NAME CHEM 4731, Final Exam

The test is divided into several sections. As noted in each section, you must answer a certain number of questions. Please answer the questions with great brevity and even greater clarity. Only use the space provided, unless noted otherwise.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

Section I. Carbohydrates. Answer 4 of the following 5 questions.

1. Draw the general structure of glycogen. Make sure to note the reducing and non-reducing ends of the molecule and the monomeric unit of which glycogen is composed (you do not have to draw out the chemical structure of this monomeric unit). Why is it important that glycogen have this general structure?

2. What are 4 key biological roles that protein glycosylation is thought to play?

3. A) The regulation of glycogen involves a cascade system. What are 2 advantages of using a cascade? B) Suppose that you force a non-coffee drinker to consume 4 large cups of coffee. How will this affect glycogen breakdown and synthesis, and why? (Note: You should not consider the effects on glycogen metabolism of their having to run to the bathroom multiple times.)

4. Suppose that you are studying a new organism, and note that it has 2 different metabolic pathways, one that converts A into B and a second metabolic pathway that converts B into A. A) What predictions would you make regarding the regulation of the two pathways? B) Will the pathways likely use the same or different enzymes? Why?

5. Glycogen phosphorylase catalyzes the following reaction. Draw a likely chemical mechanism for this reaction on the back of this page. (Note that the reaction goes with retention of stereochemistry.)

Section II. Membrane Constituents and Fatty Acid Metabolism. Answer 4 of the following 5 questions.

6. A) What is the biological role of bile acids? B) Suppose that a person was deficient in bile acids. How would this affect the composition of chylomicrons and low density lipoproteins?

7. Briefly outline the pathway of ketogenesis. Why is this pathway biologically important?

8. Inhibiting phospholipases is an effective means of preventing inflammatory responses. Describe at a molecular level why inhibiting phospholipases has this effect.

9. Briefly outline the pathway of fatty acid breakdown. Make sure to note any key cofactors and the roles they play.

10. A) What is the committed step in fatty acid biosynthesis? B) List 3 mechanisms that eucaryotic cells use to move lipids within a cell.

Section III. Amino Acid Metabolism. Answer 2 of the following 3 questions.

11. Glutamine synthase is the key regulated enzyme for nitrogen metabolism. In prokaryotes, what are the two major mechanisms of regulation, and briefly describe each mechanism.

12. A pyridoxal dependent transaminase catalyzes the following reaction. Draw a likely mechanism of this reaction on the back of this page.

13. Glutamine synthase catalyzes the following reaction. Draw a likely mechanism of this reaction on the back of this page.

Section IV. Nucleotide and DNA Metabolism. Answer 8 of the following 10 questions.

14. A) Draw the base-pair formed between dATP and 3’-TMP. B) Draw the base pair formed between guanosine and 5’-CMP.

15. Briefly outline the polymerase chain reaction. Describe why it is useful for molecular biology and list two potential problems with the products you finally obtain as compared to what you want to obtain.

16. A) What are the two major pathways used for the synthesis of pyrimidines and purines? B) What is the end product of purine degradation and why is this compound potentially problematic?

17. Methotrexate, an inhibitor of dihydrofolate reductase, and 5-fluorouracil, a compound that ultimately gives rise to an inhibitor of thymidylate synthase, are often used as cancer chemotherapeutics. However, the two drugs given simultaneously are not more toxic to the tumor cells than either drug alone and, therefore, they are not given to a patient simultaneously. In terms of the enzymes they inhibit, why is the combination of these two drugs no more toxic to cells than either drug alone?

18. In terms of the 4 major forces stabilizing DNA and nucleic acid structure, describe how each force affects a DNA helix.

19. Suppose that you inhibited the RNase H activity of reverse transcriptase. How would this affect the ability of reverse transcriptase to make a double-stranded DNA copy of double-stranded RNA and why.

20. A) Why does the UV absorbance of DNA change when it is denatured (i.e., double-stranded single-stranded. B) Briefly outline the process of DNA renaturation (ssDNA dsDNA).

21. DNA polymerase I of E. coli has three enzymatic activities. What are these three activities, and what role does each play with respect to the role(s) of DNA polymerase I during DNA replication by E. coli.

22. What are the roles of the following proteins during DNA replication. DNA primase, topoisomerase, helicase, and DNA ligase.

23. Suppose that you were studying E. coli, and inactivated the methylase activity associated with a restriction endonuclease found within the cell. Now, you allow the E. coli to continue growing. How would this affect the cellular DNA during the next two generations? Why?

Section V. Recombination, Transcription, Translation and the Unexplained Complexity of Eucaryotes. Answer 14 of the next 16 questions.

24. Suppose that you developed an E. coli that had a mutant ribosomes. These ribosomes are unusual in that when a needed tRNA-amino acid is absent, these ribosomes rapidly dissociate from the RNA template. How would this affect attenuation control and why?

25. Suppose that you are trying to develop RNA ligands that bind to leucine but not isoleucine using SELEX. For doing the SELEX, you have attached the leucine to a column via the carboxylate. After 10 rounds of SELEX, you discover that your pool of RNA molecules binds to leucine and isoleucine with approximate equal affinity. How could you modify your SELEX procedure so that the pool of RNA molecules only bound to leucine, and not to isoleucine?

26. What are 3 major differences between eucaryotic and procaryotic transcription.

27. Briefly describe how RecA and RecBCD would work together to catalyze DNA recombination. You do not have to describe how these proteins might be involved in resolution of the Holliday junction. (Hint: It would probably be easier to use the Meselson-Radding model of recombination as the basis of your description).

28. What are 3 major structural motifs of DNA binding proteins found in eucaryotes. Briefly describe each motif.

29. Briefly outline the mechanism by which translation is initiated in E. coli. Make sure to note the function of each of the initiation factors.

30. Briefly outline the mechanism by which RNA polymerase initiates transcription in E. coli.

31. What are 3 types of repetitive DNA found in eucaryotes?

32. Suppose that you developed a new l that contained a mutated cro protein. This form of cro binds 10-fold less tightly to OR1, OR2, and OR3. How and why would this A) affect the choice between a lytic and lysogenic infection when l first infects an E. coli and; B) the ability of l to escape from lysogeny?

33. You are studying how a amino acyl-tRNA synthetases recognizes its cognate tRNA. How would you determine which nucleotides in the tRNA are important for recognition by the synthetase?

34. The DNA between two identical sequences is often genetically unstable. Show why this is so.

35. There are two mechanisms by which transcription is terminated in E. coli. Outline each method.

36. Suppose that you have a cis dominant mutation in the lac operon that results in constitutive expression of this operon. What is a likely protein(s) or DNA sequence(s) where this mutation occurred, and what would be the molecular effects of this mutation.

37. A) What is chromatin and why is it important? B) Suppose that you introduced positive supercoils into eucaryotic DNA. How would this likely affect nucleosome structure?

NAME CHEM 4731, Exam 1

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. Note that some of the questions count as 2 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. Draw the structures of the following key starting materials for various pathways.

Glucose Ribulose-1,5 bisphosphate Phosphatidic Acid

2. Draw a likely mechanism for the following enzyme catalyzed reaction. (You may use the back of this page for the mechanism.)

Now, answer any 14 of the next 16 questions.

3-4. Photosynthesis contains two sets of reactions, the light and dark (or non-light requiring) reactions. Briefly outline each process. You do not need to list all intermediates, just the key starting materials and products.

5. In comparing glycolysis and gluconeogenesis, several steps are different. Why are these steps different in each process and what are these key steps?

6. Outline the pathway for fatty acid synthesis, including the release of the key final product. What is the key committed intermediate in this process?

7. Outline the pathway of N-linked protein glycosylation.

8. What is cyclic e- electron flow and why is it important for photosynthesis?

9. Draw a likely mechanism for the following enzyme catalyzed reaction. (You may use the back of this page for drawing out the mechanism)

10. Why is S-adenosylmethionine biologically important and what is it’s structure?

11. What would happen to regulation of glycogen synthesis/breakdown if you completely inhibited protein phosphatase upon treating a cell with epinephrine? Why? When a cell is treated with epinephrine, what key second messenger is produced?

12. What are the 3 major classes of N-linked oligosaccharides and how do they differ?

13. Is the rate of phospholipid movement between leaflets of a membrane slow or fast when using: (i) a purified phospholipid membrane and; (ii) in a whole cell? In each case, why?

14. What are ketone bodies and why are they important? (In terms of why they are important, make sure and talk about the starting material for their synthesis.)

15. The following molecules are regulators of glycolysis and/or gluconeogenesis in various tissues. How do they regulate each pathway (up or down) and provide a rationalization for why they have this effect.

Fructose 2,6 bisphosphate

Acetyl CoA

AMP

16. In terms of lipid metabolism, what are the key role(s) of chylomicrons, low density lipoprotein and high density lipoprotein. If you want to die of a heart attack at a very early age, which of these species should be greatly increased and which should be greatly decreased?

17. Fatty acids are oxidized in the mitochondria. What are 3 reasons why it is advantageous to oxidize fatty acids in the mitochondria rather than in the cytosol.

18. Draw the general structure of glycogen. Make sure to note the reducing and non-reducing ends of the molecule and the monomeric unit of which glycogen is composed (you do not have to draw out the chemical structure of this monomeric unit). Why is it important that glycogen have this general structure?

NAME CHEM 4731, Exam 2

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. Note that some of the questions count as 2 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

1. Draw a chemically plausible mechanism for the following enzyme-catalyzed reaction on the back of this page.

2. A key feature of ribonucleotide reductase is it’s regulation. Suppose that a cell experienced the following conditions. How would they affect the activity of ribonucleotide reductase and, consequently, the level of each dNTP within the cell. A) Due to defective deamination of deoxyadenosine, the cell accumulates a large excess of dATP. B) You treat cells with deoxythymidine such that a large excess of dTTP accumulates within the cells.

Now, answer and 14 of the next 16 questions.

3. What are 3 potential advantages provided to a cell by using multienzyme complexes for nucleotide biosynthesis?

4. Suppose a cell was under the following conditions with respect to nucleotide concentrations: high CTP, low UTP, high ATP and GTP. How would the following enzymes be regulated and why? (For why, you just have to say in a few words the key role of each enzyme).

PRPP synthetase (synthesizes PRPP)

PRPP amidotransferase (synthesizes 5-phosphoribosylamine)

Aspartate transcarbamoylase (synthesizes carbamoylaspartate)

CTP synthetase

5. What is the reaction catalyzed by nitrogenase? (You should not draw out the catalytic cycle. List the starting materials and products, including stoichiometry.) Why is it so important biologically?

6. Draw a chemically plausible mechanism for the following enzyme catalyzed reaction on the back of this page.

7. Prokaryotic glutamine synthetase is regulated by cumulative feedback inhibition. What is cumulative feedback inhibition and why is it effective with this enzyme?

8. Suppose that you introduced a PEST sequence into a protein at a position that was unimportant for protein activity or thermodynamic stability (i.e., the PEST sequence does not affect the equilibrium between the folded and unfolded states). How would this likely affect the protein in vivo and why?

9. Describe the general pathway of ubiquitin dependent protein degradation.

10. Suppose you discovered a new amino acid and wanted to determine if it was a ketogenic or glucogenic amino acid. Experimentally, how would you do this?

11. Suppose that you blocked the activity of glutamate dehydrogenase found in liver (This enzyme catalyzes the reaction: -ketoglutarate + NH3 + NADH + H+ Glutamate + NAD+ + H2O). How would this affect urea production and why?

12. Suppose that you developed an inhibitor of the enzyme tyrosine hydroxylase (catalyzes the reaction tyrosine + O2 + H4biopterin Dihydroxyphenylalanine + H2biopterin + H2O). Since you are the inquisitive type and are interested in the biological consequences of new inhibitors, you place some in your roommates morning coffee. Assuming that your roommate consumes this beverage, what biological consequences would result at the molecular level (Note: while death or severe injury are clearly biological consequences, they are not considered descriptions at the molecular level).

13. Besides causing heart attacks if present at very high levels, what are 3 biological roles of cholesterol?

14. Suppose a cell contained an enzyme that catalyzed the interconversion of NADH NADP+? How would this affect the cell and why?

15. Inhibiting phospholipases is an effective means of preventing inflammatory responses. Describe at a molecular level why inhibiting phospholipases has this effect.

16. A) Why are guanosine analogs, as opposed to adenosine, cytidine or thymidine analogs, used for treatment of Herpes infections? B) Suppose that you are a world famous oncologist, and a patient with a particularly virulent tumor comes to see you. You discover that cells from this tumor lack ribonucleotide reductase, but do have extremely high levels of the enzymes required for salvage of all four deoxynucleosides. A fellow doctor down the hall suggests that you treat this tumor with 5-fluorouracil. Is this fellow doctor a genius or an idiot, and why?

17. A) What is the biological role of bile acids? B) Suppose that a person was deficient in bile acids. How would this affect the composition of chylomicrons and low density lipoproteins?

18. Muscles secrete large amounts of alanine into the blood that is taken up by the liver. Why do muscles secrete this alanine, and why is it advantageous for muscles to use alanine as opposed to any of the other amino acids?

NAME CHEM 4731, Exam 3

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. Note that some of the questions count as 2 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

1. A) Draw the base-pair formed between dATP and 3’-TMP. B) Draw the base pair formed between guanosine and 5’-CMP.

2. What are the 3 "mechanisms" that cells use to minimize errors during DNA replication. Briefly describe each mechanism.

Now, answer and 14 of the next 16 questions.

3. How will strong base (pH=12) affect RNA and DNA? Show why this is true chemically.

4-5. Outline the mechanism of the replication fork as discussed with E. coli. Make sure to mention the DNA polymerase(s) involved, DNA primase, helicase, topoisomerase, processivity factor and single-stranded DNA binding protein, and their role(s).

6. How does ganciclovir triphosphate affect DNA synthesis and why is it toxic to eucaryotic cells?

7. What are 3 biochemical functions of single-stranded DNA binding protein?

8. In the E. coli origin of replication, there are several A:T rich regions. How would increasing the G:C content of these regions affect initiation of replication and why?

9. In terms of DNA replication, a linear chromosome can be problematic. What is the potential difficulty with linear chromosomes and what are 3 potential solutions to this problem (just list the solutions, do not describe them).

10. Briefly outline the polymerase chain reaction and describe why it is useful for molecular biology.

11. Briefly outline the chemical synthesis of DNA. List 2 factors that limit the length of oligonucleotide that can be synthesized in reasonable yield.

12. Suppose that you develop mutant E. coli strains that lack either DNA photolyase or the uvrB protein, a key component in excision repair. Which mutation would be more deleterious to E. coli and why?

13. It has been proposed that by excluding solvent from the active site of a DNA polymerase, this will enhance the ability of the polymerase to discriminate between the correct and incorrect dNTP. Is this a good or a bad idea, and why (Make sure to address hydrogen bonding and stacking interactions in your answer!)?

14. You are sequencing the following piece of DNA

using the dideoxy method: 3’- AAGCGGCTAATCC-5’.

Accidentally, you forget to include dATP in the four

reactions that contain a ddNTP. Show the band

pattern you would obtain on a gel.

15. Suppose that you eliminate the activity of DNA-adenine methylase (dam methylase), the enzyme that methylates adenine in GATC sequences. How will this affect mismatch repair in the following 2 generations of E. coli. (Make sure to describe what will happen in each generation.)

16. You discover a new protein that binds to DNA and introduces a bend in the DNA. In the region of the protein that contacts the phosphate residues on the inside of the bend, list 3 amino acids that you would expect to be found making these contacts and state why they would be there.

17. Suppose that you isolate a new topoisomerase, and want to know if it is a type I or type II topoisomerase. Provide an experimental strategy for how you would answer this question.

18. A) What are 2 mechanisms by which cells minimize the amount of uracil found in DNA? B) Eucaryotic cells contain large amounts of 5-methylcytosine. Why is this potentially problematic to the cell, as opposed to having cytosine?

Name Due in class on 9/11.

Chem 4731 Problem Set 1

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism questions, you can use the back of the page.

1. Suppose a cell lacked phosphatase inhibitor protein. How would this affect glycogen metabolism when the cell is treated with epinephrine and why?

2. Suppose that cells synthesized glycogen from the reducing end to the nonreducing end (i.e., as discussed in class and the text), but also broke down glycogen from the reducing end to the nonreducing end (i.e., in the opposite sense as it actually occurs). After a cell has been stimulated to break down glycogen, how would these changes alter the rate and pattern of glycogen breakdown?

3. Is AMP thought to be a major regulator of: (a) glycolysis in muscle cells, and (b) glycolysis and gluconeogenesis in liver cells? Why or why not?

4. The biotin dependent enzyme catalyzes the reaction: Acetyl CoA + ATP + HCO3- Malonyl CoA + ADP + Pi. Draw a likely mechanism for this reaction.

5. The enzyme phosphoenolpyruvate carboxykinase catalyzes the reaction: oxaloacetate + GTP phosphoenolpyruvate + GDP + CO2. Draw a likely mechanism for this reaction.

Name Due in class on 9/23.

Chem 4731 Problem Set 2

1. Suppose a cell contained an enzyme that could transfer a hydride between NADH and NADP? How would this affect the cell and why?

2. Suppose that the dark reactions of photosynthesis could only occur in the dark? How would this affect a plant cell and why?

3. What is a major advantage for a muscle cell to use glucose as an energy source rather than a fatty acid? Which is a better store for long term energy needs, fat or sugars? What are two reasons why this is so.

4. Fatty acids are oxidized in the mitochondria. What are 3 reasons why it is advantageous to oxidize fatty acids in the mitochondria rather than in the cytosol.

5. Draw a likely mechanism for the reaction catalyzed by 2,4 dienoyl CoA reductase.

Chem 4731 Problem Set 3/4

1. Why are inhibitors of HMG CoA reductase used to treat high blood cholesterol, as opposed to inhibitors of other enzymes in the cholesterol biosynthetic pathway?

2. Suppose you discovered a new amino acid and wanted to determine if it was a ketogenic or glucogenic amino acid. Experimentally, how would you do this?

3. Aspirin therapy is used to help prevent heart attacks. Treatment inhibits prostaglandin synthesis by the platelets, thereby inhibiting clotting and, consequently, heart attacks. In contrast, ibuprofin, which also inhibits prostaglandin synthesis by platelets is not used to prevent heart attacks. Why is aspirin used and not ibuprofen? (Hint: Platelets cannot synthesize new proteins.)

4. What are 3 methods that a cell will use to regulate the half-life of a protein.

5. For the following pyridoxal dependent decarboxylation reaction, write a chemically plausible mechanism.

6. What are 3 potential advantages are provided to a cell by using multienzyme complexes for nucleotide biosynthesis.

7. For regulation of purine biosynthesis, comment on the advantages/disadvantages of regulating PRPP synthetase and/or PRPP amidotransferase.

8. What are the advantages and disadvantages of using i) uric acid; ii) ammonia, and; iii) urea for elimination of nitrogen.

9. Suppose that a cell had excess phenylalanine hydroxylase. How would this likely affect amino acid metabolism? Suppose that this excess activity had deleterious consequences for the afflicted individual. How might you attempt to solve their problem?

10. Draw a plausible chemical mechanism for tyrosine hydroxylase.

Chem 4731 Problem Set 4

1. It has been proposed that by excluding solvent from the active site of a DNA polymerase, this will enhance the ability of the polymerase to discriminate between the correct and incorrect dNTP. Is this a good or a bad idea, and why (Make sure to address hydrogen bonding and stacking interactions in your answer!)?

2. How will i) positive and ii) negative supercoiling affect the ability of a DNA helicase to unwind DNA?

3. In double-stranded DNA, both strands of the DNA contain phosphodiester bonds. Suppose you replaced all of the phosphodiester linkages in one strand with methylphosphonate linkages. How would this affect the structure of the DNA and why? Note: Replacing the methylphosphonate linkage does not affect base-pairing or base-stacking, is chemically stable, and is has no charge.

4. Propose an experimental strategy to address if the 3’ 5’ exonuclease is important for fidelity of DNA pol I.

5. Various experimental data indicate that both topoisomerase I and II are involved in DNA replication in yeast. If you then delete all topoisomerase I activity, the yeast replicate their DNA more slowly, but they complete replication and survive. However, if you delete all topoisomerase II activity, the yeast cannot replicate their DNA and they die. Why is topo I activity not essential for DNA replication but topo II activity essential?

Chem 4731 Problem Set 5

1. Suppose that you were sequencing a piece of DNA

with the sequence: 5’-ATCGTTACG-3’. Draw the expected

gel pattern if you were sequencing using the dideoxy method.

2. Suppose that you wanted to produce a large amount of a new type II restriction endonuclease you have discovered, and you decide to do this by cloning the gene into E. coli and then using E. coli to produce the enzyme. Is this smart or stupid, and why?

3. Suppose that you changed the A:T rich regions of oriC into G:C rich regions. How would this affect initiation of replication and why?

4. What kinetic parameter(s) and concentration(s) would you need to know to predict the frequency with which the polymerase activity will misincorporate dCMP opposite a template dA at a specific site in a DNA. (Do not include effects of the proofreading exonuclease in your answer.)

5. Suppose that you had a culture of E. coli cells that was actively replicating its DNA. Now, you introduce a very potent inhibitor of DNA primase. How would this affect leading and lagging strand DNA synthesis and why?

Chem 4731 Problem Set 6

1. Suppose that a cell lacked recA. How would this affect the ability of l to escape from lysogeny and why?

2. Suppose that you made a mutant form of RNA polymerase that in the absence of a needed tRNA-amino acid, rapidly dissociated from the RNA template. How would this affect attenuation control and why?

3. You have isolated a new DNA binding protein, and want to know with which nucleotides this protein interacts and the importance of the base at each position. Very briefly, describe how you would solve these questions.

4. What is the primary difficulty with the Holliday model of recombination that is solved by the Mesolson-Radding model?

5. Suppose that you developed a l that when it infected E. coli, always gave a lytic infection. However, if the E. coli had been infected by another, wild-type l , infecting the E. coli with your mutant l did not result in a lytic infection. In what protein or DNA sequence have you likely introduced a mutation and what effect did this mutation have on the functional properties of this protein or DNA sequence?

Chem 4731 Problem Set 7

1. Will attenuation control likely be used by eucaryotes? Why or why not?

2. Suppose that you are trying to develop RNA ligands that bind to leucine but not isoleucine using SELEX. For doing the SELEX, you have attached the leucine to a column via the carboxylate. After 10 rounds of SELEX, you discover that your pool of RNA molecules binds to leucine and isoleucine with approximate equal affinity. How could you modify your SELEX procedure so that the pool of RNA molecules only bound to leucine, and not to isoleucine?

3. For the leucine-tRNA synthetase, would you expect the anticodon loop to be a major determinant for the specificity of this synthetase towards the tRNA? Why or why not?

4. What are 3 mechanisms by which the cell avoids incorporating incorrect amino acids during translation?

5. Suppose that you wanted to determine if a ribosomal protein bound to a rRNA and where it bound. Briefly outline procedures that you would use to answer each issue (Note: You do not have to describe how the procedures work, just state what the procedures would tell you.).

NAME CHEM 4731, Final Exam

The test is divided into several sections. As noted in each section, you must answer a certain number of questions. Please answer the questions with great brevity and even greater clarity. Only use the space provided, unless noted otherwise.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

Section I. Carbohydrates. Answer 4 of the following 5 questions.

1. A) In general, if you treat cells with a compound that activates gluconeogenesis, how will this compound affect glycolysis. Why? B) Draw the general structure of glycogen, making sure to note the reducing and nonreducing ends. Why is it important for glycogen to have this general structure?

2. Suppose that plant cells had very high levels of ATP and low levels of NADPH. How would this affect photosystems I and II? Why?

3. A) The regulation of glycogen involves a cascade system. What are 2 advantages of using a cascade? B) Suppose that develop a cell that has a 5-fold higher level of phosphodiesterase. How will this affect the regulation of glycogen metabolism and why?

4. Suppose that you treated cells with a specific inhibitor of N-linked protein glycosylation. What are 4 biological processes that are likely to be affected?

5. A biotin dependent enzyme catalyzes the following reaction. On the back of this page, draw a likely mechanism for this reaction.

Section II. Membrane Constituents and Fatty Acid Metabolism. Answer 3 of the following 4 questions.

6. Suppose that you developed a mutant person with a very high level of the low density lipoprotein receptor. How would this affect cholesterol metabolism and why?

7. Briefly outline the pathway of ketogenesis. Why is this pathway biologically important?

8. A) What is the general reaction catalyzed by phospholipases? B) Why are phospholipases considered attractive targets for developing new anti-inflammatory drugs?

9. Draw a likely mechanism for the following enzyme catalyzed reaction.

Section III. Amino Acid Metabolism. Answer 2 of the following 3 questions.

10. Suppose that you wanted to alter the biological half life of a protein inside of cells. What are three modifications that you could make to that protein to decrease it’s half life?

11. A pyridoxal dependent transaminase catalyzes the following reaction. Draw a likely mechanism of this reaction on the back of this page.

12. Draw a likely mechanism for the following enzyme catalyzed reaction.

Section IV. Nucleotide Metabolism, DNA Metabolism and Procaryotic Transcription. Answer 8 of the following 10 questions.

13. Briefly outline the mechanism by which procaryotic RNA polymerase initiates transcription.

14. Shown below is a hypothetical replication bubble, with the polarity of the parental DNA shown. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

15. A) What are the two major pathways used for the synthesis of pyrimidines and purines? B) What is the end product of purine degradation and why is this compound potentially problematic?

16. Suppose that you deleted the CRP (cAMP Receptor Protein) binding site from the lac operon. How would this affect the rate of transcription from the lac operon in the A) presence, and; B) absence of lactose, and why?

17. In terms of the 4 major forces stabilizing DNA and nucleic acid structure, describe how each force affects a DNA helix.

18. Replication of linear chromosomes is potentially problematic. What is the major problem and what are 3 potential solutions to this problem?

19. Suppose that you developed a form of RecBCD protein that had enhanced ability to nick single-stranded DNA in a non-sequence specific manner. How would this likely affect recombination in E. coli and why?

20. Suppose that you treat cells with a compound that destroys radicals bound to enzymes. How will this likely affect DNA replication and why?

21. During DNA replication, imbalances in the relative amounts of each dNTP are mutagenic (i.e., more mismatches are generated by the DNA polymerases). What are 2 mechanisms by which increasing the concentration of a single dNTP can lead to increased levels of mutagenesis?

22. A) What are 2 mechanisms by which uracil can be incorporated into DNA? B) Briefly describe the process of nick translation.

Section V. Translation, Eucaryotic Transcription, DNA in the Nucleus, and Signaling. Answer 15 of the next 17 questions.

23. What are three key differences between regulation of eucaryotic and procaryotic transcription?

24. With respect to transcription, what are co-activators and what are insulators?

25. Enhancers often bind to DNA at great distances from the core promoter. In terms of how they could affect transcription by RNA pol II, this could be problematic. What are two mechanisms to overcome this potential problem?

26. Suppose that you are in vitro studies of a pathway that is activated by a heterotrimeric G protein (i.e., you are using purified enzymes for the studies). Now, you replace the GTP in your studies with the non-hydrolyzable GTP analog gamma-thioGTP. How will this affect this pathway and why?

27. What are two general mechanisms by which binding of a ligand to a membrane bound receptor is thought to activate that receptor?

28. Suppose that you are working for a drug company, and are trying to discover compounds that bind to and inhibit a steroid hormone receptor (ex., the estrogen receptor). In your experiments using purified receptor, you discover two very potent inhibitors. Compound A is rather hydrophobic (the only hydrophilic groups on the molecule are two hydroxyl groups) and binds to the receptor with a KD of 1 nM, while compound B is rather hydrophilic (it contains two carboxylic acid groups) and binds to the receptor with a KD of 1 nM. Which compound is likely to be more efficient at inhibiting the receptor when treating whole cells?

29. At the molecular level, describe how the start site for RNA pol II transcription is determined.

30. Suppose that you are trying to develop RNA ligands that bind to ATP but not GTP using SELEX. For doing the SELEX, you have attached the ATP to a column via the g -phosphate. After 10 rounds of SELEX, you discover that your pool of RNA molecules binds to ATP and GTP with approximate equal affinity. How could you modify your SELEX procedure so that the pool of RNA molecules only bound to ATP, and not to GTP?

31. DNA replication and transcriptional regulation in eucaryotes are thought to be linked. What is this link?

32. What are 3 major structural motifs of DNA binding proteins found in eucaryotes. Briefly describe each motif.

33. Briefly outline the mechanism by which translation is initiated in E. coli. Make sure to note the function of each of the initiation factors.

34. In prokaryotes, there are three major mechanisms by which translation is regulated. List these three mechanisms and briefly describe them.

35. It is essential that translation occur with reasonably high fidelity. Briefly describe three major mechanisms by which translational fidelity is obtained.

36. You are studying how an amino acyl-tRNA synthetase recognizes its cognate tRNA. How would you determine which nucleotides in the tRNA are important for recognition by the synthetase?

37. Suppose that you developed an inhibitor that specifically blocked the E site of ribosomes. How would this affect translation? What would be the state of the ribosome with respect to bound tRNA species?

NAME CHEM 4731, Exam 1

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

2. In terms of products produced, what are the differences between cyclic and non-cyclic e- flow and under what conditions is each cycle used?

Now, answer 14 of the next 16 questions.

3. How will caffeine affect gluconeogenesis and why?

4. Cholesterol is further metabolized into other products. What are two major classes of products into which cholesterol is metabolized and for what are these classes of molecules used?

5. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

6. Suppose that you developed a mutant cell where b -oxidation of fatty acids occurred in the cytosol. How would this affect regulation of b -oxidation and why?

7. Why is S-adenosylmethionine biologically important and what is it’s structure?

8. Is the rate of phospholipid movement between leaflets of a membrane slow or fast when using: (i) a purified phospholipid membrane and; (ii) in a whole cell? In each case, why?

9. Outline the pathway by which N-linked oligosaccharides are synthesized.

10. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

11. Suppose that you developed a mutant cell than that had a form of protein phosphatase inhibitor that could not be phosphorylated by protein kinase. How would this affect glycogen breakdown and synthesis when a cell is stimulated by epinephrine?

12. (A) Suppose that you inhibit N-linked and O-linked protein glycosylation. What are three effects this will likely have on proteins? (B) What is/are the key regulated enzyme(s) in fatty acid biosynthesis? What are 2 key regulators of this/ese enzyme(s)?

13. In terms of lipid metabolism, what are the role(s) of: (A) albumin; (B) low density lipoprotein; and (C) high density lipoprotein.

14. What is a phospholipase? If you treated cells with an inhibitor of this type of enzyme, what are three likely biological effects this would have?

15. Briefly outline the pathway of cholesterol biosynthesis. In your description, make sure to note the basic starting material, the common building blocks that are used, and the rate limiting reaction in the process.

16. Chloroplasts contain two types of chlorophyll, antennae chlorophyll and reaction center chlorophyll. How do these two types of chlorophyll vary from one another and what is/are the role(s) of each in photosynthesis?

17. Briefly outline the pathway by which prostaglandins are synthesized. Make sure to note how the synthesis is regulated/initiated.

18. To reduce the incidence of heart attacks, aspirin therapy is often prescribed. (A) How does aspirin affect blood clotting and why? (B) Why is aspirin used instead of ibuprofen for this therapy?

NAME CHEM 4731, Exam 2

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

dUMP + Methylene-tetrahydrofolate dTMP + Dihydrofolate

2. Draw the base-pairs that would be formed between; A) dAMP and TMP, and; B) cytidine and guanine.

Now, answer 14 of the next 16 questions.

3. A key feature of ribonucleotide reductase is it’s regulation. Suppose that a cell experienced the following conditions. In each case, how would they affect the activity of ribonucleotide reductase and, consequently, the level of each dNTP within the cell. A) Due to defective deamination of deoxyadenosine, the cell accumulates a large excess of dATP. B) You treat cells with thymine such that a large excess of dTTP accumulates within the cells.

4. Prokaryotic glutamine synthetase is regulated by cumulative feedback inhibition. What is cumulative feedback inhibition and why is it effective with this enzyme?

5. A) What are PEST sequences and why are they important? B) What is the N-end rule?

6. What are the 3 primary species by which excess nitrogen is excreted from organisms? Note the relative amount of H2O required for the organism to use each species.

7. Suppose that you blocked all transport into and out of the mitochondria in a human liver cell. How and why would this affect excretion of excess nitrogen, and what species would accumulate in the cell?

8. A) How will placing DNA in a solvent that is more hydrophobic than H2O affect the stability of the helix and why? B) Drug companies are interested in generating mimics of DNA molecules where the phosphodiester linkage has been replaced by other linkages that are more stable than the phosphodiester. An example of this approach is PNA (peptide-nucleic acids) where the phosphodiesters are replaced with peptide (amide) linkages. Suppose that you replace the phosphate residues in a DNA double helix with amide linkages. How will this likely affect the structure of the DNA and why?

9. What is/are the primary difference(s) between ketogenic and glucogenic amino acids?

10. What type of structures do double-stranded RNA and DNA form? Why do they not form the same type of structure?

11. A) Suppose that you are a drug company and want to develop new anti-herpes drugs containing modified sugars. You also hope that they have minimal side effects in humans (generally considered useful if you actually want to sell your drug). To what base should you choose to attach your modified sugars and why? B) Suppose that you want a drug containing a modified sugar that only affects rapidly growing cells. To what base should you attach your modified sugar and why?

12. What is ubiquitin and briefly outline the pathway where it is important.

13. Briefly outline the pathway by which the thyroid hormones thyroxine and triiodothyronine are synthesized.

14. Suppose that you wanted to regulate; A) just purine synthesis; B) just pyrimidine synthesis, and; C) both purine and pyrimidine synthesis. In each case, propose an enzyme that you would regulate to accomplish this goal (Note: If you cannot remember the exact name of the enzyme, you can describe the reaction it catalyzes.).

15. Briefly describe the process by which two complementary strands of DNA anneal to form double-stranded DNA.

16. A) Most tissues release large amounts of glutamine into the blood stream. Why? B) Suppose that you developed a compound that would specifically destroy all bacteria that contain nitrogenase. In a fit of rage at your most recent tax bill, you released an amount of this compound sufficient to kill all of these bacteria. What would be the biological consequences of your action and why?

17. Suppose you raise the temperature of a solution containing double-stranded DNA above the TM of the DNA. How will this affect the UV absorbance of the solution and why?

18. Draw a chemically logical mechanism for the following enzyme catalyzed reaction (If needed, you can use the back of this page).

NAME CHEM 4731, Exam 3

You must answer the first 2 questions in the exam. Then, answer any 14 of the next 16 questions. In each case, answer the questions with great brevity and even greater clarity. Only use the space provided.

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. Shown below is a hypothetical replication bubble, with the polarity of the parental DNA shown. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

2. What are the 3 "mechanisms" that cells use to minimize errors during DNA replication. Briefly describe each mechanism.

Now, answer 14 of the next 16 questions.

3. Suppose that you developed a mutant form of the b subunit of E. coli DNA pol III that consisted of 2 molecules of b fused together into one molecule. Assuming that this does not alter either the overall structure of the b dimer or its’ interactions with other proteins, how would this likely affect DNA replication and why?

4. Suppose that you have a cis dominant mutation in the lac operon that results in constitutive expression of this operon. What is a likely protein(s) or DNA sequence(s) where this mutation occurred, and what would be the molecular effects of this mutation.

5. The E. coli chromosome is normally negatively supercoiled. Suppose that you developed a mutant E. coli where the chromosome was positively supercoiled. Assuming that this does not directly affect the ability of any proteins to interact with the DNA (i.e., transcription occurs normally, DNA polymerases still bind to the DNA, etc.), how would this affect initiation of DNA replication? Why?

6. How could you differentiate between a type I and type II topoisomerase?

7. Why is the polymerase chain reaction (PCR) useful? What are two potential difficulties with this technique?

8. (A) Why is it important that the DNA methylase activity associated with restriction endonucleases be able to methylate hemi-methylated DNA? (B) Suppose that you developed a restriction endonuclease where the methylase activity methylated both unmethylated and hemi-methylated DNA at equal rates. Would this be good or bad and why?

9. What are the roles of the following proteins during DNA replication. A) Primase; B) Topoisomerase; C) Helicase.

10. What are two mechanisms that cells use to prevent the incorporation of deoxyuridylate into DNA?

11. A) What are Holliday junctions and why are they important? B) What is the major difficulty of the Holliday model of recombination that is solved by the Mesolson-Radding model of recombination?

12. Briefly outline how site-specific recombination can lead to gene amplification. Either words or pictures are fine.

13. Mutational analysis and DNA footprinting (a.k.a. chemical reactivity) can be used to determine where on DNA a given protein binds. Briefly describe how each method is used.

14. During attenuation control, termination of transcription occurs opposite a series of 8-10 deoxyadenylates in the template strand. Suppose that you converted these dA residues into dG (deoxyguanylate) residues. How would this affect attenuation control and why?

15. You are sequencing a piece of DNA with the sequence No ddNTP ddA ddC ddG ddT

5’-ATCCGGAATG using the dideoxy method. Accidentally,

you include 100-fold more dATP in each reaction than you

are supposed to include. Assume that this affects neither

the fidelity of the polymerase nor inhibits the polymerase.

To the right of this question, draw the expected band pattern

you would obtain. The letters indicate the presence of

the appropriate ddNTP in each reaction.

Bottom of gel

16. Phage l produces two repressors, cI and Cro. In the helix-turn-helix motif of each protein, would you expect similar or different amino acids? Why?

17. Suppose that you eliminated chi sites from the E. coli chromosome. How would this affect recombination and why?

18. Which is better understood, eucaryotic or procaryotic DNA replication. In terms of the replication that is less well understood, what have been three major difficulties in studying this form of replication?

Name Due in class on 9/10.

Chem 4731 Problem Set 1

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism questions, you can use the back of the page.

1. Suppose that cells were unable to hydrolyze pyrophosphate (PPi). How would this affect the biosynthesis of glycogen? Why? (Make sure to consider the energetics of the reactions).

2. How will caffeine affect gluconeogenesis and why?

3. Suppose that you developed a mutant muscle cell that had decreased levels of protein phosphatase. You stimulate the cell with epinephrine and caffeine, and after a few minutes remove both drugs. How would this decreased level of protein phosphatase affect glycogen regulation, as compared to a cell that had the normal level of protein phosphatase?

4. Draw a likely mechanism for the following reaction:

5. The enzyme phosphoenolpyruvate carboxykinase catalyzes the reaction: oxaloacetate + GTP phosphoenolpyruvate + GDP + CO2. Draw a likely mechanism for this reaction.

Name Due in class on 9/17.

Chem 4731 Problem Set 2

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism questions, you can use the back of the page.

1. Suppose that you added an ionophore that allowed transport of Na+ ions to chloroplasts. How would this affect the relative amounts of non-cyclic and cyclic e- flow through photosystem I and why?

2. Suppose that you wanted to induce ketosis (i.e., a high level of ketone bodies in the blood) in a friend. Other than refusing to give them food, what kind of diet might you put them on to accomplish this and why?

3. Suppose that an organism lived in an environment where the atmosphere contained 60% CO2, rather than the 0.1% CO2 that our atmosphere contains. How would this affect the energetics of fatty acid biosynthesis and why?

4. Draw a likely mechanism for the reaction catalyzed by HMG CoA synthase.

5. Draw a likely mechanism for the reaction catalyzed by acyl CoA dehydrogenase.

Name Due in class on 10/8.

Chem 4731 Problem Set 3

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism questions, you can use the back of the page.

1. List 2 reasons why hydroxylation of phenylalanine is biologically important.

2. Suppose that we lived in an atmosphere that contained 20% H2. How would this affect nitrogen fixation by nitrogenase and why?

3. When proteins are synthesized, the N-terminal amino acid is typically methionine. This methionine residue is very frequently removed from newly synthesized proteins. Suppose that you developed a cell that could not cleave these methionine from the N-termini of proteins. How would this affect the intracellular lifetime of these proteins and why? (Assume that the presence of the methionine does not affect the folding or thermodynamic stability of the protein.)

4. Draw a plausible mechanism for the following pyridoxal dependent decarboxylation reaction.

5. Draw a plausible mechanism for the enzyme tryptophan hydroxylase.

Name Due in class on 10/13.

Chem 4731 Problem Set 4

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism question, use the back of the page.

1. Adenosine deaminase deficiency results in severe combined immunodeficiency (SCID). In patients with SCID, one notes that they have extremely low levels of dNTPs in cells from the thymus. Why does this occur, making sure to talk about ribonucleotide reductase in your answer.

2. A number of advantages are provided by using enzyme complexes that contain the active sites for sequential reactions in a pathway (i.e., substrate channeling). What are 3 potential advantages provided by substrate channeling as compared to using completely independent reactions to carry out a process?

3. 5-Fluorouracil and methotrexate are generally not given together during cancer chemotherapy. Why is this so?

4. Suppose that you developed a mutant cell that could not synthesize PRPP. How would this affect the production of the 4 bases, adenine, cytosine, guanine and uracil?

5. Draw a likely mechanism for the reaction catalyzed by adenylosuccinate synthetase.

Name Due in class on 11/5.

Chem 4731 Problem Set 5

Answer the following questions with great brevity and even greater clarity. With the exception of the enzyme mechanism questions, only use the space provided. For the enzyme mechanism, use the back of the page.

1. Suppose that you developed a mutant form of PCNA that consisted of three molecules of PCNA fused together into one large molecule. Assuming that this does not alter either the overall structure of the PCNA trimer or its’ interactions with other proteins, how would this likely affect DNA replication and why?

2. Suppose that you are interested in the role of the 3’-5’ exonuclease of a replicative DNA polymerase for ensuring fidelity. Propose a method to quantify the contribution of the exonuclease to fidelity in whole cells.

3. In general, nucleotide analogs that are absolute chain terminators are less toxic than nucleotide analogs that allow addition of the next correct dNTP. Why might this be so?

4. Suppose that you are using a DNA helicase to unwind DNA. Suppose that you now add A) negative or; B) positive supercoils to the DNA in front of the helicase. How and why will this affect the ability of the helicase to unwind the DNA?

5. Draw a likely chemical mechanism for the enzyme DNA-cytosine methyl transferase, then enzyme that catalyzes the methylation of cytosine residues in DNA. This enzyme uses S-adenosylmethionine as the methyl donor.

Name Due in class on 11/10.

Chem 4731 Problem Set 6

Answer the following questions with great brevity and even greater clarity. Only use the space provided.

1. In the replication of the T4 (and T7) DNA, replication (i.e., RNA primer synthesis) of each strand does not begin at the 3’ end of the strand. Why is this important and what would likely happen if RNA primer synthesis began exactly at the 3’ end of each strand?

2. Suppose that you developed a strain of E. coli that had levels of NAD+ that were 10-fold lower than normal cells. How would this affect DNA replication in these mutant cells and why? (Assume that these lower levels of NAD+ do not affect the levels of any of the proteins or metabolites required for replication.)

3. E. coli that lack DNA polymerase I grow slowly, but do survive. What does this tell you about Okazaki fragment processing in E. coli?

4. Suppose that you developed an E. coli that lacked DNA-adenine methylase. How would this affect repair during the next two rounds of DNA replication?

5. You are sequencing a piece of DNA with the sequence No ddNTP A C G T

5’-AATCCGGTC. To the right of this question, draw

the expected band pattern you would get using the dideoxy

method of sequencing. The letters indicate the presence of

the appropriate ddNTP in each reaction. The

bottom of this page corresponds to the bottom of the gel.

Name Due in class on 12/1.

Chem 4731 Problem Set 7

Answer the following questions with great brevity and even greater clarity. Only use the space provided.

1. Suppose that you developed an E. coli whose ribosomes bound to Shine-Delgarno sequences of MS2 mRNA at a 10-fold slower rate, but had no other effects on translation. How would this affect production of the A and Coat proteins, and why?

2. Why is chromatin important?

3. Suppose that you increase the cellular concentration of GDP by a factor of 10. How will this affect the amount of aminoacyl-tRNAs bound to EF-Tu and why?

4. To which does 5S rRNA bind more tightly: ribosomal proteins or TFIIIA. Why?

5. In general, for which tRNA synthetases will interactions with the anticodon loop of the tRNA be unimportant and why? Note: I do not want a list of the tRNAs where this will occur; rather, I want to know the common feature behind these tRNAs.

Name Due in class on 12/8.

Chem 4731 Problem Set 8

Answer the following questions with great brevity and even greater clarity. Only use the space provided.

1. Suppose that you developed a mutant form of TFIIB that interacted normally with both TBP and RNA pol II, but the size of this mutant TFIIB was increased 2-fold. How and why would this affect transciption by RNA pol II?

2. How and why will acetylation of histones affect the affinity of the histone for DNA?

3. Now, suppose that you replaced the phosphodiester linkages of DNA with amide linkages (this material is known and referred to as peptide-nucleic acid (PNA)). How and why would acetylation of histones affect their affinity for PNA?

4. Why is alternative splicing useful?

5. Suppose that you blocked 5’-capping of mRNAs. How and why would this affect transcription and translation in eucaryotes?

1. Suppose that you depleted cells of all free inorganic phosphate. How would this affect the light reactions in photosynthesis and why?

2. Suppose that you developed a mutant liver cell line that contained extremely high levels of phosphodiesterase such that cAMP levels in these cells were always very low. How would this affect glycolysis and gluconeogenesis, and why?

3. Suppose that you decreased the pH of blood by 1 pH unit. How would you expect this to affect the life-time of red blood cells and why? (Assume that this decreased pH would not affect the integrity of the cell membrane of the red blood cells, and assume that this decreased pH did not affect the ability of the hemoglobin to deliver O2.)

4. Provide a chemically logical mechanism for the following biotin-dependent, enzyme catalyzed reaction.

Phosphoenol pyruvate + ATP + HCO3- Æ Oxaloacetate + ADP + 2 Pi

5. Provide a chemically logical mechanism for the following enzyme catalyzed reaction.

Malate + NADP+ Æ Pyruvate + NADPH + CO2

1. Suppose the CO2 content of our atmosphere increased by a factor of 10, and there was also a corresponding increase in the cytosolic CO2 concentration. How would this affect fatty acid synthesis and why?

2. Suppose that you are involved in rapid, heavy exercise such that your muscles are operating anaerobically. How would a deficiency of carnitine affect your muscles ability to contract (i.e., produce energy) under these conditions and why?

3. Due to your outstanding performance, part of which was due to your deep understanding of the material covered in Chem 4731, you have become director of research for a major drug company. One of your many subordinates suggests that your company should develop inhibitors of the synthesis of phospholipids as a new class of antibacterial agents for treating infectious diseases. Is this probably a good or bad idea, and why?

4. Suppose that your cells were deficient in serine. How would this likely affect glycosphingolipid synthesis and why?

5. Provide a chemically logical mechanism for the following pyridoxal-dependent enzyme catalyzed reaction.

Glycine CO2 + methylamine

1. Suppose that you are studying a new species of whale, and discover that it contains a never-before known amino acid. How would you determine if this amino acid is ketogenic or glucogenic?

2. You discover a new enzyme that is regulated by multiple allosteric inhibitors. Experimentally, how would you determine if all these allosteric inhibitors operate via a cumulative inhibition mechanism?

3. Suppose that you treated a cell with a potent inhibitor of E2, the ubiquitin transferase. How would this affect the protein content of a cell?

4. Suppose that you mutate the muscle cells of some organism, preferably not human, such that the pyruvate dehydrogenase is extremely active at all times. In fact, under aerobic conditions there is no detectable pyruvate. How would this affect Nitrogen metabolism in these muscle cells and why?

5. Provide a chemically logical mechanism for the following pyridoxal-dependent enzyme catalyzed reaction.

Glutamate + Pyruvate Alpha-ketoglutarate + Alanine

1. Suppose that CTP did not feedback inhibit CTP synthetase. How would this affect the cellular concentrations of ATP, CTP, GTP and UTP, and why?

2. Suppose that you developed a cell containing a mutant form of ribonucleotide reductase where dTTP could not bind to the allosteric site. How would this affect the cellular concentrations of dATP, dCTP, dGTP and dTTP?

3. Suppose that you developed a cell containing a mutant form of NDP kinase that did not phosphorylate dUDP, but in all other regards was normal? How would this affect cellular concentrations of dTTP and why?

4. Several enzymes in eucaryotic nucleotide biosynthesis consist of multi-enzyme complexes such that substrate channeling occurs. What are 3 advantages of substrate channeling (versus completely independent enzymes)?

5. Provide a chemically logical mechanism for the following enzyme catalyzed reaction.

UTP + Glutamine + ATP Æ CTP + Glutamate + ADP + Pi

1. Deoxycytidine analogs are often used as cancer chemotherapeutics (ex., 2',2'-difluorodeoxycytidine). What are 4 mechanisms by which cells could become resistant to deoxycytidine analogs?

2. Suppose that you isolate a new virus containing a linear, double stranded chromosome. How could you determine which of the following general replication mechanisms are used by the virus: bidirectional replication starting at one end of the chromosome, or; unidirectional replication of each strand.

3. Suppose that you develop a mutant form of E. coli DNA pol III where the exonuclease has 10-fold higher activity. How would this affect the fidelity of DNA replication in these cells and why?

4. Suppose that you developed a form of DNA polymerase alpha that has 10-fold higher processivity than normal. How would this likely affect the fidelity of DNA replication in these cells and why?

5. In eucaryotic cells, nucleotide analogs that are chain terminators when incorporated into DNA by a polymerase are usually less toxic than nucleotide analogs that are not chain terminators when incorporated into DNA. Why might this be so?

1. Suppose that you developed a mutant form of E. coli that had dramatically elevated levels of S-adenosylmethionine. Considering DNA metabolism, how would this likely affect the ability of these cells to resist viral infection and why?

2. Suppose that you developed a mutant E. coli that had a greatly increased number of chi sites. How would this likely affect DNA metabolism in these cells and why?

3. Suppose that you develop a mutant form of E. coli whose cytosolic ionic strength is approximately 10-fold higher than in a normal E. coli. How would this affect DNA structure in these cells and why?

4. Suppose that you start with a small amount of duplex DNA that is 1000 nucleotides long, and you wanted to generate large amounts of only one of the two strands. Propose a protocol for accomplishing this goal.

5. Experimentally, how would you quantify the contribution of post-replicative repair to fidelity in E. coli?

1. Suppose that you decrease the cellular concentration of Zn+2 by a factor of 100. Assuming this does not affect the cells survival, how will this likely affect transcription in eucaryotic cells and why?

2. Suppose that you decreased the cellular concentration of cellular acetyl CoA by a factor of 100. Assuming this does not affect the cells survival, how will this likely affect transcription in eucaryotic cells and why?

3. Suppose that you treated E. coli with an agent that specifically inhibited the formation of RNA secondary structure. How would this affect transcription of the Trp operon under conditions of moderate tryptophan concentrations and why?

4. Suppose that you altered the structure of the histone such that the DNA only wrapped around it one time. Assuming that this did not affect binding of any other proteins to the DNA, how would this change affect the superhelical state of the cell's DNA and why?

5. On the back of this page, draw a chemically logical mechanism for how the sequential treatment of DNA with dimethyl sulfate and base results in the loss of guanine from dGMP residues in the DNA.

1. Suppose that you inhibit C-1 metabolism in bacteria such that cells can no longer synthesize formyltetrahydrofolate. How will this affect protein biosynthesis and why?

2. Suppose that EF-Tu hydrolyzed GTP as soon as it bound the ribosome. How would this affect protein biosynthesis and why?

3. If one takes a ribosome and proteolytically degrades all of the proteins, the remaining RNA can catalyze peptide bond formation. However, if you synthesize the ribosomal RNAs and mix them together, they cannot catalyze peptide bond formation. Why might this be so?

4. Suppose that you isolate a new protein that affects transcription of a eucaryotic gene. How could you determine whether it is either an activator or a coactivator?

5. Suppose that you are leading a drug discovery team that wants to develop a new cancer chemotherapeutic that inhibits DNA replication by binding to and inhibiting DNA polymerase d. One of the many scientists that works for you proposes to use SELEX. Do you think that this is a good or bad idea, and why?

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

2. Briefly, compare and contrast glycolysis and gluconeogenesis, making sure to comment on regulation and differences in each pathway.

Now, answer 14 of the next 16 questions.

3. Suppose that you are involved in rapid, heavy exercise such that your muscles are operating anaerobically. How would a deficiency of carnitine affect your muscles ability to contract (i.e., produce energy) under these conditions and why?

4. Cholesterol is further metabolized into other products. What are two major classes of products into which cholesterol is metabolized and for what are these classes of molecules used?

5. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

6. Suppose that you developed a compound that very potently inhibited the synthesis of hydroxymethylglutaryl CoA (HMG CoA) in bacteria. How would this affect cell wall biosynthesis in bacteria and why?

7. Why is S-adenosylmethionine biologically important and what is it’s structure?

8. Briefly outline the pathway of ketogenesis. Why is this pathway biologically important?

9. Suppose that you altered the metabolism of a plant cell such that the cell was not using any NADPH, but was still metabolizing ATP to ADP to Pi. How would this affect the cyclic and non-cyclic pathways of e- flow during photosynthesis and why?

10. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

11. Suppose that you discover a mutant person whose body has suddenly stopped producing any detectable methylmalonyl CoA mutase, the B12 dependent enzyme we talked about in class. As we discussed in class, lack of this enzyme normally results in mental retardation, ultimately due to accumulation of methylmalonyl CoA and its aberrant further metabolism. You consider two potential treatments for this person, gene therapy and dietary modification. Since no one has done any experiments regarding gene therapy with this enzyme, you immediately discard this option. Therefore, you consider a dietary solution to this problem. How could you treat this individual via diet to solve their problem, making sure to note why this dietary change would work.

12. (A) Suppose that you inhibit N-linked and O-linked protein glycosylation. What are three effects this will likely have on proteins? (B) What is/are the key regulated enzyme(s) in fatty acid biosynthesis? What are 2 key regulators of this/ese enzyme(s)?

13. List the 3 primary mechanisms by which phospholipids are moved throughout cells.

14. Briefly outline the pathway of cholesterol biosynthesis. In your description, make sure to note the basic starting material, the common building blocks that are used, and the rate limiting reaction in the process. 15. What is a phospholipase? If you treated cells with an inhibitor of this type of enzyme, what are three likely biological effects this would have?

16. Chloroplasts contain two types of chlorophyll, antennae chlorophyll and reaction center chlorophyll. How do these two types of chlorophyll vary from one another and what is/are the role(s) of each in photosynthesis?

17. How would the following changes affect cholesterol metabolism and why: A) Increase the levels of high density lipoprotein; B) Increase the level of low density lipoprotein receptor in the liver. 18. Briefly outline the pathway by which fatty acids are synthesized in eucaryotes.

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction. Glutamate + NH3 + ATP Æ Glutamine + ADP + Pi 2. In the space below, draw the base-pairs formed between; A) 3'-AMP and dTTP; B) guanosine and dCMP.

Now, answer 14 of the next 16 questions.

3. Glutamine synthase is the key regulated enzyme for nitrogen metabolism. In prokaryotes, what are the two major mechanisms of regulation, and briefly describe each mechanism.

4. Briefly describe the role that hydrogen bonds between base-pairs are thought to play in terms of stabilizing/destabilizing double-stranded DNA, and why these effects occur.

5. On the back of this page, draw a chemically logical mechanism for the following pyridoxal dependent, enzyme catalyzed reaction. (Note: The pyridoxal cofactor will be converted to pyridoxamine during the reaction.)

Phenylalanine Æ Phenylpyruvate

6. Suppose you treat some cells with 5-fluorouracil. How and why will this affect the concentrations of dTTP, dCTP, dGTP and dATP?

7. Suppose that a cell had excess phenylalanine hydroxylase. How would this likely affect amino acid metabolism? Suppose that this excess activity had deleterious consequences for the afflicted individual. How might you attempt to solve their problem?

8. Suppose that you wanted to alter the biological half life of a protein inside of cells. What are three modifications that you could make to the amino acid sequence of this protein to decrease it’s half life?

9. Suppose that you synthesize a compound that binds to aspartate transcarbamoylase (carbamoyl phosphate + aspartate Æ carbamoyl aspartate + Pi) and, importantly, prevents binding of CTP to this enzyme (Note: Binding of your compound has no effect on the enzyme.). Upon accidentally consuming this compound, how and why would the cellular levels of CTP, UTP, ATP and GTP be affected? Assume the amount that you consume is substantially less than the amount needed to kill you. 10. What are the 3 primary means by which organisms secrete excess nitrogen? Comment on the importance of H2O for secretion of each compound.

11. What is the reaction catalyzed by nitrogenase? (You should not draw out the catalytic cycle. Just the starting materials and products, including stoichiometry.) Why is it so important biologically?

12. A large number of genetic defects exist in the biosynthetic pathway for urea production. Suppose that your roomate suddenly stops producing the enzyme arginase, the enzyme that hydrolyzes arginine into urea and ornithine. Lack of this enzyme can result in severe effects on the afflicted individual (ex., mental retardation). A) What is the toxic species that will ultimately accumulate in this individual? B) Based on your interactions with your roomate and consequent knowledge of his/her/its intellect, it is abundantly clear to you that they cannot afford the loss of any further mental capacity. Since gene therapy does not work (i.e., you cannot modify their cells to get them to produce arginase), how would you treat this individual to maintain their brain?

13. Briefly outline the pathway by which the thyroid hormones thyroxine and triiodothyronine are synthesized.

14. Briefly outline the de novo purine biosynthetic pathway. Make sure to note the key regulated enzymes.

15. A) Suppose that you are a drug company and want to develop new anti-herpes drugs containing modified sugars. You also hope that they have minimal side effects in humans (generally considered useful if you actually want to sell your drug). To what base should you choose to attach your modified sugars and why? B) Suppose that you want a drug containing a modified sugar that only affects rapidly growing cells. To what base should you attach your modified sugar and why?

16. Briefly outline the process by which one chemically synthesizes oligonucleotides. What are the two factors that limit the maximum length of oligonucleotides that can be synthesized in high yield?

17. Several enzymes in eucaryotic nucleotide biosynthesis consist of multi-enzyme complexes such that substrate channeling occurs. What are 3 advantages of substrate channeling (versus completely independent enzymes)?

18. Suppose that you developed a mutant muscle cell that had the capacity to perform gluconeogenesis. Furthermore, the gluconeogenic pathway in these cells was constantly turned on and operated at an extremely high rate. How would this affect nitrogen metabolism in these cells and why? (Note: Assume that this high rate of gluconeogenesis does not affect the viability or energy levels of these cells)

1. Shown below is a hypothetical replication bubble, with the polarity of the parental DNA shown. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

2. What are the 3 "mechanisms" that cells use to minimize errors during DNA replication. Briefly describe each mechanism.

Now, answer 14 of the next 16 questions.

3. You are studying a new virus and want to know if it uses primase during its DNA replication. Propose an experimental approach to test this hypothesis.

4. During eucaryotic DNA replication, what are the likely roles of DNA polymerases a, d and e?

5. You are examining the fidelity of DNA polymerases, and are interested in modulating the intracellular dNTP concentrations in order to enhance the mutational frequency. Suppose that you want to specifically enhance the frequency with which a C is polymerized opposite the template A in the DNA sequence below when it is replicated. List 3 changes in the intracellular dNTP concentrations that would enhance the frequency with which this mutation occurred, and briefly note why each change would enhance the error frequency. 5'-CTCTAGGCC-3'

6. You discover a new protein that binds to DNA and introduces a bend in the DNA. In the region of the protein that contacts the phosphate residues on the inside of the bend, list 3 amino acids that you would expect to be found making these contacts and state why they would be there.

7. What are the primary role(s) of DNA methylation in A) procaryotes, and; B) eucaryotes?

8. What are the roles of the following proteins during repair of uracil found in DNA; A) Uracil-DNA glycosylase; B) DNA ligase, and; C) abasic endonuclease?

9. Deamination of deoxycytidine in DNA is potentially mutagenic. On the back of this page, draw a chemically logical mechanism for this reaction.

10. Briefly describe how RecA and RecBCD would work together to initiate DNA recombination. You do not have to describe how these proteins might be involved in resolution of the Holliday junction. (Hint: It would probably be easier to use the Meselson-Radding model of recombination as the basis of your description).

11. Suppose that you are studying a new virus, and are interested in how the viral DNA becomes integrated into the DNA. Your two mechanisms of choice are site specific recombination and transposition. What are two key differences between these processes that you might be able to exploit in order to determine which process your new virus uses? (Note: You do not have to describe how you would actually do the experiment, just list the specific differences you would exploit.)

12. Briefly outline the polymerase chain reaction. Describe why it is useful for molecular biology and list two potential problems with the products you finally obtain as compared to what you want to obtain.

13. Briefly outline how site-specific recombination can lead to gene amplification. Either words or pictures are fine.

14. Suppose that you develop a mutant form of E. coli whose cytosolic ionic strength is approximately 10-fold higher than in a normal E. coli. How would this affect DNA structure/stability in these cells and why?


15. Suppose that you treat cells with a compound that destroys radicals bound to enzymes. How will this likely affect DNA replication and why?

16. Suppose that you developed a mutant form of HIV reverse transcriptase that had greatly enhanced RNaseH activity. In fact, it is so active, it destroyed any and all RNA bound to DNA. How would this likely affect the ability of HIV to infect new cells and why?

17. DNA polymerase I of E. coli has three enzymatic activities. What are these three activities, and what role does each play with respect to the role(s) of DNA polymerase I during DNA metabolism in E. coli.

18. Replication of linear chromosomes is potentially problematic. What is the major problem and what are 3 potential solutions to this problem.

Section I. Carbohydrates. Answer 4 of the following 5 questions.

1. The overall efficiency of photosynthesis for carbon storage is only approximately 4%. Suppose that you wanted to reengineer plants such that the overall efficiency of carbon storage increased significantly. What are 3 ways that you would modify plants to increase this efficiency?

2. Briefly outline the pathway of N-linked protein glycosylation. You need only consider the processes that occur in the Endoplasmic reticulum.

3. In photosynthesis, what is cyclic electron flow and why is it important?

4. In terms of general principles of regulation and the enzymes involved, how are gluconeogenesis and glycolysis similar and different?

5. A biotin dependent enzyme catalyzes the following reaction. On the back of this page, draw a likely mechanism for this reaction.

Section II. Membrane Constituents and Fatty Acid Metabolism. Answer 3 of the following 4 questions.

6. A) Why are phospholipases considered an attractive target for anti-inflammatory agents in humans? B) What are the 3 general mechanisms by which fatty acids are moved within cells?

7. Suppose that you developed a mutant person with a very low level of the low density lipoprotein receptor. How would this affect cholesterol metabolism and why?

8. Briefly outline the pathway of b-oxidation of fatty acids. For long chain fatty acids, how is their oxidation controlled?

9. Draw a likely mechanism for the following enzyme catalyzed reaction.

Section III. Amino Acid Metabolism. Answer 2 of the following 3 questions.

10. Briefly describe the pathway where ubiquitin is important.

11. In humans, there are two primary compounds by which nitrogen is excreted. What are the two compounds, and what general class of molecules gives rise to each compounds? 12. A pyridoxal dependent transaminase catalyzes the following reaction. Draw a likely mechanism of this reaction on the back of this page.

Section IV. Nucleotide and DNA Metabolism. Answer 8 of the following 10 questions.

13. In terms of the 4 major forces stabilizing DNA and nucleic acid structure, describe how each force affects a DNA helix.

14. Suppose that you developed a mutant cell that had very low levels of dUTPase. How would this affect DNA replication and why?

15. In terms of DNA replication in E. coli, what are the roles of the following proteins: DNA polymerase I, DNA polymerase III, and single-stranded DNA binding protein.

16. Briefly outline the regulation of ribonucleotide reductase.

17. Various experimental data indicate that both topoisomerase I and II are involved in DNA replication in yeast. If you then delete all topoisomerase I activity, the yeast replicate their DNA more slowly, but they complete replication and survive. However, if you delete all topoisomerase II activity, the yeast cannot replicate their DNA and they die. Why is topo I activity not essential for DNA replication but topo II activity essential?

18. Deoxycytidine analogs are often used as cancer chemotherapeutics (ex., 2',2'-difluorodeoxycytidine). What are 4 mechanisms by which cells could become resistant to deoxycytidine analogs?

19. Repeated DNA sequences are often genetically unstable. Show why this is thought to be so.

20. A) What is the key difficulty of the Holliday model of recombination that is solved in the Mesolson-Radding model? B) Suppose that you develop a mutant E. coli that has greatly increased levels of RecBCD. How will this likely affect recombination in these cells and why?

21. Shown below is a hypothetical replication bubble, with the polarity of the parental DNA shown. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

22. Suppose that you inhibited the RNase H activity of reverse transcriptase. How would this affect the ability of reverse transcriptase to make a double-stranded DNA copy of double-stranded RNA and why?

Section V. Transcription, Translation, DNA in the Nucleus, and Signaling. Answer 15 of the next 17 questions.

23. Briefly outline the mechanism by which RNA polymerase initiates transcription in E. coli.

24. You are studying the lac operon of E. coli, and discover a new mutation that is trans-dominant such that the lac operon is always turned off. In what protein or region of the DNA is the mutation most likely located, and what are the molecular effects of the mutation?

25. Which are more highly processed, RNAs produced in eucaryotes or RNA produced in procaryotes? For those RNAs that are more highly processed, list 3 major types of processing that occur.

26. A) What is chromatin and why is it important? B) Suppose that you introduced positive supercoils into eucaryotic DNA. How would this likely affect nucleosome structure and why?

27. What are 3 mechanisms by which translation can be regulated. Very briefly describe each method.

28. Briefly outline the process by which translation is initiated in E. coli.

29. Suppose that you treat E. coli cells with a compound that disrupts RNA secondary structure. How will this affect transcription and why?

30. Briefly outline the process of RNA splicing.

31. The vast majority of all hormone receptors are found on the cell surface. An exception to this rule is the estrogen receptor, which is found in the cytosol. What property of estrogen makes it feasible for its receptor to be located in the cytosol and why?

32. Suppose that you decreased the rate of GTP hydrolysis by EF-Tu. How would this likely affect protein synthesis and why?

33. What are 3 major forms of repetitive DNA found in eucaryotes?

34. Suppose that you developed a compound that inhibited all RNases in E. coli. How would this likely affect protein biosynthesis and why? (Note: Assume cells are not dividing, hence effects on DNA replication are not important)

35. Eucaryotic activators typically contain 3 different domains. What are these 3 domains and what is their function?

36. What is combinatorial regulation and why is it important in eucaryotes?

37. What is chromatin remodeling and why is it important?

38. Suppose that you developed an inhibitor that specifically blocked the E site of ribosomes. How would this affect translation? What would be the state of the ribosome with respect to bound tRNA species?

39. Briefly describe the mechanism(s) by which translational fidelity is obtained.

 

Section I. Carbohydrates. Answer 2 of the following 3 questions.

1. Briefly outline the pathway of N-linked protein glycosylation. You need only consider the processes that occur in the Endoplasmic reticulum.

2. In photosynthesis, what is cyclic electron flow and why is it important?

3. Suppose that you are studying a new organism, and note that it has 2 different metabolic pathways, one that converts A into B and a second metabolic pathway that converts B into A. A) What predictions would you make regarding the regulation of the two pathways? B) Will the pathways likely use the same or different enzymes? Why?

Section II. Membrane Constituents and Fatty Acid Metabolism. Answer 2 of the following 3 questions.

4. A) What is the biological role of bile acids? B) Suppose that a person was deficient in bile acids. How would this affect the composition of chylomicrons and low density lipoproteins?

5. Briefly outline the pathway of b-oxidation of fatty acids. For long chain fatty acids, how is their oxidation controlled?

6. Draw a likely mechanism for the following enzyme catalyzed reaction.

Section III. Amino Acid Metabolism. Answer 2 of the following 3 questions.

7. Glutamine synthase is the key regulated enzyme for nitrogen metabolism. In prokaryotes, what are the two major mechanisms of regulation, and briefly describe each mechanism.

8. In humans, there are two primary compounds by which nitrogen is excreted. What are the two compounds, and what general class of molecules gives rise to each compounds?

9. A pyridoxal dependent transaminase catalyzes the following reaction. Draw a likely mechanism of this reaction on the back of this page.

Section IV. Nucleotide and DNA Metabolism. Answer 7 of the following 9 questions.

10. What kinetic parameter(s) and concentration(s) would you need to know to predict the frequency with which the polymerase activity will misincorporate dCMP opposite a template dA at a specific site in a DNA. (Do not include effects of the proofreading exonuclease in your answer.)

11. Suppose that you developed a mutant cell that had very low levels of dUTPase. How would this affect DNA replication and why?

12. In terms of DNA replication in E. coli, what are the roles of the following proteins: DNA polymerase I, topoisomerase, and single-stranded DNA binding protein.

13. In terms of the 4 major forces stabilizing DNA and nucleic acid structure, describe how each force affects a DNA helix.

14. Briefly outline the process by which translation is initiated in E. coli.

15. Two important features of DNA structure are the major and minor grooves, and the highly regular structure of the phosphate backbone. Biologically, why are these features important?

16. You are studying the regulation of the lac operon in E. coli, and discover a mutant form of the operon. You obtain the following data regarding the mutation. (1) It is trans dominant. (2) When cells are grown in the absence of lactose (+ or - glucose), the operon is turned off. (3) When the cells are grown in the presence of lactose (+ or - glucose), the operon is turned on at a very slow rate. Where did the mutation likely occur ? Explain the reasoning behind your answer.

17. A) What is the key difficulty of the Holliday model of recombination that is solved in the Mesolson-Radding model? B) Suppose that you develop a mutant E. coli that has greatly increased levels of RecBCD. How will this likely affect recombination in these cells and why?

18. Shown below is a hypothetical replication bubble. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

Section V. Eucaryotic transcription and beyond. Answer 13 of the next 15 questions.

19. Suppose that you wanted to determine if a ribosomal protein bound to a rRNA and where it bound. Briefly outline procedures that you would use to answer each issue (Note: You do not have to describe how the procedures work, just state what the procedures would tell you.).

20. Eucaryotic transcriptional activators can be grouped into 3 different classes, depending upon the structural motif they use to bind DNA. What are these 3 classes, and briefly describe each class.

21. Which are more highly processed, RNAs produced in eucaryotes or RNA produced in procaryotes? For those RNAs that are more highly processed, list 3 major types of processing that occur.

22. Briefly describe how DNA is packaged in eucaryotes.

23. What are 3 mechanisms by which translation can be regulated in E. coli. Very briefly describe each method.

24. Briefly outline the process by which translation is initiated in E. coli.

25. Briefly describe how chromatin can affect trancription, and how the transcriptional machinery affects chromatin structure.

26. Substrate specificity is a significant issue with respect to many kinases involved in signaling. What is potentially problematic about the substrate specificity of these kinases, and what are two mechanisms by which the cell overcomes these problems?

27. How and why will the following molecules affect steroid biosynthesis? A) Caffeine, a molecule of which I am very fond, and which is a potent inhibitor of phosphodiesterase, an enzyme that hydrolyzes cAMP. B) Compound X, a potent inhibitor of the STAR protein (steroidogenic acute regulator).

28. Eucaryotic activators typically contain 3 different domains. What are these 3 domains and what is their function?

29. What is combinatorial regulation and why is it important in eucaryotes?

30. A) Other than increasing the frequency of cancer, what are two key biological functions of the p450 family of enzymes? B) Briefly outline how aryl hydrocarbons can regulate p450 levels.

31. The vast majority of all hormone receptors are found on the cell surface. An exception to this rule is the estrogen receptor, which is found in the cytosol. What property of estrogen makes it feasible for its receptor to be located in the cytosol and why?

32. Suppose that you increased the rate of GTP hydrolysis by Ras. How would this likely affect cell growth and why?

33. What are 3 major forms of repetitive DNA found in eucaryotes?

1. Suppose that you developed a mutant person that transferred the methyl ester of sialic acid to proteins on

on the surface of erythrocytes, instead of sialic acid. Assuming that this change did not affect the activity, stability, etc. of these erythrocyte proteins or of the membrane, how would this affect the half-life of the erythrocytes and why?

2. Suppose that you developed a mutant liver cell line that contained extremely low levels of phosphodiesterase such that cAMP levels in these cells were always very high. How would this affect glycolysis and gluconeogenesis, and why?

3. Suppose that you added to plant cells a Mg+2 ionophore that became incorporated only in thylakoid membranes. How would likely this affect the dark reactions of photosynthesis and why?

4. Provide a chemically logical mechanism for the following biotin-dependent, enzyme catalyzed reaction.

Phosphoenol pyruvate + ATP + HCO3- ® Oxaloacetate + ADP + 2 Pi

 

5. Provide a chemically logical mechanism for the following enzyme catalyzed reaction.

Malate + NADP+ ® Pyruvate + NADPH + CO2

1. Suppose that the glucagon concentrations in your blood affect 10-fold. How will this affect fatty acid synthesis and why?

2. Suppose that you are involved in rapid, heavy exercise such that your muscles are operating anaerobically. How would a deficiency of carnitine affect your muscles ability to contract (i.e., produce energy) under these conditions and why?

3. Suppose that you develop a mutant person who has an extremely high level of the enzymes propionyl CoA carboxylase, methylmalonyl CoA epimerase and methylmalonyl CoA mutase. How would this affect the membrane composition of this individual and why?

4. Provide a chemically logical mechanism for the reaction catalyzed by a phospholipase.

5. Provide a chemically logical mechanism for the following pyridoxal-dependent enzyme catalyzed reaction.

Glycine à CO2 + methylamine

1. Deoxycytidine analogs are often used as cancer chemotherapeutics (ex., 2',2'-difluorodeoxycytidine). What are 4 mechanisms by which cells could become resistant to deoxycytidine analogs?

2. Suppose that you reduced the levels of S-Adenosylmethionine in E. coli by a factor of 10. How would this likely affect DNA metabolism and why?

3. Suppose that you treat cells with a compound that inhibits the reduction of ADP to dADP by ribonucleotide reductase to the extant that dATP levels are 5-fold lower than normal (Note: This concentration is still 5-fold higher than the KM of the DNA polymerases for dNTPs). How would this likely affect DNA repair and why?

4. Suppose that you developed a form of DNA polymerase alpha that has 10-fold higher processivity than normal. How would this likely affect the fidelity of DNA replication in these cells and why?

5. In eucaryotic cells, nucleotide analogs that are chain terminators when incorporated into DNA by a polymerase are usually less toxic than nucleotide analogs that are not chain terminators when incorporated into DNA. Why might this be s

1. When doing DNA protection ("footprinting") studies, two commonly used reagents are DNase and hydroxyl radical. Which reagent is likely to give more detailed information in this type of study and why?

2. Suppose that you developed a mutant E. coli that had a greatly increased number of chi sites. How would this likely affect DNA metabolism in these cells and why?

3. Suppose that you develop a powerful inhibitor of integrase from HIV. How and why would this affect the HIV in; (A) cells already infected with HIV, and; (B) cells to which you add HIV and the inhibitor simultaneously?

4. Suppose that you eliminated all r binding sites from the E. coli chromosome. How would this likely affect transcription and why?

5. Experimentally, how would you quantify the contribution of post-replicative repair 1. Suppose that you decrease the cellular concentration of Zn+2 by a factor of 100. Assuming this does not affect the cells survival, how will this likely affect transcription in eucaryotic cells and why?

2. Suppose that you decreased the cellular concentration of acetyl CoA by a factor of 100. Assuming this does not affect the cells survival, how will this likely affect transcription in eucaryotic cells and why?

3. Suppose that you treat rapidly growing bacterial cells with methotrexate. How will this likely affect translation and why?

4. What is combinatorial control and why is it essential in eucaryotes?

5. Suppose that you increase the cellular concentration of GDP by a factor of 20. How will this likely affect trans1. Suppose that you treated your roomate with a drug that inhibited dihydrobiopterin reductase. How would this likely affect their amino acid metabolism and why?

2. Suppose that we lived in an atmosphere that contained 20% H2. How would this affect nitrogen fixation by nitrogenase and why? Please assume that atmospheric conflagration is not an issue.

3. When proteins are synthesized, the N-terminal amino acid is typically methionine. This methionine residue is very frequently removed from newly synthesized proteins. Suppose that you developed a cell that could not cleave these methionine from the N-termini of proteins. How would this affect the intracellular lifetime of these proteins and why? (Assume that the presence of the methionine does not affect the folding or thermodynamic stability of the protein.)

4. Suppose that you developed a mutant form of E. coli where pyrimidine synthesis was regulated at the level of carbamoyl phosphate synthetase. How would this likely affect the cells and why?

5. Draw a plausible mechanism for the following pyridoxal dependent decarboxylation reaction.

Alanine + Formaldehyde - -> Pyruvate + Methylamine

6. Suppose that CTP did not feedback inhibit CTP synthetase. How would this affect the cellular concentrations of ATP, CTP, GTP and UTP, and why?

7. Suppose that you developed a cell containing a mutant form of ribonucleotide reductase where dTTP could not bind to the allosteric site. How would this affect the cellular concentrations of dATP, dCTP, dGTP and dTTP, and why?

8. Suppose that you are studying DNA stability in aqueous solutions, and make the solvent more hydrophobic. How will this likely affect the stability of the DNA duplex and why?

9. Suppose that you develop a small, cationic molecule that binds very tightly to the sugar-phosphate backbone of DNA, thereby neutralizing the - charges on the phosphate. How will this likely affect the structure of the DNA and why?

10. Provide a chemically logical mechanism for the following enzyme catalyzed reaction.

UTP + Glutamine + ATP CTP + Glutamate + ADP + Pi

1. Suppose that you reduced the intracellular concentration of NAD+ by 10-fold. How would this affect the toxicity of deptheria toxin and why?

2. Suppose that EF-Tu hydrolyzed GTP as soon as it bound the ribosome. How would this affect protein biosynthesis and why?

3. If one takes a ribosome and proteolytically degrades all of the proteins, the remaining RNA can catalyze peptide bond formation. However, if you synthesize the ribosomal RNAs and mix them together, they cannot catalyze peptide bond formation. Why might this be so?

4. Suppose that you developed a potent inhibitor of DNA polymerase a. Ignoring the effects of this compound on DNA replication, how would this compound likely affect telomere structure in A) stem cells, and; B) muscle cells, and why?

5. Suppose that you developed a form of transducin that could not hydrolyze GTP. How would this liEach question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

2. Glutamine synthase is the key regulated enzyme for nitrogen metabolism. In prokaryotes, what are the two major mechanisms of regulation, and briefly describe each mechanism.

Now, answer 14 of the next 16 questions.

3. Briefly outline the pathway of ketone body formation. Make sure to note why this process is important physiologically.

4. Cholesterol is further metabolized into other products. What are two major classes of products into which cholesterol is metabolized and for what are these classes of molecules used?

5. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

6. (A) The precursors for protein and lipid glycosylation are nucleotide-sugars. Why is it important for the sugar to be attached to the nucleotide? (B) You are trying to develop novel insecticides, and develop a compound that blocks the biosynthesis of dolilchol. Would you expect this compound to be toxic to insects and why?

7. As we discussed in class, the two leaflets of a membrane often contain very different compositions of phospholipids. However, when the membranes are initially synthesized, they often contain equal amounts of each phospholipid in both leaflets. What enzyme(s) is responsible for allowing the membranes to assume an asymmetric leaflet composition? From the standpoint of energetics, what maintains the asymmetry?

8. Suppose that you altered the metabolism of a plant cell such that the cell was not using any NADPH, but was still metabolizing ATP to ADP and Pi. How would this affect the cyclic and non-cyclic pathways of e- flow during photosynthesis and why?

11. Chloroplasts contain two types of chlorophyll, antennae chlorophyll and reaction center chlorophyll. How do these two types of chlorophyll vary from one another and what is/are the role(s) of each in photosynthesis?

9. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

10. Briefly outline the pathway of cholesterol biosynthesis. In your description, make sure to note the basic starting material, the common building blocks that are used, and the rate limiting reaction in the process.

12. How would the following changes affect cholesterol metabolism and why: A) Decrease the levels of high density lipoprotein; B) Decrease the level of low density lipoprotein receptor in the liver.

13. Suppose that you developed an inhibitor of nitrogenase, and sprayed it on your neighbors farm fields once every year (Note: Your compound was completely stable in the soil for precisely 1 year, and then completely broke down.). How would this likely affect the productivity of your neighbors field and why? How could he overcome this problem, assuming that he does not terminate your existence and/or your stock of this inhibitor?

14. Outline the pathway for fatty acid synthesis, including the release of the key final product. What is the key committed intermediate in this process?

15. In terms of general principles of regulation and the enzymes involved, how are gluconeogenesis and glycolysis similar and different?

16. Aspirin therapy is used to help prevent heart attacks. Treatment inhibits prostaglandin synthesis by the platelets, thereby inhibiting clotting and, consequently, heart attacks. In contrast, ibuprofin, which also inhibits prostaglandin synthesis by platelets is not used to prevent heart attacks. Why is aspirin used and not ibuprofen?

17. What is a phospholipase? If you treated cells with an inhibitor of this type of enzyme, what are three likely biological effects this would have?

18. Suppose that you developed a mutant prairie dog that contained a modified form of acyl carnitine translocase, such that the mutant form of the enzyme can no longer bind malonyl CoA. How would this likely affect fatty acid metabolism in your new creature?

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. On the back of this page, draw a chemically logical mechanism for the following enzyme catalyzed reaction.

Aspartate + NH3 + ATP ® Asparagine + ADP + Pi

2. DNA polymerase I from E. coli contains 3 enzymatic activities. What are these 3 activities, and what is their function during DNA synthesis?

Now, answer 14 of the next 16 questions.

3. Suppose that you develop a small, cationic molecule that binds very tightly to the sugar-phosphate backbone of DNA, thereby neutralizing the - charges on the phosphate. How will this likely affect the structure of the DNA and why?

4. Briefly describe the role that hydrogen bonds between base-pairs are thought to play in terms of stabilizing/destabilizing double-stranded DNA, and why these effects occur.

5. On the back of this page, draw a chemically logical mechanism for the following pyridoxal dependent, enzyme catalyzed reaction. (Note: The pyridoxal cofactor will be converted to pyridoxamine during the reaction.)

Lysine ® 2-Keto-6-amino-hexanoic acid

6. Suppose you treat some cells with 5-fluorouracil. How and why will this affect the concentrations of dTTP, dCTP, dGTP and dATP, and why?

7. A) What are PEST sequences and why are they important? B) Suppose that you depleted a cell of ubiquitin. How would this likely affect protein metabolism in cells and why?

8. In humans, there are two primary compounds by which nitrogen is excreted. What are the two compounds, and what general class of molecules gives rise to each compounds?

9. A) A large number of guanosine analogs are of potential interest as cancer chemotherapeutics. However, there is a fundamental problem with using guanosine analogs to treat tumors. What is this problem? B) Unlike many cancer chemotherapeutics, 5-fluorouracil is given as the free base. What are two potential mechanisms by which 5-fluoruracil could be converted to the nucleoside?

10. Briefly outline the pathway by which the thyroid hormones thyroxine and triiodothyronine are synthesized.

11. A large number of genetic defects exist in the biosynthetic pathway for urea production. Suppose that your roomate suddenly stops producing the enzyme arginase, the enzyme that hydrolyzes arginine into urea and ornithine. Lack of this enzyme can result in severe effects on the afflicted individual (ex., mental retardation). A) What is the toxic species that will ultimately accumulate in this individual? B) Based on your interactions with your roomate and consequent knowledge of his/her/its intellect, it is abundantly clear to you that they cannot afford the loss of any further mental capacity. Since gene therapy does not work (i.e., you cannot modify their cells to get them to produce arginase), how would you treat this individual to maintain their brain?

12. A) Why is aromatic amino acid synthesis considered an especially attractive target for new herbicides? B) As a test of your organic chemistry skills, you attempt to develop compounds that are structurally very similar to tetrahydrobiopterin. Much to your surprise, one of the compounds, to which you gave the name killall, is an extremely potent inhibitor of dihydrobiopterin reductase. Suppose that, with their consent, you repeatedly gave this compound to your roomate. How would you expect this compound to affect them and in terms of the general reactions affected, why?

13. Suppose that you have developed an enzyme that unwinds DNA. How will i) positive and ii) negative supercoiling affect the ability of this enzyme to unwind DNA and why? (Assume that the presence of supercoils does not affect the ability of this enzyme to interact with the DNA.)

14. Briefly outline the de novo pyrimidine biosynthetic pathway. Make sure to note the key regulated enzymes.

15. Two important features of DNA structure are the major and minor grooves, and the highly regular structure of the phosphate backbone. Biologically, why are these features important?

16. Briefly outline the process by which one chemically synthesizes oligonucleotides. What are the two factors that limit the maximum length of oligonucleotides that can be synthesized in high yield?

17. In the space below, draw the base-pairs formed between; A) 3'-AMP and dTTP; B) guanosine and dCMP.

18. Suppose that you developed a mutant muscle cell that had the capacity to perform gluconeogenesis. Furthermore, the gluconeogenic pathway in these cells was constantly turned on and operated at an extremely high rate. How would this affect nitrogen metabolism in these cells and why? (Note: Assume that this high rate of gluconeogenesis does not affect the viability or energy levels of these cells)

Each question is worth 7 points. If you are not sure of the answer, you can just write "I don’t know", and receive 2 points.

1. Shown below is a hypothetical replication bubble, with the polarity of the parental DNA shown. The origin of bidirectional replication is noted by an X each strand. On both strands, show the expected products making sure to note their polarity, presence/absence of RNA, etc.

2. Briefly outline the mechanism by which RNA polymerase initiates transcription in E. coli.

Now, answer 14 of the next 16 questions.

3. Suppose that you were studying E. coli, and inactivated the methylase activity associated with a restriction endonuclease found within the cell. Now, you allow the E. coli to continue growing. How would this affect the cellular DNA during the next two generations? Why?

4. Deoxycytidine analogs are often used as cancer chemotherapeutics (ex., 2',2'-difluorodeoxycytidine). What are 4 mechanisms by which cells could become resistant to deoxycytidine analogs?

5. You are examining the fidelity of DNA polymerases, and are interested in modulating the intracellular dNTP concentrations in order to enhance the mutational frequency. Suppose that you want to specifically enhance the frequency with which a C is polymerized opposite the template A in the DNA sequence below when it is replicated. List 3 changes in the intracellular dNTP concentrations that would enhance the frequency with which this mutation occurred, and briefly note why each change would enhance the error frequency.

 

5'-CTCTAGGCC-3'

6. A major class of regulatory proteins are the "Helix-Turn-Helix" proteins. Please comment on where they are important, and the important features of the proteins in terms of their general structure and function.

7. What are the primary role(s) of DNA methylation in A) procaryotes, and; B) eucaryotes?

8. The trp operon (i.e., it controls the genes involved in biosynthesis of tryptophan) in E. coli uses attenuation control. Suppose that you deleted the trp repressor protein from E. coli. How would this likely affect control of the trp operon and why?

9. DNA polymerase b catalyzes the removal of abasic sites from DNA in the reaction shown below. On the back of this page, draw a chemically logical mechanism for this reaction.

10. Briefly describe how RecA and RecBCD would work together to initiate DNA recombination. You do not have to describe how these proteins might be involved in resolution of the Holliday junction. (Hint: It would probably be easier to use the Meselson-Radding model of recombination as the basis of your description).

11. You are studying the regulation of the lac operon in E. coli, and discover a mutant form of the operon. You obtain the following data regarding the mutation. (1) It is cis dominant. (2) When cells are grown in the absence of lactose (+ or - glucose), the operon is turned off. (3) When the cells are grown in the presence of lactose (+ or - glucose), the operon is turned on at a very slow rate. Where did the mutation likely occur ? Explain the reasoning behind your answer.

12. Briefly outline the polymerase chain reaction. Describe why it is useful for molecular biology and list two potential problems with the products you finally obtain as compared to what you want to obtain.

13. Briefly outline how site-specific recombination can lead to gene amplification. Either words or pictures are fine.

14. Briefly outline the pathway of post-replicative repair of mismatches in procaryotes.

15. There are two mechanisms by which transcription is terminated in E. coli. Outline each method.

16. In terms of DNA replication, a linear chromosome can be problematic. What is the potential difficulty with linear chromosomes and what are 3 potential solutions to this problem (just list the solutions, do not describe them).

17. You have isolated a new DNA binding protein, and want to know with which nucleotides this protein interacts and the importance of the base at each position. Very briefly, describe how you would solve these questions.

18. What are two mechanisms that cells use to prevent the incorporation of deoxyuridylate into DNA?