COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #1

                                                                                                Thursday, January 26, 2006

                                                                                                9:00 a.m. to 11:00 am

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.                  WRITE YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.                  NUMBER ALL PAGES.

3.                  EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER.

4.                  WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.                  USE PEN.

6.                  STAPLE PAGES FOR EACH QUESTION SEPARATELY.

PAPER #1

TWO-HOUR QUESTIONS:          

ANSWER ONE OF THE FOLLOWING FOUR QUESTIONS:

Q1.    

ATP-dependent chromatin remodeling complexes possess a variety of biochemical activities in vitro, including chromatin disruption, nucleosome sliding, nucleosome spacing, and chromatin assembly activities. Imagine you are in a chromatin remodeling lab and are doing nucleosome remodeling experiments. First, you label one end of the DNA that has nucleosome positioning properties and reconstitute the DNA into nucleosomes with histone octamers. This DNA is 250 bp long and has two restriction enzyme sites (RE1 and RE2) located at sites 30 and 105 respectively (Figure 1A).

a)      Next you analyze the material by native gel electrophoresis (Figure 1B). You isolate the bands N1 and N2 from the native gel and perform DNase I footprinting (Figure 1C). Interpret these data and defend your interpretation.

b)      You then add BRG1, the catalytic subunit of the Swi/Snf chromatin remodeling complex. To detect BRG1 binding you add a 5 fold molar excess of BRG1 per nucleosome. The resulting DNase I footprinting is altered by BRG1 such that hypersensitivities (thicker bands) and additional footprints are observed at specific regions in the gel. You find that the hypersensitivities require the ATPase domain. What does this tell you about BRG1-nucleosome interactions?

c)      Now you want to observe BRG1-dependent nucleosome remodeling. First you digest the end-labeled nucleosomes -/+ BRG1/ATP (5 fold less than Figure 2) with RE1 or RE2, strip the histones, and compare this digestion to DNA on a gel (Figure 3A). You also do the remodeling reaction and run the native nucleosomes on a non-denaturing gel as in Figure 1B. You detect no significant loss of histones. What did the BRG1 do to the nucleosome?

d)     Finally, you set up a triple helix assay as in Figure 4 to observe displacement of the labeled third strand (squiggly line). You notice the labeled strand can be displaced in set up A but not B which has a 5 base gap on the top strand. However, strand displacement occurs in C which has a 5 base gap on the bottom strand. Interpret these results.

e)      Summarize your results with a model.

f)       How does the nucleosome remodeling reaction you describe facilitate transcription?

OR

Q2.   

A newly discovered enzyme (Cas-H) is shown to catalyze transformation of highly toxic substrate A to form a highly valuable fuel source termed product B. At 5 °C, the time it takes for a known amount of enzyme Cas-H to convert half of substrate A to B (t_1/2) is 1 hour, but upon increasing the temperature to 60 °C, the time it takes for half of A to convert to B decreases to 1 minute.

Valuable Equations:

(1)        ln([A]_t/[A]₀) = -kt       or         [A]_t = [A]₀exp(-kt)

             where [A]_t is concentration of A at time t and [A]₀ is concentration of A at time t = 0.

(2)        DG^‡ = -RTln(hk/k_BT)

(3)        DG^‡ = DH^‡ - DTDS^‡

(4)        °K = °C + 273°

Constant Values:

R = 1.99 ´ 10^-3 kcal mol^-1 °K^-1

h = 1.58 ´ 10^-37 kcal s

k_B = 3.3 ´ 10^-27 kcal °K^-1

a)      Calculate the enthalpy of activation (DH^‡, kcal/mol) of this valuable reaction (Hint: slope = Dy/Dx and proper units are crucial).

b)      Calculate the entropy of activation [DS^‡, kcal/(mol °K)] of this valuable reaction (Hint: DS^‡ may be calculated with eq 3, using the DH^‡ value calculated from above and DG^‡ calculated from either temperature. You may check your answer by making sure you get the same answer for DS^‡ using the DG^‡ value calculated at either 5 °C or 60 °C).

c)      Calculate free energy of activation (DG^‡) of the system at room temperature (21 °C) (Hint: You now have DH^‡ and DS^‡; go figure!).

d)     If the starting concentration of toxic substrate A is [A]₀ = 2 mM, calculate the concentration of the highly valuable fuel source B after 10 minutes at room temperature (21 °C)  (Hint: [B] = [A]₀ - [A]_t=10min).

e)      If the reaction is carried out in a total volume of 200 mL and the value of B is $300/mmol, calculate the total value of product B formed.

OR

Q3.    

One of your colleagues has performed a systematic screen for synthetic interactions between deletions of every “nonessential” gene in the budding yeast, S. cerevisae. One interaction was identified that inhibits growth on some carbon and nitrogen sources under aerobic conditions. This interaction was between deletions of two genes; the protein product of one of these has a role in feeding amino acids into the TCA cycle, whereas the other deletion is in a previously uncharacterized ORF.

a)      How would you determine whether the synthetic interaction involved loss of a protein encoded by the ORF and not a DNA sequence or RNA product of the ORF?

b)      Given that you determined that the phenotype depends on loss of the ORF protein product, how would you determine the function of this protein? Describe the steps you would take in sufficient detail to satisfy your thesis committee that you are on the right track.

c)      In your analysis, you determined that the protein is a member of a protein family with at least one domain common to a class of “oxidases” that use an FAD cofactor. Many of the related oxidases convert proline to pyrroline-5-carboxylate (P-5-C) as a product. Some of the bacterial enzymes in this family also convert P-5-C to Glutamate in a NAD-dependent reaction (this activity is called “P-5-C dehydrogenase”). Describe the steps you would take IN DETAIL to determine if “ORF protein” is a proline oxidase and/or a P-5-C dehydrogenase. Include the type of assays you would employ, the equipment required to run the assays, and the form the data would take.

d)     Given that you have confirmed that FAD is a cofactor and is converted to FADH₂ during the reaction, describe how you will determine K_m and k_cat of the enzyme for the amino acid substrate(s). How would you use this kinetic information to determine if the enzyme is specific for proline, if it uses other amino acid substrates, and which is the best substrate?

OR

Q4.    

A recent paper by Lindorff-Larsen et al. “Simultaneous determination of protein structure and dynamics”, Nature (2005) 433, 128-132 (attached) presents a new protocol for understanding protein dynamics.  Please answer the following questions after reading this paper (it is not necessary to read the supplementary information or any references quoted in the paper).

a)      What is S²?

b)      How does S² differ from NOE?

c)      How do the authors validate their method for determining the protein structure and dynamics?

d)     The authors define a “q”-factor.  What would be the value of this factor, if the authors had managed to exactly and perfectly replicate the dynamics of their test protein? How close did they come?

e)      Under ideal conditions, how should the curves in Figure 1 look like? (sketch this out).

f)       What does the DER method say about the carboxy terminus of ubiquitin?

g)      The authors state that the unrestrained MD ensemble is worse than the DER ensemble.  What evidence do they provide for this statement?

h)      What timescale of motions are the authors looking at with their DER approach?  Will this approach give us clues about the folding mechanism of this protein?

i)        Do the author’s findings contradict the notion that most proteins have a hydrophobic core?

j)        Are all the atoms in the test protein in a liquid-like state?  How do the authors quantitate this? What kinds of motions are reported as being characteristic of the liquid-like state of ubiquitin?

(END OF PAPER #1)
 

COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #2

                                                                                                Thursday, January 26 2006

                                                                                                1:00 p.m. to 3:00 p.m.

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.      WRITE  YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.      NUMBER ALL PAGES.

3.      EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER.

4.      WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.      USE PEN.

6.      STAPLE PAGES FOR EACH QUESTION SEPARATELY.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q5.    

It appears that RNA localization is one way that most, if not all, cells use to localize protein.   Describe three mechanisms that could account for how RNAs get localized.  How would you experimentally distinguish which mechanism was operating in a given cell?

OR

Q6.

Apical localization of membrane proteins in polarized cells appears to occur by several different mechanisms.  One of these mechanisms proposes that N-glycosylation is involved.  Suppose you have cloned an apically oriented glycoprotein with 2 N-glycosylation sites.  Design and describe a set of experiments to test the hypothesis that a specific N-glycoside participates in the apical localization of this protein.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q7.

A novel heart-specific gene was identified by microarray analysis comparing mRNA expression in heart muscle versus skeletal muscle cells.

1) Describe experiments you could perform to verify the heart-specific expression of this gene, and to examine whether this gene is also expressed in other, non-muscle tissues (10 minutes).

2) Genomic sequences containing approximately 2 kb of sequence upstream of the transcriptional start site of this gene were isolated by screening a genomic library with a gene-specific cDNA probe. How would you go about identifying potential heart-specific cis-acting gene regulatory elements within this genomic region (20 minutes).

OR

Q8.     

Most loss-of-function mutations are recessive, but there are a number of ways a mutation can be dominant. 

 (a)  You construct an expression vector in which a short segment of a given gene is deleted.  You overexpress that deletion mutant protein a mammalian cell (which you assume has either one or two copies of the natural [wild type] gene).  Suppose you see mutant phenotype (deletion mutation is dominant). Give at least two mechanisms by which this could occur. Suppose you do not see mutant phenotype. What additional experiment is needed to conclude that the deletion mutation is recessive?

(b)  Many RNA tumor viruses have been shown to carry oncogenes that are derived by mutation of cellular genes that have a normal function in nontransformed cells.  When a tumor virus infects a nontransformed cell and causes it to become transformed, the oncogene is expressed in the presence of the natural [wild type] cellular gene.  This means that the oncogene must act as a dominant mutation.  Give at least two mechanisms (in addition to the two given in your answer to part a) that could explain why the viral oncogene is dominant.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q9.   

There is much interest in identifying essential genes in the model organism Escherichia coli K-12 because it is likely that essential genes in this laboratory organism are also essential in closely-related enteric pathogens such as Yersinia pestis and Salmonella typhi. Genes that are essential for growth (on rich media at 37°C) are attractive targets for new antibiotics. A recent study by Svetlana Gerdes et al. attempts a global identification of K-12 essential and non-essential genes by sequencing a large number of random chromosomal transposon insertions. Their logic is that genes with insertions are non-essential and that genes with no insertions are essential. Their results have ended up in various databases as fact. The problem is that the method apparently is flawed. Numerous well-characterized essential genes are identified in the Gerdes study as non-essential. Likewise many so-called essential genes are E. coli genes that have been successfully knocked out by targeted mutagenesis and therefore cannot be essential. The transposons used are designed to be non-polar, so assume that the transposon insertion mutations do not affect the expression of downstream (or upstream) genes.

a)      Give two different reasons why Gerdes et al. might have found insertions in essential genes. Hint 1: The insertion events are recA⁺-independent, but the stability of some transposons’ antibiotic resistance markers is recA⁺-dependent, i.e., some antibiotic-sensitive cells segregate out of some mutant populations spontaneously in the absence of the antibiotic, but only if the cells are recA⁺.

(Hint 2: some “non-essential” genes had only two or three insertion events mapped into the coding region.)

b)      Give two different reasons why some known non-essential genes did not accumulate insertions.

OR

Q10.

You perform a transposon mutagenesis experiment using:

1) E. coli strain L1 deleted for the lac operon but otherwise wild type       

(phenotype =Ara⁺ Lac^-)

2) A replication defective Lambda phage containing a Tn5(LacZ), a transposon that encodes an active transposase, the gene for kanamycin resistance and a portion of the lacZ gene that lacks a promoter, lacks a Shine-Dalgarno sequence and lacks the first couple of codons from the 5’-end of lacZ.

You isolate a mutant you name strain A2 that has the following phenotype:

Kan^R (resistant to kanamycin)

Ara^- (unable to use arabinose as a sole carbon source)

LacZ^- (no b-galactosidase activity when there is no arabinose in the growth medium)

LacZ⁺ (high b-galactosidase activity when there is arabinose in the growth medium)

1. Describe the most likely cause of this phenotype and the specific role of the transposon in this phenotype. Your answer needs to account for all the molecular details required to explain the loss of arabinose catabolism and the conditional b-galactosidase activity.

2. You grow phage P1 on strain A2 and perform a backcross of Tn5(LacZ) to strain L1, selecting for Kan^R. To your surprise you note two classes of transductants. One class has the same phenotype as A2. However, the plates also show a second class, which is phenotypically Ara⁺, LacZ^-, Kan^R. You go back through your previous days’ calculations and realize the MOI you calculated for the transposon mutagenesis experiment that gave rise to the A2 mutant was 5, instead of the 0.1 you had aimed for. Interpret the results of this experiment by describing the apparent genotype of your original mutant strain A2. Propose two possible reasons why the second unexpected class appeared in the backcross.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q11.

Northern analysis of mRNAs from CHO cells indicated that mRNA for protein X is present at 5-fold higher amounts than the mRNA for protein Y. Yet, nuclear run-on experiments indicate that the transcription rates for the two messages are essentially equal. Provide an explanation for this observation and devise an experiment to test your hypothesis.

OR

Q12.

Table I

Summary of purification of protein X

                                       (units)                            (mg)                         (units/mg)     

1. Crude extract             1000                             100                                   10

2. DEAE-Sephadex       3000                               20                                  150

3. Hydroxyapatite          2000                                0.5                              4,000

4. Sephacryl S-300         1000                                0.1                            10,000

Answer the following questions based on Table I:

a) What is the apparent level of purification of protein X?

b) What might explain the increase in enzyme activity from step 1 to step 2 and how   might you test this point?

c) Based on your answer to “b”, how would this affect your answer to “a”?     

(END OF PAPER #2)

COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #3

                                                                                                Friday, January 27, 2005

                                                                                                9:00 a.m. to 11:00 a.m.

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.      WRITE  YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.      NUMBER ALL PAGES

3.      EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER

4.      WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.      USE PEN.

6.      STAPLE PAGES FOR EACH QUESTION SEPARATELY.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q13. 

Describe the basic principles that govern the operation of the MALDI-TOF mass spectrometer and explain how this instrument can be used to determine the amino acid sequence of a short peptide.

OR

Q14. 

Give brief definitions of ORD and CD and explain why they provide the same information about protein structure. Why is CD generally preferred to ORD? How and why can these techniques be used to estimate the secondary structure composition of a protein molecule?

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q15. 

Describe how the structure of bacteriorhodopsin (the purple membrane protein) was determined to 3 Ǻ resolution with the use of the electron microscope. Why has this technique not proven universally applicable for the determination of structures of membrane proteins ?

OR

Q16. 

The initial model of a protein molecule constructed from an electron density map calculated using MIR (multiple isomorphous replacement) phases is frequently of poor quality. What parameter is used to determine the level of agreement between the model and the experimental X-ray diffraction data? What steps can be taken to improve the accuracy of the model (i.e., refine the structure)?

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q17.  

Draw the molecular structure of histidine in its unprotonated neutral form. Describe two different 'general' mechanisms by which the unprotonated form of histidine may function to accelerate the rate of its corresponding enzymatic reaction. Finally, describe two different ways that the molecular architecture of an enzyme active site may serve to lower the pK_a of the catalytic histidine in order to ensure that it is fully deprotonated.

OR

Q18. 

The following data represent measurements of the initial velocities of an enzyme-catalyzed reaction as a function of substrate concentration:

                       [s] mM                 V mMoles / min.

                   ___________           _____________

                           1                                   30

                           2                                 100

                           3                                 230

                           4                                 450

                           5                                 810

                           6                                 930

                           7                                 990

                           8                               1010

                           9                               1020

Using two alternative plots of these data, what conclusions can you draw about the mechanism of action of this enzyme?

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q19. 

You have identified two potential binding sites for a microRNA in the 3’-UTR of protein X mRNA. Protein X is expressed in fibroblasts but is absent from adipocytes.  You hypothesize that the downregulation of protein X is essential for the differentiation of fibroblasts into fat cells and that the expression of this specific microRNA is instrumental in this process. Design an experiment to test your hypothesis.

OR

Q20. 

You are studying the regulation of expression of protein Y. This protein is expressed in most cells and is even expressed under stress conditions (heat shock) where cap-mediated translation typically does not occur. You suspect that the translation of protein Y occurs from an internal ribosome entry site (IRES). How would you test this hypothesis? Design an experiment to find out if cap-mediated translation of this protein occurs at all, or if all translation occurs from the IRES.

(END OF EXAM)