General Biochemistry II with Kevin Ahern

Video Lectures

Displaying all 25 video lectures.
Lecture 1
Citric Acid Cycle I
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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.
Lecture 2
Citric Acid Cycle II
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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.
Lecture 3
Lipids & Membranes
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Lipids & Membranes













Lecture 4
Membrane Transport
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Membrane Transport
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.


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).
Lecture 5
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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.
Lecture 6
Oxidative Phosphorylation
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Oxidative Phosphorylation
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.

Oxidative Phosphorylation


Lecture 7
Lipid Metabolism, Fat Transport in the Body
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Lipid Metabolism, Fat Transport in the Body

a. Intact inner mitochondrial membrane
b. Electron transport creates a proton gradient



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


O goes to L
L goes to T
T goes to O






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.
Lecture 8
Fatty Acid Oxidation
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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.
Lecture 9
Fatty Acid Synthesis
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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.
Lecture 10
Fatty Acid Synthesis/Prostaglandins
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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.



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.






Lecture 11
Nucleotide Metabolism I
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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.
Lecture 12
Nucleotide Metabolism II
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Nucleotide Metabolism II

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.


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.






Lecture 13
DNA Replication, Recombination, Repair I
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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.



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.
Lecture 14
DNA Replication, Recombination, Repair II
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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).
Lecture 15
DNA Replication, Recombination, Repair III
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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.






Lecture 16
General Biochemistry Review
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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.



Lecture 17
Transcription I
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Transcription I


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.



Lecture 18
Transcription II
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Transcription II







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.
Lecture 19
Protein Synthesis I
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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.
Lecture 20
Protein Synthesis II
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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.
Lecture 21
Gene Regulation I
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Gene Regulation I


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.





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.




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)
Lecture 22
Gene Regulation II
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Gene Regulation II







8. Histones of the octamer have strong structural similarity to each other.








Lecture 23
Sensory Systems
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Sensory Systems

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.




Lecture 24
Immune System
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Immune System

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.





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.
Lecture 25
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This information is provided for all of you who love learning.


2. The innate system uses a Toll-like receptor that binds to the PAMP lipopolysaccharide structure on the surface of Gram negative bacteria.


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.


7. IgM antibodies are the first responders of the humoral immune system.


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.