redundancy) of the genetic code to the wobble hypothesis. · Wobble hypothesis: proposed that complete set of 61 sense codons can be read by fewer than 61 distinct tRNAs because of particular pairing properties of bases in anticodons. Pairing of anticodon with first two nucleotides of codon is always precise but anticodon has more flexibility in pairing with the third nucleotide of the codon · Special purine “inosine” allows “wobble” by allowing the tRNA to pair with codons that have one of U C an A in third position (degeneracy idea) Explain the universality of the genetic code. · With few exceptions, the same codons specify the same amino acids in all living organisms and even in viruses · Also indicates that it was established very long ago (evolution has remained unchanged) Explain what is meant by the “reading frame” of mRNA. · Only one correct reading frame for each mRNA, code can be read correctly only by starting at the right place (at start codon and reading 3 nucleotides at a time) Describe the basic structure of a gene. · Gene consist of two main parts promoter (control sequence for transcription) and transcription unit (the section of gene that Is copied into RNA molecule) Describe the process of transcription, including the steps and key proteins involved. · 1)Initiation- the molecular machinery that carries out transcription assembles at the promoter and begins synthesizing an RNA copy of the gene · 2)Elongation- , in which the RNA polymerase moves along the gene extending the RNA chain · 3)Termination - in which transcription ends and the RNA molecule—the transcript—and the RNA polymerase are released from the DNA template. Compare and contrast the processes of DNA replication and transcription vs. RNA/mRNA •for a given gene, only one of the two DNA nucleotide strands acts as a template for synthesis of a complementary copy, instead of both, as in replication. •only a relatively small part of a DNA molecule—the sequence encoding a single gene—serves as a template, rather than all of both strands, as in DNA replication. •RNA polymerases catalyze the assembly of nucleotides into an RNA strand, rather than the DNA polymerases that catalyze replication. •the RNA molecules resulting from transcription are single polynucleotide chains, not double ones, as in DNA replication. •wherever adenine appears in the DNA template chain, a uracil is matched to it in the RNA transcript instead of thymine, as in DNA replication. Describe the different eukaryotic RNA polymerases in terms of their functions. In eukaryotes, RNA polymerase II transcribes protein-coding genes to yield mRNA RNA polymerase III transcribes tRNA genes and the gene for one of the four rRNAs, and RNA polymerase I transcribes the genes for the three other rRNAs. Determine the sequence of mRNA transcribed from a given DNA template or coding strand sequence. -mRNA is written 5’-->3’ (use antisense of DNA and just replace T with U) Describe the posttranscriptional modifications that occur in the production of eukaryotic mRNAs. -At the 5’ end of the pre-mRNA is the 5’ guanine cap, consisting of a guanine- containing nucleotide that is reversed so that its 3’ −OH group faces the beginning rather than the end of the molecule. A capping enzyme adds this 5’ cap to the pre- mRNA at the beginning of transcription -the enzyme poly(A) polymerase adds a chain of 50 to 250 adenine nucleotides, one nucleotide at a time, to the newly created 3’ end of the pre-mRNA. The string of adenine nucleotides, called the poly (A) tail, enables the mRNA produced from the pre-mRNA to be translated efficiently and protects it from attack by RNA-digesting enzymes in the cytoplasm. -mRNA splicing in the nucleus (by ribonucleoprotein snRNP’s “snurps” joined together in a spliceosome) removes introns from pre-mRNAs and joins exons together Describe a spliceosome in terms of composition and function -a spliceosome, a complex formed between the pre-mRNA and a handful of small ribonucleoprotein particles (a complex of RNA and proteins, called snRNPs). --Complementary base-pairing between regions of snRNA and mRNA ensures that the cutting and splicing are so exact that not a single base of an intron is retained in the finished mRNA, nor is a single base removed from the exons. Without this, reading frames would be changed and the wrong codons would be produced. Explain how introns contribute to variability of proteins encoded by the same gene. --introns may provide a selective advantage to organisms by increasing the coding capacity of existing genes through a process called alternative splicing and in a process that generates new proteins called exon shuffling. - different introns can be removed in each mRNA = increase in gene coding List and describe the roles of different forms of RNA in converting the information in a gene to synthesize a protein. ○ mRNA – messenger RNA, encodes amino acid sequence of a polypeptide ○ tRNA – transfer RNA; brings amino acids to ribosomes during translation ○ rRNA – ribosomal RNA; with ribosomal proteins, makes up the ribosomes ○ snRNA – Small nuclear RNA; with proteins, forms complexes that are used in RNA processing in eukaryotes Describe the process of translation, including the key proteins and nucleic acids involved. Describe how proteins can be trafficked to different cellular locations. Three destinations: 1. cytosol → happens directly from ribosomes 2. endo-membrane system → uses signal sequences near the N-terminal end (proteins going into the ER) → this is considered cotranslational import 3. other membrane bound organelles (mitochondria, chloroplast, microbodies, nucleus)→ uses transport sequences and nuclear localization signals (for proteins going to the nucleus) → this is considered posttranslational import Identify the type of point mutation that has occurred, provided with original and new sequences and a genetic code table. Determine the potential impact of each type of mutation on the final protein product (phenotype). · Base-Pair substitution mutation- involves change of one base to another in genetic material; this will cause a change in base in a codon in mRNA (protein can still potentially work properly) · Missense mutation- mutation alters codon to specify a different amino acid; resulting in protein will have a different amino acid sequence (depends on which amino acid it is changed to and what it was originally) · Nonsense mutation- mutation changes sense (amino acid coding) codon to nonsense (termination) codon in mRNA; this results in premature stop and a short than normal polypeptide ★ Silent mutation - does not alter amino acid · Frameshift mutation: single base pair deleted or inserted in coding region results in reading frame of resulting mRNA altered ( after that point ribosome reads codons that are not the same as for the normal mRNA typically producing completely different amino acid sequence; resulting in nonfunctional amino acid sequence Explain how phenotype is related to genotype at a molecular level. - Genotypes determine the phenotype Explain why most mutations in a multicellular organism do not result in phenotypic change. - wobble / redundancy - some codons can stand to be changed by a single base and not have any negative effect; some changes may even result in the same amino acid CYCLE 7: Regulation of Gene Expression 1. Describe the phenomenon and function of “differential gene expression” in general terms. - So basically the number of genes doesn’t determine the complexity and the differences between organisms; how the gene is EXPRESSED is the determinant 2. Compare and contrast the regulation of gene expression in E. coli for the lac operon versus the trp operon. LAC Operon (prokaryotes) ● Metabolism of lactose as an energy source involves three genes adjacent to one another on the chromosome in the order; lacZ, lacY, and lacA. ● These genes are transcribed as a unit into a single mRNA ○ 1. The lacZ gene encodes the enzyme B- Galactosidase, which catalyses the conversion of the disaccharide sugar, lactose, into the monosaccharide sugars, glucose and galactose. ○ 2. The lacY gene encodes a permease enzyme that transports lactose actively into the cell. ○ 3. The lacA gene encodes a transacetylase enzyme, the function of which is more relevant to metabolism of compounds other than lactose. ● When lactose is added to the medium, the lac operon is turned on and all three enzymes are synthesized rapidly. This occurs by lactose entering the cell and low levels of B-galactosidase molecules present covert some of it to allolactose, an isomer of lactose. Allolactose is an inducer for the lac operon. It binds to the Lac repressor, altering its shape so that the repressor can no longer bind to the operator DNA. Now RNA polymerase is able to freely bind to the promoter and transcribe the three genes at a dramatically elevated rate ● Lac operon is called an inducible operon ● The lac repressor protein, encoded by LacI gene, is expressed in the absence or presence of lactose ● In the absence of lactose, the lac repressor binds to the lac operator site. ● Repressor binding to the operator blocks progression of RNA polymerase, like a DNA roadblock. ● Since RNA polymerase is unable to transcribe the lac structural genes, the corresponding proteins are not made. ● Lactose: ABSENT —-> Result: lac operon repressed ● When lactose is present in the cell medium, it binds to the allosteric site of the lac repressor. This changes the conformation of the repressor. ● In this conformation, the repressor can no longer bind to the lac operator site. ● Without the repressor blocking its way, RNA polymerase is able to transcribe the structural genes. ● When lactose is present the lac structural genes are expressed. The proteins encoded by the Z and Y genes are required for the metabolism of lactose. ● The lac operon is controlled by a regulatory protein known as the lac repressor. The lac repressor is encoded by regulatory gene lacl, and is synthesized in its active form. There is always a low concentration of lac operon gene products in the cell. TRP Operon (prokaryotes) ● There are five genes in this operon, trpA to trpE, that encode the enzymes for the steps in the tryptophan biosynthesis pathway. Expression of the trp operon is controlled by the TRp repressor, a regulatory protein encoded by the trpR gene. ● When tryptophan is absent from the medium and must be made by the cell, the trp operon genes are expressed. (default state) Since the trp repressor is inactive and cannot bind to the operator, RNA polymerase can bind to the promoter and transcribe the operon. The resulting mRNA is translated to produce the five tryptophan biosynthetic enzymes that catalyze the reaction for tryptophan synthesis. ● If tryptophan is present, there is no need for the cell to make it, so the trp operon is shut off. This occurs because the tryptophan entering the cell binds to the Trp repressor and activates it. The active Trp repressor then binds to the operator of the trip operon and blocks RNA polymerase from binding to the promoter. ● Trp operon is an example of a repressible operon. Contrast ● In the trp operon, the repressor is synthesized in an inactive form. ● In the lac operon, the repressor is synthesized in an active form. When the inducer (allolactose) is present, it binds to the repressor and inactivates it. Describe the DNA binding domain of a regulatory protein and why such domains are present in proteins involved in regulation of gene expression. Regulation of gene expression in prokaryotic cells occurs primarily at the transcriptional level Eukaryotes, coordinated synthesis of proteins with related functions also occurs but without the need to organize the genes under the control of a single promoter in an operon (gene expression in eukaryotes is more complex. it involves promoter proximal regions, promoter proximal enhancer, enhancers etc…) - Short term regulation: gene sets are quickly turned on or off in response to changes in environmental or physiological conditions (most similar to prokaryotic) - Long term regulation: multicellular eukaryotes involve changes in gene expression that are associated with the development and differentiation of an organism Identify the outcome of specific situations discussed in this chapter given particular conditions (e.g., expression of lac operon in E. coli grown in medium containing glucose and lactose). Lac Operon LACTOSE PRESENT, GLUCOSE ABSENT: 1. Lactose converted to allolactose (inducer) which inactivates lac repressor 2. Active adenylyl cyclase synthesizes cAMP to high levels. cAMP binds to activator CAP, activating it. Activated CAP binds to CAP site in the promoter 3. RNA polymerase binds to promoter 4. Genes of operon are transcribed to high levels 5. Translation produces high amount of enzymes LACTOSE PRESENT, GLUCOSE PRESENT: 1. Lactose converted into allolactose, inactivates lac repressor 2. Breakdown of glucose leads to inactivation of adenylyl cyclase, amount of cAMP in cell drops to a level too low to activate CAP. Inactive CAP can’t bind to CAP site 3.RNA polymerase unable to bind to promoter 4. Transcription occurs at low rate, rate is higher than when lactose is absent but lower than when lactose present and glucose absent Trp Operon TRYPTOPHAN ABSENT: 1. The Trp repressor is inactive, can’t bind to operator 2. RNA polymerase is able to bind to the promoter and transcribe the structural genes into a single mRNA molecule 3. Translated in 5 enzymes of the tryptophan biosynthesis pathway TRYPTOPHAN PRESENT: 1.Tryptophan entering cell acts as a corepressor by binding to the inactive Trp repressor and activating it 2. Active Trp repressor binds to operator 3.RNA polymerase cannot bind 4. Operon’s genes are not transcribed Compare and contrast the epigenetic mechanisms of feedback loops and chromatin packaging. The appropriate pattern of gene expression is inherited from parent cells rather than having to be independent
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