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Yang
Buelow
Backer
Levy
Shechter
100
List 2 reasons that the cell prefers to store energy in the form of triglycerides.
What is: 1) Greater ATP yield per carbon compared to glucose. 2) Anhydrous.
100
Which sugar molecule is depicted in the picture? (Be specific)
What is L-Glucose
100
2. The endogenous GTPase activity of heterotrimeric G-proteins serves to: (1 point) a. Synthesize cGMP as a second messenger. b. Hydrolyze GTP returning the G protein to a pre-stimulated level of activity. c. Stimulate the activity of enzymes by producing energy. d. Synthesize GTP as an energy source.
What is B
100
How can the on rate of LacI for its operator be faster than the rate of diffusion (2pts)? (Answers longer than two sentences will receive no credit)
What is The non-specific binding of LacI to DNA allows it to slide along the DNA in search of the operator. This turns a 3D search into a 1D search.
100
What region of the genome would be free of nucleosomes? A) The gene body B) Intergenic regions C) Heterochromatin D) The transcription start site E) None of the above, there is no naked DNA in the cell
What is D
200
The major rate-limiting step in the cholesterol biosynthesis pathway is catalyzed by HMG- CoA synthase, which makes HMG-CoA from acetyl-CoA. True or False ? (If false, please provide a brief explanation.)
What is False. The rate-limiting step is catalyzed by HMG-CoA reductase, which makes Mevalonate from HMG-CoA.
200
Name the disaccharide shown in the image (your name should include the sugar monomers, and information about the disaccharide linkage).
What is Glcβ1-6Glc (Do not accept Gentiobiose)
200
Which statement is most accurate when describing the specificity of SH2 domain containing proteins? a) There is little specificity, all SH2 domain containing proteins will bind to all pY residues with similar affinities. b) Residues that neighbor the pY residue are important for the specificity of SH2 domain containing proteins. c) Multiple SH2 domains in the same protein can be occupied simultaneously, leading to increased affinity and specificity. d) Post-translational modification of the SH2 domain is the key to specificity. e) Both B and C are correct
What is E
200
Name the enzymes which catalyze the addition of amino acids to tRNA, and briefly explain the mechanism.
What is Aminoacyl-tRNA synthetases (AARs). 2 step rxn: 1) Use ATP to "charge" the amino acid creating Aminoacyl-AMP and releasing PPi. 2) Attach amino acid to the tRNA, releasing AMP. Class 1 AARs attach through the 2' OH of tRNA. Class 2 attach through 3' OH of tRNA.
200
(8 points total) a. (2pts) What is the aberrant DNA structure shown below (Letter C) and what typically causes it? b. (3pts) What repair pathway is typically used to repair this damage and how does this repair machinery work? Concepts are more important than the names of the enzymes, although they are helpful. c. (3pts) What might happen if a DNA replication fork encountered this damage?
What is a) Cyclobutane pyrimidine dimer b) Nucleotide Excision Repair (NER). XPC recognizes damage, TFIIH unwinds the DNA, XPA & RPA confirm damage, XPG (nuclease) removes 27-30nt ssDNA fragment, templated repair using the normal DNA replication machinery. c) These alter the structure of the DNA inhibiting DNA polymerase and arresting replication.
300
Which is false regarding the long chain fatty acid oleate (18:1n­9 double bond between carbons 9 and 10)? (3 points) A) 3 rounds of β­oxidation can occur until a cis­delta­3 double bond is formed. B) The complete oxidation of oleate to acetyl­CoA proceeds by dehydration, hydration, dehydration and cleavage. C) The cis­delta 9 double bond creates a 30 degree bend in the fatty acid. D) The first step in β­oxidation of oleate involves dehydrogenation requiring FAD.
What is B
300
How are N-Glycans linked to proteins?
What is via an Asparagine residue (Bonus if they say, followed by X , Ser/Thr).
300
In yeast, a scaffolding protein (Ste5) is known to enhance MAP kinase signaling by physically coupling the kinase cascade. Predict what would happen to signaling pathways in a yeast strain which overexpresses Ste5? Explain why.
What is: Reduced signaling through the MAPK pathway. Stoichiometry is critical for efficient coupling, overexpression would act to separate members of the MAPK pathway.
300
Briefly explain the antibacterial mechanism of puromycin.
Puromycin inhibits translation of bacterial mRNA by acting as a mimic for Tyrosine-tRNA. This causes premature termination of translation when the ribosome encounters a tyrosine codon. Note this is only true for bacterial ribosomes.
300
(9 points total) A former labmate performed the experiment below (Letter D) to assay DNA replication in vitro with a recombinant DNA polymerase. You are trying to repeat his experiment. He is unreachable and his notebook handwriting is illegible. a. (2pts) What is “component x”? b. (2pts) Why is component “x” necessary in this reaction? c. (2pts) What other enzymes require “component x”? Why? d. (3pts) Describe the catalytic mechanism of DNA polymerase.
What is a) Magnesium b) Stabilizes/positions phosphate groups on incoming dNTP. c) RNA polymerases (basically the same reason). d) Conserved Asp residues in the catalytic pocket coordinate Mg2+ positioning the incoming dNTP. This promotes nucleophilic attack by the 3' OH to the alpha phosphate group (PPi leaves). Specificity is conferred mainly by geometry of the incoming dNTP.
400
Briefly describe the effects of cholesterol on the physical properties of biological membranes. (3 points)
What is: Cholesterol is a rigid molecule that “rigidifies” biological membranes. At low concentrations, it increases bilayer thickness, while at high concentrations it often forms cholesterol-rich domains. Cholesterol also broadens the transition peak from the gel state to the liquid crystalline state, but does not change the melting temperature.
400
Name four different Glycosaminoglycans.
What is: i) Hyaluronic acid, ii) chondoitin sulfate, iii) dermatan sulfate, iv) heparan sulfate, v) keratan sulfate
400
Under basal conditions, Src is inhibited by a phosphorylated tyrosine residue in its C-terminus that binds to its SH2 domain. When the “best” phosphotyrosine motif for the Src SH2 was determined, it was noticed that the Src C-terminal tyrosine does not fit the optimal consensus sequence for binding to the Src SH2 domain. Why might this mismatch be important for Src activation in vivo? Why does this make sense from a biological perspective?
What is: The C-terminal phosphotryosine acts as an autoinhibitory motif for Src, effectively putting Src in an off mode. To become active Src has to use its SH2 domain to bind to phosphotyrosine residues on other proteins. If its own C-terminal phosphotyrosine was ideal, then this would never happen and Src would remain off.
400
In the handout (Letter A) are data from a dot blot experiment involving a radiolabeled RNA and 2 proteins, protein A and protein B. For each experiment, trace (<0.1nM) radiolabeled RNA was incubated with increasing concentrations of protein and then applied to a dot blot apparatus containing a nitrocellulose membrane stacked on top of a positively charged nylon (H+) membrane. The last blot contains an equimolar mixture of proteins A and B. The protein concentration is indicated above each dot. An empty circle indicates no binding on that membrane, and a dark circle indicates binding. The darker the dot, the more bound RNA. Write the answers in your exam book. 1) Which of the following statements in correct (1pt) a. The RNA binds the nitrocellulose membrane in the absence of protein through charge-charge interactions. b. The RNA binds to the nitrocellulose membrane only when it is bound to protein. c. RNA binds to the nylon membrane through hydrophobic interactions. d. RNA will not bind the positively charged nylon membrane. 2) Based on the data, approximate the observed binding constant for (6pts): a. Protein A b. Protein B c. Proteins A and B combined 3) Based on the data, which statement is true? (1pt) a. Protein B binds Protein A in the absence of RNA b. Protein A also binds DNA c. A and B act cooperatively to bind the RNA d. The combination of protein A and B has little effect on the apparent binding constant. e. None of the above 4) Which answer is correct? (1pt) a. The mass of protein A is greater than protein B b. The mass of protein B is greater than protein A c. The mass of protein A and B cannot be determined from this data 5) Which method might have been used to radiolabel the RNA used in this experiment? (1pt). a. The RNA was made by transcription using a dsDNA template, T7 RNA polymerase and a mixture of all four NTPs containing gamma-32P ATP. b. The RNA was made by transcription using a dsDNA template, T7 RNA polymerase and a mixture of all four NTPs containing alpha-32P ATP. c. The RNA was labeled at the 3’ end using T4 polynucleotide kinase and gamma-32P ATP. d. The RNA was labeled at the 5’ end using alkaline phosphatase and alpha-32P ATP.
What is 1) b 2) Protein A = 32nM, Protein B = 16nM, combined = 4nM 3) C 4) C 5) B
400
Micrococcal nuclease treatment of a chromatin sample revealed the digestion pattern in lane #2 (Letter E) of the adjacent gel. Which of the following is true? A) This is not actually a chromatin sample B) Theses nucleosomes contain 200 bp of DNA C) There is no linker DNA on the gel D) The nucleosomes likely contained only one of each of the core histones E) This chromatin was likely bound to linker histone
What is E
500
14C acetate labeled on both carbons is added to cells that avidly synthesize cholesterol and cholesteryl esters. If 14C acetate were added and then oxygen immediately removed from the cell environment making it anaerobic, what is the likely end product in the cholesterol biosynthetic pathway that you would purify from these cells? (3 points)
What is Squalene
500
Describe (in detail) how UDP-GlcNAc reports on cellular metabolic state.
What is UD -> Nucleotide Metabolism P -> Overall Energy Glc -> Glucose Metabolism N -> Nitrogen Metabolism Ac -> Fatty Acid Metabolism
500
cyclic AMP (cAMP) was the first identified secondary messenger. How is cAMP made in the GPCR signaling pathways? What is the major downstream target of cAMP in these pathways? Briefly explain the mechanism of target activation.
What is: Adenylyl Cyclase (AC) is activated by the GTP-bound alpha subunit of heterotrimeric G-proteins. AC converts ATP to cAMP. cAMP binds to the regulatory subunit of Protein Kinase A (PKA) causing dissociation of regulatory subunits from catalytic subunits, and leading to the activation of PKA.
500
In a second set of experiments, you measure the binding constants for proteins A and B at increasing concentrations of salt (see Letter B). 6) Which protein likely contains fewer ionic interactions with the RNA? (1pt). 7) Which two amino acid residues would most likely be involved in ionic interactions with the RNA? (1pt) a. N and Y b. Y and S c. W and F d. R and K
What is 6) Protein A 7) D
500
(10 points total) Chromatin is the physiological form of the genome in eukaryotic cells. Briefly but thoroughly answer: a. (3pts) What are the constituents of the fundamental repeating unit of chromatin and how are they arranged? b. (4pts) What are the two current models, and supporting evidence, for the arrangement of the 30nm-fiber? c. (3pts) What is the molecular structure shown at right (Letter F) and what role can it serve within chromatin? There are multiple correct answers for its role.
What is a) 2 copies of each H2A, H2B, H3, and H4 + approx. 147bp of DNA wrapped in a left-handed superhelix around the histone octamer (approx 1.7X). b) 1-start model (solenoid): EM using long repeat length (typical in cells) 2-start model (zig-zag): Tetranucleosome crystal structure, EM using short repeat length. c) Tri-methyl Lysine (Kme3). Modification to histone tails. On H3 at promoters it can activate (H3K4me3) or repress (H3K9me3) transcription of a gene.