|Citric Acid Cycle I
The following is a summary of my lecture. I provide it (and subsequent ones) for your information and not as a mechanism of dumping more information on you. Use them if they help you to recall the material. Otherwise, don't bother.
1. Both oxidative decarboxylation (in higher cells) and non-oxidative decarboxylation (in yeast) use an enzymatic activity called the pyryvate dehydrogenase complex to convert pyruvate from glycolysis into acetyl-Coa for the citric acid cycle. This enzyme complex is in the mitochondrion and requires that pyruvate from the cytoplasm be transported to the mitochondrion. This complex includes the following:
Pyruvate decarboxylase (your book calls it "Pyruvate Dehydrogenase Component" (E1)
Dihyrolipoamide transacetylase (E2)
Dihyrolipoamide dehydrogenase (E3)
It also uses the coenzymes, Thiamine Pyrophosphate (TPP), Lipoamide, NAD, FAD, and Coenzyme A (also called CoASH or CoA).
2. The mechanism of the reaction catalyzed by the complex is very similar to that catalyzed by the alpha-keto-glutarate dehydrogenase complex of the Citric Acid Cycle. Both involve oxidation of alpha-keto acids.
3. In aerobic higher organisms, the reaction mechanism involves binding of pyruvate by an ionized TPP, decarboxylation, transfer to the lipoamide molecules, linkage of the acetyl group to CoASH to form acetyl-CoA, transfer of the electrons from the oxidation to FAD (forming FADH2) and transfer of electrons from FADH2 to NAD+ to form NADH.
4. In yeast fermentation, the reaction that occurs stops at the decarboxylation step with resolution to form acetealdehyde without loss/gain of electrons (no oxidation/reduction). Thus, enzyme activities E2 and E3 above are not needed in yeast fermentation. Acetaldehye in yeast fermentation is converted to ethanol. Note that when oxygen is present, fermentation in yeast does not occur and activities E2 and E3 catalyze reactions just like animal cells, producing acetyl-CoA.
5. Mitochondria are the "power plants" of the cell and are the places where much oxidation occurs. Byproducts of this oxidation can result in damaged mitochondria. Mitochondria have an outer membrane (fairly permeable) an inner membrane (only permeable to water, carbon dioxide, oxygen, carbon monoxide) and a matrix (liquid component). Infoldings of the inner membrane are called cristae.
6. The citric acid cycle occurs in the mitochondrial matrix and is found in almost every cell. In the cycle, two carbons are added from acetyl-CoA and two carbons are released as carbon dioxide.
7. Biological oxidations in the citric acid cycle involve NAD+ (reduced to NADH) and FAD (reduced to FADH2). In the citric acid cycle, three NADH and one FADH2 are produced, along with one high energy phosphate (GTP in animals, ATP in plants and bacteria) per acetyl-CoA that enters the cycle (Remember that one molecule of glucose yields two acetyl-CoAs for the cycle).
8. The two carbons added from acetyl-CoA in the beginning of the cycle do NOT become oxidized to CO2 until beginning in the second time around the cycle.
|Citric Acid Cycle II
1. The citric acid cycle consists of two main parts - release of CO2 (first part) and conversion to oxaloacetate (second part). You are responsible for the structures of molecules in the citric acid cycle and the names of the enzymes.
2. In the "first" reaction of the citric acid cycle, citrate synthase catalyzes the joining of the acetyl group from acetyl-CoA to oxaloacetate to make citrate. This reaction is VERY energetically favorable, due to breaking of the thioester bond in acetyl-CoA. The energetically favorable reaction helps to "pull" the relatively unfavorable reaction preceding it.
3. Aconitase catalyzes the rearrangement of citrate to isocitrate. For your information - Aconitase is inhibited by fluorocitrate. Fluoroacetate is a poison that can be used by citrate synthase to make fluorocitrate.
4. The first decarboxylation of the citric acid cycle is catalyzed by isocitrate dehydrogenase and the reaction is strongly favored to the right. The products of this reaction are NADH and alpha ketoglutarate.
5. Alpha ketoglutarate is an important intermediate for its involvement in anaplerotic reactions related to transamination (we'll talk about these later). The mechanism of the enzyme acting on alpha ketoglutarate (alpha ketoglutarate dehydrogenase complex) is virtually identical to the mechanism of action of the pyruvate dehydrogenase complex and involves all of the same coenzymes. The products of this reaction are succinyl-CoA and NADH
6. The only substrate level phosphorylation in the citric acid cycle is catalyzed by succinyl-CoA synthetase. The products of this reaction in the citric acid cycle are GTP and succinate. Note that the enzyme is named for the reverse reaction.
7. Succinate dehydrogenase contains a covalently-linked FAD electron carrier. The Delta G zero prime of zero allows the reaction to be readily reversed to produce succinate, when needed. The products of this reaction in the forward direction of the citric acid cycle are FADH2 and fumarate (trans double bond). This reaction is similar to the first oxidation reaction for a fatty acid.
8. Addition of water to fumarate (catalyzed by fumarase) yields L-malate.
9. Oxidation of L-malate by malate dehydrogenase yields NADH and oxaloacetate. This reaction is a rare oxidation reaction that is energetically unfavorable. Conversion of malate to oxaloacetate is the only energy "bump" to be gotten over in the citric acid cycle and that is readily accomplished thanks to the 'pulling' of the citrate synthase reaction, which keeps oxaloacetate concentrations low.
10. The citric acid cycle can be regulated allosterically in several places, but the most important regulation of the cycle is probably the amount of NAD+ and FAD that is available. NAD+ (and FAD) is essential for the cycle to operate and it is essential for the pyruvate dehydrogenase complex reaction to occur. This relates to metabolic control, as we shall see in discussions later of electron transport and oxidative phosphorylation.
11. When all of the NADHs and FADH2s of the citric acid cycle are converted to ATP, the cycle yields 30- 38 ATPs per molecule of glucose (depending on how you count them - we'll talk about this later), compared to 2 for glycolysis (under anaerobic conditions). The citric acid cycle is thus an incredibly efficient producer of energy for the cell.
12. Many factors combine to regulate metabolism through the citric acid cycle. All of these ultimately come down to energy needs. Most are manifested through the availability or lack of NAD+. When NAD+ is lacking (high NADH levels), the cycle will be inhibited. When NAD+ levels are high (low NADH levels), the cycle is favored. Oxygen is a limiting reagent needed to keep the citric acid cycle turning. This is because oxygen is required ultimately for the conversion of NADH back to NAD+. Remember that NAD+ is required for three reactions of the citric acid cycle. If any one of these reactions is stopped, the cycle grinds to a halt. While glycolysis can use fermentation to get around conditions lacking oxygen, the citric acid cycle cannot.
|Lipids & Membranes
1. Membranes are composed of a lipid bilayer and other molecules, such as cholesterol. The lipid bilayer contains two main categories of molecules - glycerophospholipids and sphingolipids.
2. Glycerophosphlipids contain glycerol, phosphate and one or more fatty acids. The fatty acids in the molecules of the lipid bilayer are either saturated (no double bonds) or unsaturated (contain one or more double bonds). Double bonds in biological fatty acids are almost exclusively in the cis configuration, resulting in a bent shape for unsaturated fatty acid.
3. Glycerophospholipids (also called phosphoglycerides by your book) are related to fats in having a glycerol backbone and two fatty acids, but they differ from fat in having a phosphate located in position #3 of the glycerol. Naming of glycerophospholipids is generally as "phosphatidyl-X" where X is the name of the molecule attached to the phosphate. Examples include phosphatidylserine, etc.
4. If there is no other molecule attached to the phosphate on the glycerophospholipids described above in #3, you have phosphatidic acid. Phosphatidic acid is an important intermediate in synthesis of phosphatidyl lipids, as well as fats.
5. Sphingolipids are molecules related to glycerophospholipids that are based on sphingosine. Sphingomyelin is a component of the myelin sheath of nerve cells. Sphingolipids containing a single sugar are called cerebrosides and sphingolipids containing a complex carbohydrate moiety are called gangliosides. Sphingolipids are prominent components of the membranes of nerves and brain tissue.
6. Steroids are lipids that are not derived from fatty acids. In animals, steroids are derived from cholesterol. Cholesterol is found in the membrane of cells and is important for membrane stability. Cholesterol is prominent in brain membranes - up to 14% of the dry weight of brain.
7. A lipid bilayer has a polar exterior facing water and a non-polar interior. As such, the membrane provides a good barrier to both polar and non-polar substances. In contrast to the lipid bilayer, fatty acids aggregate into a micelle.
8. In addition to glycerophospholipids and sphingolipids, membranes contain proteins, glycoproteins, and glyolipids. Four types of membrane proteins are integral (protein projects through both sides of the membrane), peripheral (protein projects into only one side of the membrane), anchored (protein is linked to a molecule embedded in the lipid bilayer, or associated (protein associates by hydrogen bonding with an integral membrane protein.
9. Integral membrane proteins are difficult to remove from membranes, but peripheral and associated membrane proteins are not.
10. Membranes provide a barrier between the cell and the external environment. Membranes provide a barrier to passage of many molecules, including molecules, such as sugar that the cell could use for food.
11. Integral membrane proteins span into and/or across the plasma membrane and thus must have both hydrophilic and hydrophobic portions that interact appropriately with the same portions of the plasma membrane. By contrast, peripheral membrane proteins are found in association with membranes, such as by interacting with an integral membrane protein. Anchored membrane proteins are proteins attached to a molecule (like a fatty acid). The molecule is embedded in the lipid bilayer and thus the protein is anchored to it.
12. Bacteriorhodopsin in an integral membrane protein that uses light, chemistry, and mechanics to move protons across a membrane barrier.
13. One can assemble artificial lipid bilayers containing compounds as a means of delivering materials into cells. These artificial systems are called liposomes. Liposomes can carry substances and when the membrane of the liposome fuses with a cell membrane, the contents of the liposome are delivered into the cell. This is a useful way of getting compounds into cells that are not easily transported across the cell membrane in other ways.
14. Membrane-spanning proteins have alternating regions of non-polar membrane crossing regions interrupted by polar short sections joining the non-polar regions. Consequently, one can use a computer to examine the amino acid sequence of a protein and predict reasonably accurately if the protein is a membrane protein or not.
1. Diffusion is a process in solutions where molecules move from a high concentration to a low concentration.
2. We break transport across membranes into two main categories - 1) passive transport (diffusion driven, so materials move only from high concentration to lower concentration and don't require outside energy), and 2) active transport (an energy-requiring process that moves at least one molecule from a low concentration to a higher concentration - this is contrary to simple diffusion).
3. Active transport moves at least one molecule in the opposite direction of where diffusion would operate (that is, active transport moves at least one molecule from a low concentration to a higher concentration).
4. ATP is a primary energy source for active transport, but there are other sources, as well (see below). The term 'pump' is used to describe the protein component of an active transport system. Pumps that move two molecules in the same direction across a membrane are called symports (or synports), whereas pumps that move two molecules in opposite directions across a membrane are called antiports. Pumps are called electroneutral if their action does not result in a net change in charge and electrogenic if their action changes the charge across the membrane as a result of their action.
5. An example of a passive transport system is a glucose transporter in blood cells that simply lets glucose diffuse into cells. No energy is required for that particular transporter. Other glucose transporters in other cells are active in that they use energy to move glucose against a concentration gradient.
6. An example of a passive transport system is a glucose transporter in blood cells that simply lets glucose diffuse into cells. No energy is required for that particular transporter. Other glucose transporters in other cells are active in that they use energy to move glucose against a concentration gradient.
7. P-type ATP-using transport systems use phosphoaspartate as a covalent intermediate in their mechanism of action.
8. The mechanism of transport of the Ca/ATPase pump includes binding of ATP and the relevant ions (calcium, in this case), transfer of phosphate from the ATP to the protein (making phosphorylaspartate), conformational change in the protein causing movement of the ions across the membrane, hydrolysis of the phosphate from an asparatic acid side chain in the protein, a second conformational change to bring the protein back to its original state. The Ca/ATPase pump is called a symport because all of the molecules are being moved in the same direction across the membrane.
9. Another P type ATPase is the Na/K ATPase. The Na/K ATPase transports three sodiums out of the cell and two potassiums in for each cycle. This is an electrogenic transport mechanism and uses hydrolysis of ATP to drive the process. Movement of Na and K is essential for the cell being able to maintain osmotic balance. The Na/K ATPase is called an antiport because it moves molecules in opposite directions.
10. Another class of transporter proteins that use ATP to move molecules are the ABC transporters. An example is the Multidrug Resistance Protein that is involved in the resistance of cancer cells to chemotherapy agents. They act by binding the compound first. This causes a conformational change in the protein that allows ATP to bind. Binding of ATP causes the protein to 'evert' (move its opening from one side of the membrane to the other). This has the effect of moving the bound compound to the outside of the cell. After this happens, ATP is hydrolyzed to change the protein to evert again and change back to its original conformation (opening facing inwards).
1. An interesting transporter is the Na+/Ca++ exchange pump (not shown in a figure in class, but described). It uses movement of Na+ in to cells to be a driving force for pumping Ca++ out. Remember than Ca++ stimulates muscular contraction. If Ca+ is not pumped OUT, its concentration in muscle cells remains high, stimulating contraction. Digitoxigenin is a compound from foxglove that binds the Na+/K+ ATPase, preventing development of a Na+ gradient. As a consequence, digitoxigenin increases Ca++ concentration, since Ca++ pumping requires a Na+ gradient. Digitoxigenin is used as a heart stimulant.
2. Nerve cells use the gradient of Na+ and K+ built up by the Na+/K+ pump to transmit signals. In nerve transmission, special "gates" open and close to allow Na to diffuse into nerve cells and K to diffuse out of nerve cells.
3. The first step in nerve transmission involves opening of Na+ gates. These allow Na+ to diffuse into the cell, since Na+ concentration is higher outside of cells than inside. Movement of the positively charged sodium ion causes a change in the electrical potential of the cell near the Na+ gate. To compensate for the voltage change, the K+ gates open and Na+ gates close, allowing K+ to flow out of the cell. This results in an overcompensation of the voltage. The K+ gates close and the region where the original movement of ions occurred recovers. During this time, no nerve signal can be transmitted at that point.
4. The nerve signal is transmitted as a consequence of the initial movement of Na+ into the cell. Before it can be pumped out, some of the sodium diffuses down to the next Na+ gate and the change in the voltage environment causes it to open and trigger the same events as occurred in the last step. Thus, the signal moves from one junction to another to another, ultimately arriving at the end of the axon.
5. Tetrodotoxin is a neurotoxin because it inhibits the action of nerve cells. It is found in the puffer fish and it blocks the Na+gates.
6. Channels (gates) are made by protein molecules in the membranes of cells. Channels are generally very specific for what they will allow to pass through them. Glucose channels, for example are fairly specific for glucose. Sodium and potassium channels are very specific for each respective ion.
7. Ion specificity is accomplished by two mechanisms. The first is physical. If an ion is too big to fit in a channel, it is excluded. This is the case of the sodium channel, which excludes potassium ions because they are too big.
8. The second mechanism of specificity is energy. An example is the potassium channel, which excludes sodium ions. In this case, the channel allows larger ions (potassium) to pass through, but blocks smaller ions, like sodium ions.
9. The mechanism of exclusion of the potassium channel relates to the energies of solvation of each ion. For potassium ions, the energy of desolvation of the ion as it enters the channel is overcome by the energy of resolvation as it enters the channel. Thus, entry of potassium ions is energetically favored. This is due to the geometry of the potassium channel closely matching the dimensions of the potassium ion.
10. When sodium ions try to enter the channel, their energy of desolvation is greater than is realized by their resolvation in the channel. Thus, they do not enter. Their energy of resolvation not as favorable due to their ion sizes not matching the dimensions of the potassium channel.
11. Movement of ions across a membrane is accomplished by controlled gates. Two control mechanisms include voltage controlled gates (open/close with voltage changes) and gates controlled with both voltage and a ball/chain. The latter are found in nerve cells.
12. As noted, the first gate that opens in nerve transmission is the sodium gate. Movement of sodium ions in creates the action potential that begins the transmission of the nerve signal (see above). The "ball" at the sodium gate quickly plugs the hole after the sodium ions start leaking in.
13. After the nerve has "fired" the gradient must be restored and this is the job again of the Na/K ATPase.
1. For every oxidation, there is an equal and Loss of elecrons by one molecule means gain of them by another one. Oxidation is a process that involves the loss of electrons. Reduction is a process that involves the gain of electrons.
2. Electrons are carried to the electron transport system in the mitochondria by NADH and FADH2.
3. Mitochondria are the site of electron transport and oxidative phosphorylation.
4. Electrons from NADH enter the electron transport system through complex I.
5. Electrons from FADH2 enter the electron transport system through complex II.
6. Coenzyme Q (CoQ) accepts a pair of electrons from either complex I or complex II and passes electrons singly to cytochrome c through complex III. Thus, coenzyme Q acts as a "traffic cop" for electrons.
8. Oxygen is thus the terminal electron acceptor and is a limiting compound during periods of heavy exercise.
9. If oxygen is not available, electrons will NOT pass through the electron transport system and NADH and FADH2 will not be reoxidized. For these reasons, the citric acid cycle will not run either. This is part of metabolic control.
10. Several compounds inhibit electron transport - rotenone (an insecticide) and amytal block all action of Complex I. Antimycin A blocks all action of Complex III. Cyanide, azide, and carbon monoxide block all action of complex IV.
11. Movement of electrons through Complex III is known as the Q cycle. This cycle begins with the binding of two molecules of CoQ (QH2 and Q) to Complex III. QH2 has two electrons and two protons. Q has neither.
12. After QH2 and Q bind, QH2 sends one electron to Q, creating Q- and one electron to cytochrome C. The two protons QH2 was carrying are expelled into the intermembrane space. This converts QH2 to Q. Both cytochrome C and Q leave the complex, but Q- remains behind.
13. Next, another QH2 and another cytochrome C binds to Complex III. QH2 sends one electron to Q-, creating Q-2 and one electron to cytochrome C. It also expels its two protons to the intermembrane space and becomes Q. Then Q-2 extracts two protons from the matrix and becomes QH2. Last, cytochrome C, QH2, and Q all leave the complex. (As you can see, words describing the process are complicated. The figure shows it much more clearly).
14. Electron transfer through complex IV occurs one electron at a time (since one electron arrives at a time from cytochrome c). Interruption of electron flow can result in production of reactive oxygen species. Cellular enzymes, such as superoxide dismutase and catalase (see below) help to deactivate superoxides.
15. In electron flow through complex IV, the first electron is transferred to copper and the second one is transferred to iron. Oxygen then binds to the iron first, followed by formation of a peroxide bridge between the iron and copper atoms. Addition of a third electron (to the oxygen on the copper) and binding of a proton from the matrix causes the O-O bond to be cleaved. A fourth electron then reduces the oxygen on the iron and a proton binds from the matrix as well. Last, two protons from the matrix bind to the hydroxyls on the iron and copper, forming two water molecules, which are released and the cycle is complete.
16. During electron movement through Complex IV, four protons are taken from the matrix and combined with oxygen to form two water molecules. In addition, four other protons are taken from the matrix by the complex and pumped outside the mitochondrial matrix. As a consequence, the proton numbers in the matrix decrease by 8 during the process. The proton numbers outside the mitochondrion INCREASE by four in the process, so the net difference is 12 protons just for movement through complex IV.
17. Superoxide dismutase (SOD) acts in a two step fashion to deactivate superoxides (O2-). In the first step, the oxidized form of SOD accepts an electron from O2, creating molecular oxygen and a reduced SOD. In the second step, the reduced SOD combines its extra electron with that of another O2- and two protons to create hydrogen peroxide and the oxidized form of SOD. Hydrogen peroxide (H2O2) is converted to oxygen and water by the enzyme catalase.
1. ATP is created in oxidative phosphorylation by the movement of protons back into the mitochondrial matrix through complex V (also called the ATP synthase).
2. Two essential functions of electron transport - 1. Pump protons out of mitochondrial matrix and 2. Reoxidize NADH and FADH2 to NAD and FAD, respectively. In healthy, normal cells, oxidative phosphorylation is tightly coupled to electron transport.
|Lipid Metabolism, Fat Transport in the Body
1. The chemiosmotic hypothesis, originally proposed by Peter Mitchell, explains how mitochondria make ATP in oxidative phosphorylation. Important aspects of it include:
a. Intact inner mitochondrial membrane
b. Electron transport creates a proton gradient
c. ATP is made by movement of protons back into the mitochondria
2. Coupling of electron transport and oxidative phosphorylation at a practical level means that the mitochondrial inner membrane remains impermeable to protons, except for those that enter via the ATP synthase and result in ATP production.
3. The ATP synthase consists of a turbine-like structure containing 3 sites called Loose (L), Tight (T), and Open (O). Functions of these forms include
L - Holds ADP and Pi in preparation for ATP formation
O - Releases ATP formed in T and binds ADP + Pi
T - Causes ADP and Pi to join and form ATP
4. Movement of protons through the ATP synthase cases rotation/conformational changes in the complex that result in formation of ATP from ADP and Pi. Conversions in the process occur as follows:
O goes to L
L goes to T
T goes to O
5. Mitochondria which are "tightly coupled" have intact membranes AND the only way protons get back into the matrix is by passing through Complex V. If you poke a hole in the membrane (using DNP or an uncoupling protein, such as found in brown fat), protons can leak back in without making ATP. This has the effect of generating heat AND burning up energy sources (like glucose and fat). As noted, DNP is a dangerous compound that killed people who tried to use it to lose weight.
6. When mitochondria are tightly coupled, metabolic (respiratory) control exists. This means that electron transport will stop if oxidative phosphorylation stops, since the protons don't come back int and the proton gradient gets very high, stopping the pumping of protons. When electron transport stops, NADH accumulates and the citric acid stops. Conversely, if one stops electron transport with cyanide, oxidative phosphorylation will stop very shortly because the proton gradient is lost when no protons are being pumped.
7. When mitochondria are uncoupled (by poking a hole in them to let protons leak back into the matrix without passing through Complex V), electron transport is no longer limited by oxidative phosphorylation and runs amok. That is why heat is generated. Protons are pumped, but they fall back in throught the hole in the mitochondrial inner membrane. No ATP is made. NADH is rapidly converted to NAD+, so the citric acid cycle and other pathways run rapidly.
8. Things that affect these processes are ADP (necessary for the Complex V to function), oxygen (necessary for electron transport to function), NADH (source of electrons for electron transport), and NAD+ (needed for citric acid cycle).
9. NADH cannot cross the inner membrane of the mitochondrion, as there is no protein to move it. Electrons make it into the mitochondria by means of shuttles. Insect muscles a glycerol-3phosphate/DHAP shuttle that transfers electrons from NADH ultimately to FADH2. Mammalian systems, by contrast, use a malate/aspartate system that converts oxaloacetate to malate (carrier of electrons) that then gets transported by a transport protein. Once inside the matrix, malate transfers electrons to NAD+, creating NADH and oxaloacetate.
Glycerolipid, Sphingolipid, and Cholesterol Metabolism
1. Glycerophospholipids typically contain a saturated fatty acid at position #1 and an unsaturated fatty acid at position #2 on the glycerol backbone.
2. Glycerophosphlipid biosynthesis occurs most commonly through the synthesis of phosphatidic acid. This can be made from glycerol-3-phosphate by esterifying fatty acids to positions 1 and 2 of glycerol-3-phosphate, yielding phosphatidic acid. Phosphatidic acid is a branch point between the synthesis of fats and glycerophospholipids.
3. For the synthesis of glycerophospholipids, phosphatidic acid is an immediate precursor of CDP-Diacylglycerol, which is a precursor of the various glycerophospholipids (see Figure 19.2). CTP combines with phosphatidic acid to yield a pyrophosphate and CDP-Diacylglycerol. Activation by CDP yields a high energy intermediate that can be readily converted to phosphatidyl glycerophospholipids.
|Fatty Acid Oxidation
1. Fats are broken down to fatty acids and glycerol by enzymes known as lipases. One of these, hormone sensitive triacylglycerol lipase, is the only regulated enzyme of fat or fatty acid breakdown. It is located in fat-storing cells called adipocytes.
2. Triacylglycerol lipase action cleaves the first fatty acid off of a fat and this step is necessary before the other lipase can act to remove the other fatty acids from a fat.
3. Glycerol, is the only part of a fat that can be made into glucose (via gluconeogenesis). Fatty acids travel in the bloodstream carried by serum albumin.
4. Fatty acid oxidation occurs in the matrix of the mitochondrion. In the cell, fatty acids are attached to CoA and then at the mitochondrion, the CoA is replaced by carnitine. Inside the mitochondrial matrix, the carnitine is replace by CoA again.
5. Steps in fatty acid oxidation include dehydrogenation, hydration, oxidation, and thiolytic cleavage. The dehydrogenation and oxidation reactions yield reduced electron carriers (FADH2 and NADH). The double bond formed in the first dehydrogenation reaction is in the trans form. The hydration yields a hydroxyl group on the third carbon from CoA end in the "L" configuration. Thiolytic cleavage is catalyzed by the enzyme called thiolase. Two enzymes you should know include acyl-CoA dehydrogenase, which come in three forms (specialized for long, medium, and short chain fatty acyl-CoAs) and thiolase, which catalyzes the thiolytic cleavage to release acetyl-CoA.
6. The first reaction of fatty acid oxidation involves a set of enzymes know as acyl dehydrogenases. These are specific for fatty acids with long, medium, or short chains. The medium chain acyl dehydrogenase has been implicated in some instances of sudden infant death syndrome.
7. The long chain acyl dehydrogenases are found in peroxisomes and this is where oxidation of long chain fatty acids (longer than 16 carbons) begins (not in the mitochondrial matrix). Oxidation here involves transfer of electrons to oxygen to make hydrogen peroxide, instead of FADH2. Peroxisomal fatty acid oxidation is therefore LESS efficient than mitochondrial beta oxidation.
8. The first step of oxidation generates a trans-intermediate plus FADH2. The second step involves addition of water across the trans double bond to create an intermediate in with an OH on carbon 3 in the L configuration. The third step involves oxidation of the hydroxyl intermediate to a ketone on carbon 3. The last step involves cleaving off of an acetyl-CoA and production of a fatty acyl-CoA with two fewer carbons. The last step is catalyzed by the enzyme thiolase.
9. The reactions of beta oxidation up to the thiolase reaction chemically mirror the reactions of the oxidation of succinate up to oxaloacetate.
10. Seven cycles of beta oxidation of palmitoyl-CoA in the matrix yield 8 acetyl-CoAs.
11. Oxidation of biologically occurring fatty acids with cis double bonds requires two additional enzymes compared to oxidation of saturated fatty acids. These enzymes are enoyl-CoA-isomerase and 2,4-dienoyl-CoA-reductase.
12. Enoyl-CoA-isomerase converts cis or trans bonds between carbons 3 and 4 to trans bonds between carbons 2 and 3. Since beta oxidation normally has trans bonded intermediates between carbons 2 and 3, this enzyme is sufficient for conversion of many naturally occurring fatty acids to be oxidized.
13. 2,4-dienoyl-CoA reductase acts on intermediates that have double bonds between carbons 2-3 and 4-5. It uses NADPH to reduce the two double bonds to one double bond and the resulting double bond is placed in a cis configuration between carbons 3-4. Enoyl-CoA-isomerase then can convert this intermediate to one with a trans double bond between carbons 2-3, thus allowing beta oxidation to continue.
|Fatty Acid Synthesis
1. The process occurs similar to beta-oxidation, though in reverse. Important distinctions are noted below in a-f.
* a. Fatty acid synthesis up to palmitate occurs in the cytoplasm, but beta oxidation occurs in mitochondrial matrix.
* b. Fatty acids are built using an acyl carrier protein (ACP), but beta oxidation uses CoA.
* c. NADPH is used to donate electrons in synthesis, but NAD+ or FAD are used to accept electrons in oxidation in the mitochondrion.
* d. A three carbon molecule, malonyl-ACP donates two carbons to the growing fatty acid chain - a carbon dioxide is lost in the process. Beta oxidations yield two carbon acetyl-CoA units.
* e. Synthesis of fatty acids longer than 16 carbons occurs in endoplasmic reticulum or mitochondrion. Oxidation of fatty acids longer than 16 carbons begins in peroxisomes.
* f. In fatty acid biosynthesis, a D-hydroxyl intermediate is formed at carbon #3. In fatty acid oxidation, an L-hydroxyl intermediate is formed at carbon #3.
2. Acetyl-CoA carboxylase catalyzes the addition of a carboxyl group to acetyl-CoA to form malonyl-CoA. The enzyme is regulated allosterically (inhibited by palmitoyl-CoA and activated by citrate) and by covalent modification (phosphorylation inhibits, dephosphorylation activates).
3. Fatty acid biosynthesis occurs in the cytoplasm and the fatty acids are built on a 'carrier' known as acyl carrier protein (ACP). The Co-A above is swapped for ACP to start the synthesis process. Acetyl-CoA gets into the cytoplasm from the mitochondrion by the citrate shuttle. Acetyl-CoA is linked to oxaloacetate in the mitochondrion to make citrate, which is transported out and then citrate is cleaved to yield acetyl-CoA and oxaloacetate in the cytoplasm.
4. Malonyl-ACP is the "adding block" for fatty acid biosynthesis. Acetyl-ACP is the starting block for fatty acid biosynthesis.
5. During fatty acid synthesis, decarboxylation of malonyl-ACP yields a two carbon addition to the growing fatty acid chain. The two carbons from malonyl-ACP go onto the carboxyl end of the growing chain.
6. The numerous enzymes of fatty acid biosynthesis are contained in a multi-enzyme complex called fatty acid synthase.
7. Fatty acids up to 16 carbons long are synthesized in the cytoplasm by the fatty acid synthase. Synthesis of fatty acids longer than 16 occurs in the endoplasmic reticulum (or mitochondrion) catalyzed by enzymes called elongases.
|Fatty Acid Synthesis/Prostaglandins
1. Enzymes that produce unsaturation in fatty acid biosyntheis are called desaturases. They employ an unusual electron transferring process initiated by donation of electrons from NADH and accepted by oxygen. Desaturases are found in the endoplasmic reticulum.
2. Essential fatty acids are those that must be provided in the diet of an organism, because the organism cannot synthesize them. In mammals, linoleic and linolenic acids are essential fatty acids because they cannot make double bonds closer to the end than the Delta-9 position (oleic acid is an omega-9 fatty acid). Thus, linoleic acid (Delta 9,12 double bonds = omega 6 for an 18 carbon fatty acid) and linolenic acid (Delta 9,12,15 double bonds = omega 3 for an 18 carbon fatty acid) must be provided in the diet of mammals.
3. Fatty acids longer than 16 carbons are produced by action of enzymes called elongases. These are found in the endoplasmic reticulum and the mitochondrion.
4. Trans fatty acids are produced by partial hydrogenation of vegetable oil. Hydrogenation of vegetable oil saturates its double bonds, raising its melting point. This chemical treatment is done for fats/oils in many processed foods and a byproduct of this action is creation of fatty acids with trans (instead of the natural cis) double bonds. Trans fatty acids in fats (called trans fats) are associated with increasing LDLs, lowered HDLs and atherosclerosis. The reason is not fully known.
5. Prostaglandins are hormone-like compounds made from arachidonic acid by action of an enzyme known as prostaglandin synthase. There are several prostaglandin synthases in the body. The reactions they catalyze are forming cyclic oxygen-containing compounds (that's what prostaglandins are), so the enzymes are also known as cyclooxygenases (or COX for short). The COX enzymes are known as COX-1 and COX-2.
6. Prostaglandins are involved in numerous physiological effects, including control of vasodilation/constriction, uterine contractions, aggregation/stickiness of platelets, inflammation/pain, and maintenance of stomach tissue, among others. Inhibitors of COX enzymes are called COX inhibitors. Aspirin and ibuprofen are non-steroidal drugs (called NSAIDs) that inhibit COX-1 and COX-2.
7. Prostaglandins produced by COX-2 enzymes appear to have no role in stomach maintenance, so inhibitors specific to them were sought. Examples include Celebrex and Vioxx, but they also appear to have negative side effects on the heart.
8. Arachidonic acid is produced from linoleic acid released from glycerophospholipids by action of an enzyme known as phospholipase A2 (PLA2). PLA2 can be inhibited by corticosteroids, so action of these compounds can also prevent prostaglandin formation indirectly. Corticosteroids are important for treatment of severe inflammation or pain.
9. Leukotrienes can also be produced from arachidonic acid. The pathway that leads to them does not involve cyclization, so that pathway is called the linear pathway to distinguish it from the cyclic pathway that leads to prostaglandins. Leukotrienes are involved in mucus production and bronchial constriction and play important roles in causing asthma attacks.
10. Another class of molecules made from prostaglandins is the thromboxanes. These molecules help to make platelets "sticky", favoring aggregation. Thus, taking aspirin reduces synthesis of prostaglandins, which in turn reduces amounts of thromboxanes, which reduces stickiness of platelets, which makes it harder for blood to clot. It is for this reason that people prone to clotting problems are advised to take aspirin daily.
|Nucleotide Metabolism I
1. Nucleotides consist of a) sugar, b) nitrogenous base, and c) phosphate
2. Nucleosides consist of a a) sugar and b) nitrogenous base
3. The sugars of nucleosides and nucleotides are either ribose (found in ribonucleotides of RNA) or deoxyribose (found in deoxyribonucleotides of DNA).
4. Unless otherwise specified, the term "nucleotide" will be used in this class to indicate either ribonucleotides (contain ribose) or deoxyribonucleotides (contain deoxyribose).
5. Unless otherwise specified, the term "nucleoside" will be used in this class to indicate either ribonucleosides (contain ribose) or deoxyribonucleosides (contain deoxyribose).
6. The term nucleoside phosphate is equivalent to a nucleotide (nucleoside + phosphate + base = nucleotide). This is true whether it is a monophosphate, diphosphate, or triphosphate.
7. The nitrogenous bases found in nucleotides include adenine (purine), guanine (purine), thymine (pyrimidine), cytosine (pyrimidine), and uracil (pyrimidine).
8. The bases adenine, guanine, and cytosine are found in both ribonucleotides and deoxyribonucleotides. Thymine is almost always found in deoxyribonucleotides. Uracil is found primarily in ribonucleotides and rarely in DNA, but does appear as a deoxyribonucleotide intermediate in thymidine metabolism.
9. Nucleosides are named according to the base they contain. Nucleosides containing purines are named by adding "os" before the "ine." Thus, nucleosides containing guanine are called guanosine. Nucleosides containing pyrmidines are named with the suffix "idine" at the end of the name of the base they contain. Thus, the pyrimidine nucleosides are cytidine, uridine, and thymidine.
10. Ribonucleotides are the building blocks of RNA and deoxyribonucleotides are the building blocks of DNA.
|Nucleotide Metabolism II
1. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotides from ribonucleotides. The substrates are ribonucleoside diphosphates (ADP, GDP, CDP, or UDP) and the products are deoxyribonucleoside diphosphates (dADP, dGDP, dCDP, or dUDP).
2. RNR has two pairs of two identical subunits - R1 (large subunit) and R2 (small subunit). R1 has two allosteric binding sites and the active site of the enzyme. R2 forms a tyrosine radical necessary for the reaction mechanism of the enzyme.
3. Ribonucleotide reductase is allosterically regulated via two binding sites - a specificity () binding site (controls which substrates the enzyme binds and which deoxyribonucleotides are made) and an activity binding site (controls whether or not enzyme is active - ATP activates, dATP inactivates). Specificity sites act in a generally complementary fashion. Binding of deoxypyrimidine triphosphates to the specificity site tends to inhibit binding and reduction of pyrimidine diphosphates at the enzyme's active site and stimulates binding and reduction of purine diphosphates at the active site. Binding of deoxypurine triphosphates tends to inhibit reduction of purine diphosphates and stimulates reduction of pyrimidine diphosphates. Don't confuse the active site with the activity site. The ACTIVE SITE is where the reaction is catalyzed, whereas the ACTIVITY SITE is the allosteric binding site for ATP or dATP.
4. Synthesis of dTTP by the de novo pathway takes a convoluted pathway from dUDP to dUTP to dUMP. The last reaction is catalyzed dUTPase.
5. The de novo pathway for thymidine synthesis converts dUMP to dTMP, using a tetrahydrofolate derivative and the enyzme thymidylate synthase. In the process, dihydrofolate is produced and must be converted back to tetrahyrdolate in order to keep nucleotide synthesis occurring.
6. The enzyme involved in the conversion of dihydrofolate to tetrahydrofolate, dihydrofolate reductase (DHFR), is a target of anticancer drugs which inhibit the enzyme. An inhibitor of DHFR is methotrexate or aminopterin.
7. ATCase is regulated allosterically by ATP (activates) and CTP (inactivates). It is the most important regulatory enzyme in de novo pyrimidine biosynthesis and it helps to balance the relative amounts of purines and pyrimidines. Another important regulatory enzyme in the pathway is CTP synthase, which is inhibited by CTP. This enzyme helps balance the relative amounts of CTP and UTP.
8. PRPP amidotransferase is an important regulatory enzyme for purine biosynthesis. It is inhibited by AMP, GMP, and IMP. If AMP is low and GMP is high (or vice-versa), the enzyme is reduced in activity, but still can function. This is important to help increase the amount of the other one, thus helping to balance AMP and GMP.
9. Salvage of purine nucleotides is important metabolically - perhaps more so than salvage of pyrimidines. The enzyme HGPRT is involved in the direct salvage of guanine nucleotides and indirectly involved in salvage of adenine nucleotides through IMP and hypoxanthine.
10. Breakdown of purines results in production of xanthine. Oxidation of xanthine yields uric acid. This compound serves an excretory role in birds and dalmations (among other organisms). Uric acid is not very water soluble and can precipitate out, cause the painful condition known as gout. Gout often strikes in the big toe. Uric acid acts as an antioxidant and may have protective roles against diseases, such as multiple sclerosis. The disease is successfully treated with allopurinol, which acts as a suicide inhibitor of the xanthine oxidase enzyme.
|DNA Replication, Recombination, Repair I
1. DNA consists of a double helix. Each strand of the helix is a polymer of nucleotides joined together in phosphodiester linkages that have alternating sugar-phosphate-sugar-phosphate links. On the inside of the double helix are the complementary base pairs held together by hydrogen bonds. The arrangement of the double helix is in an 'anti-parallel' fashion, meaning that one strand oriented in the 5' to 3' direction is directly paired to a complementary strand oriented in the 3' to 5' direction. Phosphodiester bonds involve linkage between the 5' phosphate group of the incoming nucleotide and the 3' hydroxyl of the previous nucleotide in the chain.
2. DNA contains four bases - A,T,C, and G arranged with A paired with T and G paired with C on the internal portion of the double helix. Hydrogen bonds stabilize these base pairs - two for the A-T pair and three for the G-C pair. Thus, G-C pairs are harder to break than A-T pairs.
3. DNA has a major and a minor groove arising from asymmetric glycosidic linkages between the deoxyribose sugar and each base in the double helix.
4. DNA has three major forms - A,B, and Z. The A and B forms are right-handed helices, whereas the Z form is a left-handed helix. The B form of DNA is the most prevalent one and contains about 10.5 bases per turn of the helix.
5. Z-DNA may have roles in marking the location of genes in eukaryotic chromosomes.
6. Another DNA form is the A form (actually discovered by Rosalind Franklin), which is more "compressed" and is also a right handed helix. The A form is the form assumed by double strand RNA or RNA-DNA duplexes as well. RNA cannot exist in the B form due to steric hindrance arising from the oxygen on carbon number 2 of ribose, which is not present in the deoxyribose of DNA.
7. DNA consists of a double helix. Each strand of the helix is a polymer of nucleotides joined together in phosphodiester linkages that have alternating sugar-phosphate-sugar-phosphate links. On the inside of the double helix are the complementary base pairs held together by hydrogen bonds. The arrangement of the double helix is in an 'anti-parallel' fashion, meaning that one strand oriented in the 5' to 3' direction is directly paired to a complementary strand oriented in the 3' to 5' direction. Phosphodiester bonds involve linkage between the 5' phosphate group of the incoming nucleotide and the 3' hydroxyl of the previous nucleotide in the chain.
8. DNA contains four bases - A,T,C, and G arranged with A paired with T and G paired with C on the internal portion of the double helix. Hydrogen bonds stabilize these base pairs - two for the A-T pair and three for the G-C pair. Thus, G-C pairs are harder to break than A-T pairs.
9. The linking number (L) of a DNA is the sum of the number of twists (T) of a DNA plus the number of writhes (W). Thus, L = T + W. The twists are the number of times two the two helices cross each other. The writhe is the number of superhelical turns found in a DNA. Writhes can be positive or negative and in either case, when the W is a non-zero value, the molecule is said to be superhelical = to have superhelicity.
10. Writhing of DNA occurs in an attempt by a DNA molecule to "relax." A DNA molecule is relaxed when its number of base pairs (bp) per twist (T) is that of B-DNA (10.4-10.5 bp per turn). Thus, if one takes a circular DNA, opens it and removes two twists from it and then closes it, the number of twists will decrease, but the number of base pairs remains the same. In this case, the numbers of bp per twist will INCREASE. This causes a tension that is relieved by the DNA TWISTING two turns. This will cause the writhe to compensate by forming two negative superhelical turns, giving W a value of negative two. Note that the linking number remains the same.
|DNA Replication, Recombination, Repair II
1. 1.DNA polymerase I has three enzymatic activities - a 5' to 3' DNA polymerase activity, a 3' to 5' exonuclease activity (also called proofreading), and a 5' to 3' exonuclease activity.
2. All DNA polymerases require a primer to start DNA synthesis. The primer is formed inside of cells by a special RNA polymerase known as primase. (RNA polymerase does not require a primer)
3. DNA replication proceeds by two distinct mechanisms (both 5'-3', however)- one on each strand. Leading strand and lagging strand synthesis occur by different mechanisms, but both are catalyzed by the same DNA replication complex (Pol III, in the case of E. coli).
4. Leading strand synthesis is continuous in the 5' to 3' direction. Lagging strand synthesis can only occur when the leading strand synthesis opens up a new single stranded region for replication. The 5' to 3' syntheses of the lagging strand are discontinuous. The many pieces of lagging strand synthesis are called Okazaki fragments.
5. Okazaki fragments must be combined together ultimately. First, the RNA primer must be removed from each one. The 5' to 3' exonuclease activity of DNA Polymerase I is needed to remove the initial RNA primer of leading strand synthesis, but is needed frequently to remove the primers of lagging strand synthesis.
6. DNA ligase is an enzyme that creates phosphodiester bonds between adjacent nucleotides between Okazaki fragments. Biotechnologists use this enzyme to join DNA fragments together to create recombinant molecules.
7. E. coli DNA replication occurs at 1000 base pairs per second. At 10 base pairs per turn, this represents a machine turning at 5000 to 6000 rpm. E. coli's helicase protein (DNA B - part of the BC complex) unwinds DNA at a rate of at least 5000 rpm. The protein separates strands ahead of the DNA Pol III so as to make single strands accessible for replication. Unwinding of strands causes superhelical tension to increase ahead of the helicase. Topoisomerase II (gyrase) relieves the tension created by the helicase and is essential for replication to proceed efficiently.
8. DNA Polymerase III is very processive in its action, meaning that once it gets onto a DNA molecule, it stays on it for a long time replicating it. DNA Polymerase I is NOT very processive.
9. In E. coli DNA replication, a dimer of DNA Polymerase III is at the replication fork and performs most of the DNA replication in the cell. One portion of it replicates the leading strand and the other replicates the lagging strand. Leading strand synthesis is faster, so the lagging strand template sometimes loops out in a trombone-like fashion when the lagging strand replication falls behind.
10. Proteins at/near the replication fork and their functions described so far include primase (makes RNA primers necessary for the DNA polymerase to act on), SSB (single stranded binding protein - protects single-stranded DNA and interacts with the replication proteins), DNA gyrase (topoisomerase II - relieves the superhelical tension created by helicase), Pol I (removes RNA primers), DNA ligase (joins DNA fragments together by catalyzing synthesis of phosphodiester bonds at nick sites), and helicase (unwinds double helix).
|DNA Replication, Recombination, Repair III
1. Initiation of replication in E. coli occurs at a specific site on the E. coli genomic DNA, known as OriC, in the cell's circular chromosome. The OriC site contains three repeats of an AT rich sequence near some sequences bound by the DNA A protein.
2. Replication initiation begins with binding of the several copies of the DNA A protein to the OriC site. Bending and wrapping of the DNA around DNA A proteins causes the AT-rich sequences noted above to become single-stranded.
3. Next, the DNA BC complex binds the DNA B protein (helicase) to each of the single strands in opposite orientations. The DNA C protein is released in the process. Next, SSB and primase bind the exposed single-stranded regions and cause DNA A protein to be released. The primases begin synthesizing RNA primers (remember - 5' to 3' RNA synthesis only also) in opposite directions on each strand. The primases DO NOT require a pre-existing primer to function.
4. Note that replication is bi-directional - two replication forks pointed in opposite directions from the origin. They meet later at a termination site on the other side of the genomic DNA.
5. Eukaryotic DNA replication is coordinated tightly with the cell cycle. Checkpoints during the cell cycle ensure that progression through the cell cycle does not occur if there are problems with the DNA. When such conditions arrive, the repair process can be initiated and if repair cannot be performed, a series of events resulting in cellular death may start to occur.
6. Eukaryotic chromosomes differ from prokaryotic DNAs in being linear. The linear ends of the chromosomes are called telomeres. Telomeric sequences have thousands of copies of repeats of short sequences.
7. The enzyme that builds telomeres is called telomerase and is found predominantly in fetal and cancer cells, as well as fertilized eggs. Differentiated cells for the most part do not appear to have an active telomerase.
8. With each round of DNA replication, linear chromosomes in eukaryotes shorten. Thus, the more telomeric sequences a chromosome has, the more divisions it can undergo before the telomeres are "eaten up".
9. Telomerase is a reverse transcriptase - an enzyme that uses an RNA template (a circular RNA that it carries) to synthesize DNA. Other reverse transcriptases are found in retroviruses, such as HIV.
10. Damage to DNA can occur chemically (deamination of adenine to form hypoxanthine), by oxidation (creation of 8-oxo-guanine by reactive oxygen species reaction), by reaction with an aflatoxin metabolite, by reaction with a cross-linking reagent, such as psoralen, and by dimerization of adjacent thymines stimulated by ultraviolet light. These systems require repair - deescribed below. Another system requiring repair is DNA sliding, which can occur amid repeating sequences. Lack of a repair system for these leads to Huntington's disease.
|General Biochemistry Review
1. Base excision repair can removed damaged based from DNA. It differs from nucleotide excision repair in removing the damaged base first, followed by removal of a segment where the base was.
2. Disruption of error correction systems can have severe consequences.
3. Error-related systems associated with cancer include HNPCC (colon cancer) and BRC-A (not mentioned in class), which is involved in DNA repair. A critical protein for monitoring DNA for damage prior to division is p53. It can stop the cell cycle if it senses damage and initiate repair. If repair is unable to be performed, p53 can induce cellular suicide - apoptosis.
4. An Ames test uses a selectable marker that can give a readily observable phenotype (such as growth on antibiotic) when mutation happens. By comparing the number of cells with the observable phenotype in a the presence of a test compound to the number of cells in another tube lacking that compound, the mutagenicity of a compound can be determined.
5. Recombination of DNA results in mixing and matching of DNA sequences. The process occurs most often between homologous sequences on different chromosomes. The process can be quite active during meiosis.
6. Recombination proceeds through formation of a Holliday junction. Holliday junctions form as a result of alignment of homologous sequences, followed by cleavage of strands on each chromosome, invasion of the strands into the opposite chromosome, movement of the junction, another cleavage reaction, followed by reformation of phosphodiester bonds.
7. Enzymes involved in recombination are called recombinases and are similar in function to the integrase of HIV.
1. Transcription is the process where RNA is made using DNA as a template. Students should ABSOLUTELY not mix up or misuse the terms DNA Replication, Transcription, and Translation.
2. RNA polymerization requires an enzyme called RNA polymerase. It can start a chain without a primer, incorporates nucleotides into a growing chain in the 5' to 3' direction using phosphodiester bonds, and uses ATP, GTP, CTP, and UTP as starting compounds. The product of RNA polymerization is called a transcript.
3. The 5' -most nucleotide in RNA has three phosphates on it. All other nucleotides in RNA have only the single phosphate of a phosphodiester bond. Synthesis of the phosphodiester bond arises from nucleophilic attack of the 3' oxygen on the internal phosphate (closest to carbon 5 of the ribose) of the incoming 5' nucleotide.
4. Cells have three main types of RNA - mRNA (carries message to be translated into protein), tRNA (carries amino acids to ribosomes for incorporation into protein), and rRNA (components of ribosomes).
5. In E. coli, all of the RNAs are made by a single polymerase, known as RNA Polymerase. Eukaryotic cells have three RNA polymerases - RNA Polymerase I (rRNAs), RNA Polymerase II (mRNAs and snRNAs), and RNA Polymerase III (tRNAs).
6. E. coli RNA Polymerase has five distinct polypeptide subunits we discussed in class - alpha, beta, beta prime, and sigma.
7. Footprinting is a technique for determining where on a DNA molecule a protein is bound. The figure I gave in class explains the technique better than I can here in words.
8. Promoters in E. coli (RNA polymerase binding sites adjacent to genes) function with widely varying rates of efficiency - some stimulating initiation of transcription every few seconds, others only one or twice per life cycle. One way for a promoter to control such events is via variation in conserved sequences. E. coli genes have two conserved sequences - one at -10 relative to the transcription start site (TATAAT) and another at -35 relative to the transcription start site (TTGACA).
9. The more closely a given promoter's sequence matches the consensus sequence of the -10 sequence, the more active the promoter is at initiating transcription. RNA Polymerase slides along the DNA and when the sigma subunit identifies a promoter site, it stops.
10. Different sigma factors (such as the one made during heat shock) allow the cell to turn on sets of genes with different sequences in the -10/-35 sequences as needs arise.
1. The factor involved in factor dependent transcription termination in E. coli is called rho. It binds to the 5' end of an RNA being made and (using ATP energy) "climbs" the RNA until it reaches the RNA polymerase. There it destabilizes the RNA/DNA duplex, favoring the release of the RNA polymerase from the DNA and the RNA from the DNA, as well.
2. In prokaryotes, tRNAs are the most altered (processed) RNAs. Modifications start with their being cleaved from a larger RNA containing both tRNAs and rRNAs. Ribonuclease P is a ribozyme (catalytic RNA) that cleaves the 5' end of tRNAs from the larger RNA. Ribonuclease III catalyzes excision of rRNAs from the larger molecule.
3. Eukaryotes and prokaryotes differ significantly in the relationship between transcription and translation. Prokaryotes have no nucleus. In them, translation starts oftentimes WHILE a message is being transcribed. There are no significant modifications to mRNAs in prokaryotes.
4. In eukaryotes, transcription and translation are spacially separated. Transcription occurs in the nucleus, whereas translation occurs in the cytoplasm. In addition, eukaryotic mRNAs are modified at the 5' end (capping), the 3' end (polyadenylation) and even in the middle (editing and splicing).
5. Eukaryotes have 3 specialized RNA polymerases. They differ in their sensitivity to alpha-amanitin (a poison from some mushrooms). RNA polymerase II (makes mRNAs) is the most sensitive. RNA polymerase III (makes tRNAs and small rRNA) has moderate sensitivity and RNA polymerase I (makes large rRNAs) has low sensitivity.
6. Sequence elements that affect transcription of eukaryotic genes. They include the TATA box (positioned approximately -30 to -100), and a CAAT box and GC box (-40 to -150).
7. The TATA box is not found in front of all eukaryotic genes, but is essential for strong transcription.
8. The promoters for each RNA polymerase are different in structure. I will not hold you responsible for their structures.
9. Enhancer sequence elements are DNA sequences that about bound by enhance (transcription factor) proteins. Enhancer proteins act in this way to enhance transcription of genes located up to many thousands of base pairs upstream (ahead of), downstream (down from ) or even in the middle of genes.
10. RNA Polymerase II in eukaryotes differs from RNA polymerase in E. coli in not binding to the DNA directly, but rather, it must bind to another protein that binds to the promoter first.
|Protein Synthesis I
1. In contrast to prokaryotic mRNAs, eukaryotic mRNAs are extensively modified. Modifications include
* Addition of a 5'-5' cap of a methyl guanosine to protect from degradation
* Addition of a poly-A tail at the 3' end under the control of the sequence AAUAAA near the end of the mRNA (also to protect from degradation)
* Editing - modification of bases chemically, such as was described in class for the Apo-B proteins
* Splicing - removal of introns between exons.
2. Splicing is a modification to eukaryotic mRNAs that occurs in the middle of the mRNA. Splicing also occurs to tRNAs and rRNAs in eukaryotes.
3. Splicing involves removal of internal sequences from RNA followed by joining of ends. The removed sequences are called introns. The segments that make it into the final RNA are called exons.
4. The only sequences common to all spliced RNAs are a GU sequence at the 5' end of the intron and an AG at the 3' end of the intron. A third sequence - an A residue surrounded by pyrimidines also is common.
5. Protein/RNA complexes called snRNPs mediate the splicing process in higher eukaryotes. snRNPs contain small nuclear RNAs (snRNAs) and proteins.
6. In splicing, the hydroxyl of the A residue attacks the phosphate of the phosphodiester bond at the 5' end of the intron, creating a 5'-2' bond (part of the lariat structure). Attack by the released 3' end of the exon on the 3' end of the intron joins the two exon ends and releases the intron as a lariat.
7. Exon shuffling (occurs in splicing in different tissues) allows cells to make many versions of protein from a single sequence. This is important in immunology and in fine-tuning cellular needs that are tissue specific.
8. Lower eukaryotes are able to excise introns by an autocatalytic mechanism. At least one prokaryotic gene is spliced autocatalytically.
9. In splicing, the U1 snRNA forms base pairing with the 5' end of the intron sequence.
10. In splicing, the U2 snRNA froms base pairs with the pyrimidine-rich region in the intron and with the snRNA of U6. Pairing with the intron forces outwards the 'A' residue that attacks the phosphate, as noted in class.
1. Translation is performed by ribosomes on mRNA and occurs in the 5' to 3' direction. The rate of translation in bacteria is about the same as the rate of transcription (45-50 bases or 15-17 amino acids per second). The 5' end of the coding region corresponds to the amino end of the protein. The 3' terminus of the coding region corresponds to the carboxyl end of the protein.
2. Translation is performed by ribosomes on mRNA and occurs in the 5' to 3' direction. The rate of translation in bacteria is about the same as the rate of transcription (45-60 bases or 15-20 amino acids per second). The 5' end of the coding region corresponds to the amino end of the protein. The 3' terminus of the coding region corresponds to the carboxyl end of the protein.
3. As polypeptides are being synthesized, the previously synthesized chain is attached to the free amine of the incoming (new) amino acid and the entire complex is, as a result, attached to the 'new' tRNA. Thus, polypeptides are synthesized in the amino to carboxyl terminus.
4. Transcription and translation are coupled together in bacteria, but not in eukaryotes.
5. Translational accuracy is about one error per thousand to ten thousand amino acids. Greater accuracy would slow translation down, so a balance is struck between the need for accuracy and the need to synthesize proteins reasonably rapidly.
|Protein Synthesis II
1. The anticodon loop has three bases complementary to the codon in the mRNA. tRNAs provide the translation function between nucleic acid sequence and amino acids. The anticodon loop frequently contains the inosine base. The base at the 3' end of the codon of the mRNA (corresponds to the base at the 5' end of the anticodon in the tRNA) is called the wobble base because it is less important for specifying the amino acid to be inserted than the first two bases.
2. Aminoacyl-tRNA synthetases have the ability to recognize and correct errors in joining of amino acids to tRNAs. For example, if one puts the wrong amino acid on the end of a tRNA and then adds an appropriate aminoacyl-tRNA synthetase, the amino acid is readily removed.
3. Two regions of aminoacyl-tRNA synthetases are important for editing - called the activation site and the editing site.
4. There are two classes of amino acid tRNA synthetases. They differ in the way they bind tRNAs and in which hydroxyl of the ribose ring they attach the amino acid to. Class I enzymes attach the amino acid to the hydroxyl on carbon #2. Class II enzymes attach the amino acid to the hydroxyl on carbon #3.
5. Base pairings in RNA are slightly different than in DNA. For example, G-U base pairs are not unstable. "I" (inosine) can also pair with C,U, or A.
6. In the genetic code, there are 64 possible combinations of the bases of the codon. Three of the possibilities (UAA, UGA, and UAG) are used as 'stop' codons. They tell the ribosomes where to stop making protein. A start codon is AUG and it codes for methionine. Since there are 61 codons used to code for amino acids and there are 20 amino acids, there is therefore 'redundancy' in the genetic code.
7. The Shine-Dalgarno sequence (GGAGG) is located near the AUG start codon in prokaryotic sequences. It is complementary to a sequence in the 16S rRNA and serves to help align the ribosome with the start site for translation in prokaryotes.
8. In prokaryotes, the first amino acid incorporated into a protein is a formylated form of methionine called fMet. The formyl group is put onto methionine after it is in the tRNA by a transformylase enzyme. Formylation of methionine in prokaryotes protects the otherwise free amino end from reacting intramolecularly and terminating transcription.
9. Peptides exit the ribosome as they are being synthesized via a tunnel in the structure.
10. Ribosomes have three sites for binding/holding/releasing tRNAs. They are called the A,P, and E sites, corresonding to the order in which tRNAs move through them (except for the very first one).
11. Initiation of protein syntheis starts with binding of IF1 and IF3 to the 30S ribosomal unit.
12. IF2 (when bound to GTP) acts to carry the Met-tRNAf to the P site of the 30S subunit and base pairs it with the AUG start codon. IF3 departs in the process. The complex of mRNA, IF1, IF2, and Met-tRNAf is called the 30S initiation complex.
13. Hydrolysis of the GTP in IF2 results in release of the IF2 and IF1 from the initiation complex. That, coupled with binding of the 50S subunit yields the 70S initiation complex with Met-tRNAf in the P site and the A and E sites open.
14. The process of elongation begins on the 70S initiation complex. EF-Tu (a G protein coupled to GTP) carries a charged tRNA to the A site of the complex. If the tRNA anti-codon base pairs properly with the codon in the mRNA, it stays matched with the codon and GTP is hydrolyzed on EF-Tu and EF-TuGDP is released. If the tRNA anti-codon does not form a stable base pairing with the complex, the entired charged tRNA-EF-Tu-GTP complex dissociates.
15. Next, the peptide group on the tRNA in the P site is transferred and covalently linked via peptide bond to the amino acid on the tRNA in the A site. This reaction is catalyzed by an enzymatic activity called peptidyltransferase - a ribozyme activity of the 23S rRNA in the 50S subunit.
16. The tRNA in the A site along with the peptide it is covalently attached to is transferred to the P stie as the "empty" tRNA in the P site is moved to the E site. EF-G-GTP is involved in the process and GTP is hydrolyzed in the process. EF-G-GTP has a similarity to the tRNAaminoacid-EF-Tu-GTP complex and may act to displace it.
17. As the old tRNA is released from the E site, the empty A site accepts the aminoacyl tRNA corresponding to the next codon. The net result of one turn of this cycle is that the polypeptide has grown by one amino acid residue and the ribosome has moved along the mRNA by three nucleotide residues. The process is repeated until a termination signal is reached.
|Gene Regulation I
1. Gene expression refers to the processes that result in the production of functional protein. Gene expression can be controlled at the levels of transcription, processing (splicing in eukaryotes), translation, mRNA stability, and protein stability. Tissue-specific gene expression is essential for multcellular, differentiated organisms.
2. Transcription factors, as noted previously, are proteins that bind to DNA and affect the transcription of genes located near where they bind. Common DNA-binding structures are found in the diverse set of transcription factors that are know. They include motifs (motifs - structural features) for helix-turn-helix, homeodomains, leucine zippers, and zinc fingers.
3. Leucine zipper structures are found in adjacent alpha helices and contain regions with leucine residues appearing about every 7 amino acids. The leucine interact with each other to hold the strands together and in doing so allow other portions of the helix to bind DNA properly.
4. Zinc fingers are structures with cysteine residues that hold zinc ions and create a finger-like structure that can stick into the DNA helix.
5. Proteins that bind to specific DNA sequences must "read" the sequence of bases inside the helix, usually by inserting a region into the major groove of the DNA and "checking" the hydrogen bonding molecules inside. Since different base pairs have unique hydrogen bonding orientations, the proteins that find and bind to specific base sequences.
6. Control of gene expression is also essential for prokaryotic organisms to be able to respond properly to their environments. For example, E. coli prefers glucose for energy, but must be able to use other sugars, like lactose, when they are available.
7. An operon is a prokaryotic system for organizing genes all under the same transcriptional control. Genes on the same operon in prokaryotes are all synthesized on the same mRNA. mRNAs containing multiple gene coding sequences are referred to as polycistronic.
8. The lactose operon consists of three linked structural genes that encode enzymes of lactose utilization, plus adjacent regulatory sites. The three enzymes --z, y, and a--encode beta-galactosidase, beta-galactoside permease (a transport protein), and thiogalactoside transacetylase (an enzyme of still unknown metabolic function), respectively.
9. Transcription of the lac operon commences at a promoter (lacP) before lacZ and transcribes a 5,200 nucleotide messenger RNA molecule (mRNA), ending at a terminator beyond lacA.
10. X-Gal is a synthetic substance used to study lac operon expression. X-Gal has the useful property that it turns blue when acted on by beta-galactosidase, giving a measure of how much the operon has been induced by the amount of blue color produced.
11. Negative transcriptional regulation of the lac operon is accomplished by a protein known as the lac repressor. It binds the operon's operator region and inhibits transcription.
12. In the absence of inducer molecules, the lac repressor tightly binds to the operator and inhibits transcription of the operon. When inducer molecules are present, they bind to the lac repressor and change its shape and reduce its ability to bind the operator, thus allowing the RNA polymerase to bind the promoter and start transcription.
13. The promoter sequence of the lac operon differs somewhat from the ideal consensus sequence of an E. coli promoter. Consequently, in the absence of positive acting elements, the lac promoter does not function well on its own. A protein that acts positively to help activate the lac operon is the CRP (cAMP Receptor Protein). (more on this next time)
|Gene Regulation II
1. Negative transcriptional regulation of the lac operon is accomplished by a protein known as the lac repressor. It binds the operon's operator region and inhibits transcription.
2. In the absence of inducer molecules, the lac repressor tightly binds to the operator and inhibits transcription of the operon. When inducer molecules are present, they bind to the lac repressor and change its shape and reduce its ability to bind the operator, thus allowing the RNA polymerase to bind the promoter and start transcription.
3. CAP (also called CRP) must bind to cAMP in order to function. When CRP binds cAMP, its affinity increases for the lac operon adjacent to the RNA polymerase binding site (-68 to -55). This binding facilitates transcription of the lac operon by stimulating the binding of RNA polymerase to begin transcription.
4. When both CAP and the lac repressor are bound to the lac operon, the repressor 'wins', shutting down transcription of the operon.
5. In eukaryotic cells, DNA is wrapped up (coiled up) with basic proteins called histones. Histone sequences are strongly conserved from yeast to humans.
6. Four histones form a core around which DNA is wrapped. This core contains two copies each of histones H2A, H2B, H3, and H4. This core of proteins is called an octamer.
7. The appearance of chromatin DNA is that of beads on a string, with the octamer wrapped with DNA composing the beads and the DNA strand coated with histone H1 (and H5) composing the string.
8. Histones of the octamer have strong structural similarity to each other.
9. Wrapping of DNA around the histone octamer provides only partial compression of the length of a DNA molecule. Additional compression occurs as a result of coiling of octamer/DNA complexes as well, forming higher order structures.
10. Enhancer sequences are bound by enhancer proteins and are found only in eukaryotes. Multiple enhance sequences may be present before the start site of a particular gene. Binding of enhancer proteins to enhancer sequences allows for tissue specific expression of genes if the enhancer proteins themselves are expressed tissue specifically. Enhancer proteins help to "clear" out the histones from a region of a chromosome to allow transcription to occur.
11. Nuclear hormone receptors, such as the estrogen receptor, have DNA binding domains and ligand binding domains. The binding of the estradiol (and estrogen) ligand to the estrogen receptor causes a conformational change in the protein, but does not change the binding of the protein to DNA. Binding of the estradiol DOES appear to activate the protein and thus activate transcription of the genes that the receptor binds to the promoter of.
12. The key to action of the nuclear hormone receptor that binds estradiol is that binding of estradiol favors binding of the receptor to co-activator proteins. These co-activator proteins help to turn on transcription of the relevant genes. Binding of co-activator proteins by transcriptional factors, such as the estrogen receptor is called recruitment.
13. An antagonist of the estrogen receptor is the drug tamoxifen. Antagonists bind proteins and prevent them from acting. Binding of tamoxifen by the estrogen receptor stops the receptor from activating transcription of genes that it normally activates.
14. Tamoxifen appears to act by binding the estrogen receptor (I use the terms estrogen receptor and nuclear hormone receptor here as the same thing), with a part of the molecule extending into the region of the protein that normally binds to co-activators. Thus, tamoxifen acts by stopping recruitment by the receptor of co-activators. Tamoxifen is used to treat tumors that are stimulated by the binding of estrogens to the receptor.
15. Altering chromatin structure is an essential function for transcriptional activation in eukaryotes. Co-activator proteins appear to play a role in this process by catalyzing the acetylation of lysine residues in histones. Acetylation of histone lysines neutralizes their positive charge, changing the affinity of histones for DNA and changing the nature of their interaction with DNA, thus allowing more proteins to be able to gain access to the DNA where the acetylation has occurred.
16. Proteins involved in transcriptional control often have bromodomains. These regions of protein recognize and bind to acetylated lysine residues in histones.
1. Smell arises from nerve signals originating in nasal epithelia. Molecular components of this process include 7TM proteins that bind odorants, which activates a G protein called Golf . Golf, in turn, binds GTP, activates adenylate cyclase, stimulating cAMP synthesis. cAMP binds to a cAMP-gates ion channel in the cell membrane allowing cations to enter the cell, starting the nerve signaling process.
2. Humans have only about 30% of their odorant rcceptors active, whereas rodents a large percentage of their 1000 receptors active. Olfactory receptors (ORs) are similar in slightly structure to the beta-adrenergic receptor involved in epinephrine signaling. Each olfactory neuron synthesizes only a single OR. This differs from individual taste buds, which each synthesize several receptors for tastes.
3. We are able to perceive a VERY wide range of smells, due to the combinatorial mixing of signals from the many different 7TMs at the end of olfactory cells.
4. OR signaling proceeds via 7TM receptors that synthesize cAMP when an odorant binds to the 7TM (through the usual mechanisms). cAMP binds to a channel protein that opens when cAMP binds to it, allowing Ca++ and Na+ into the cell, thus starting the signal.
5. Smell neurons terminate in very different regions of the brain.
6. Taste sensing is related to smell. Not all tastes have odors, however. In contrast to smell, we only have 5 primary tastes we detect through taste buds located on distinct areas of the tongue. The five primary tastes are sweet, sour, bitter, salty, and umami.
1. Taste sensing is related to smell. Not all tastes have odors, however. In contrast to smell, we only have 5 primary tastes we detect through taste buds located on distinct areas of the tongue. The five primary tastes are sweet, sour, bitter, salty, and umami.
2. Taste buds contain about 150 cells with microvilli projections that are rich in taste receptors. About 50-100 7TM receptors are involved in taste. Not all primary tastes are associated with 7TM receptors, however.
3. Salty tastes are detected by passage of the ions directly through ion channels on the tongue. One class of these channels is sensitive to amiloride, which obscures the taste of salt and lowers neuron activity relating to sodium.
4. A G protein associated with the taste receptors that use 7TM receptors is called gustducin.
5. Whereas individual olfactory receptors are uniquely expressed on each olfactory neuron, with direct 'wiring' to distinct areas of the brain, taste receptors are not uniquely expressed on taste buds and the entire set of taste buds for primary tastes link to a single area of the brain.
6. Vision arises from signaling initiating in rod and cone cells of the eye. Rod cells (sense light, but not distinct for color) contain the pigment rhodopsin, which consists of a 7TM protein called opsin linked to a vitamin A derivative called retinal (linked via a lysine bond). Retinal is light sensitive and can flip between the 11-cis form and the all-trans form when exposed to light. This slight change in structure of retinal also changes the rhodopsin protein, ultimately activating a vision-related G protein called transducin.
7. Transducin binds GTP when active and this causes transducin to activate a specific phosphodiesterase to break down cGMP to GMP. Lowering cGMP concentration causes a cation ion channel to stop the movement ions of ions into the cell, starting the nerve signal.
8. Cone cells of humans have pigment-specific receptors for red, green, and blue light. We differ from more closely related organisms by virtue of the fact that we have evolved red receptors from our green receptor. Dogs and rodents, for example do not have red receptors.
9. The protein that binds the retinal in each of these is similar to the opsin of rod cells (40% identity). Color blindness (usually in males) arises due to recombination between related red and green receptor genes on the X chromosome.
10. Hearing arises as a result of signaling in the ear arising from hair cell micromanipulation (movement) arising from sound waves. Tipping of the cells causes little "tethers" to pull open ion channels that initiate a nerve signal.
11. Touch is the least understood sense. There are receptors for pressure, temperature and other sensations. Touch receptors can be linked to pain centers called nociceptors located in the spinal cord and brain. Interestingly, the compound capsaicin, which causes hot sensations in the mouth stimulates the same nociceptors.
12. The capsaicin receptor is a transmembrane protein. This protein contains a pore that opens to allow calcium ions in when capsaicin binds, thus initiating a nerve signal. Mice lacking the capsaicin receptor do not respond at all to capsaicin.
This information is provided for all of you who love learning.
1. The immune system contains the innate immunity system and the adaptive immunity system.
2. The innate system uses a Toll-like receptor that binds to the PAMP lipopolysaccharide structure on the surface of Gram negative bacteria.
3. The adaptive immune system system contains two major groups of lymphocytes (immune system cells), B cells and T cells. B cells are involved in the production of antibodies and T cells are involved in both cellular killing, as well as stimulation of the B cells.
4. Immunoglobulin G (IgG) is one of five major antibody classes made by the B lymphocytes of the humoral immune system (cellular immune system described below). IgG is the most abundant antibody in the blood serum. Others include IgA (in mucus), IgM (early responder), IgD (function uncertain), and IgE (parasite protection).
5. The structure of antibodies has several common features. First, they are composed of two sets of Heavy (H) and light (L) chains arranged in a Y shape. Both the H and L chains have constant and variable regions. The variable regions of the H and L chains are adjacent to each other and the variation in these regions are responsible for antibody diversity (108 different shapes). The different classes of antibodies vary in the H chains in the constant region.
6. Molecules bound by antibodies are called antigens. Specific structural regions of an antigen bound by an antibody are called epitopes.
7. IgM antibodies are the first responders of the humoral immune system.
8. Antibody diversity arises from recombination of DNA sequences and splicing of mRNA sequences for coding for the variable regions of H and L chains.
9. For the light chains, the coding sequences are on human chromosome 2 and involve three protein domains - V, J, and C regions. V is the variable portion, J is the portion that joins the V to the C, which is the constant region. Mixing and matching of domains by recombination generates tremendous diversity - over 1000 antibodies per base pair of the human genome.
10. In H chains (chromosome 14), recombination occurs between segments V,D, J and C. Recombination within the constant regions of the H chains can result in "class switching" in which a desired variable segment is swapped among to the various segments to put the binding site onto regions to make IgG, IgE, etc.