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A. First you reconstitute nucleosomes with salt dialysis on
COMPREHENSIVE
EXAMINATION
IN
BIOCHEMISTRY AND
MOLECULAR BIOLOGY
PAPER #1
Thursday, February 1, 2007
PLEASE USE THE
FOLLOWING INSTRUCTIONS:
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TWO-HOUR QUESTIONS:
Q1.
A novel protein X contains
the consensus motif PXXP known to be a target for SH3 domains. It is thus
conceivable that the protein X exerts its function, at least in part, through
its ability to recognize downstream targets containing SH3 domains. To test the
hypothesis that protein X binds SH3 domains in a specific manner, you undertake
to clone the corresponding gene of interest in a bacterial expression vector
with a N-terminal polyhistidine tag (His-tag), purify the recombinant fusion
protein to apparent homogeneity and subsequently carry out a series of
experiments to determine its function. After purification, you notice that the
protein yield is too low; in fact a vast amount of protein ends up in inclusion
bodies. You have two options before you. One is to purify the protein X from
inclusion bodies, or alternatively, you could also consider re-designing
your vector so as to add a stability tag at the N-terminus that may prevent the
formation of inclusion bodies. Please provide your answers to the following
questions:
a)
What are
inclusion bodies? What properties of proteins render them more likely to be
expressed as inclusion bodies? [10 points]
b)
What is the
function of His-tag? What are pros and cons of purifying recombinant proteins
expressed with an His-tag? [10 points]
c)
How would you
purify the recombinant His-tagged protein X from soluble fraction on an affinity
column (starting with cultured bacterial cells)? Summarize major steps involved
in a logical and coherent manner. [10 points]
d)
Name TWO suitable
tags that could be used to stabilize protein X. Give arguments for and against
the use of each tag. [10 points]
e)
Name TWO suitable
protease sites that could be added to your expression construct to remove the
tag(s) after purification of protein X? Give pros and cons of each protease. [10
points]
f)
Protein X is less
than 75% pure after it has been passed through an affinity column. Provide TWO
further protein purification steps/procedures that could be carried out to
enhance the purity of protein X? Give arguments for and against each
step/procedure. [10 points]
g)
Before you can
begin your functional analysis, you are asked to dialyze protein X in the
Minimal Buffer (50mM Tris, 200mM NaCl, 5mM EDTA and 5mM
b-mercaptoethanol
@ pH 8.0). Provided you have access to 1M Tris, 4M NaCl, 0.1M EDTA and 14.3 M
b-mercaptoethanol
stock solutions, determine the volume (in ml) of each of these stock solutions
required to make up 5L of the Minimal Buffer solution. What is the role of Tris,
NaCl, EDTA and
b-mercaptoethanol
in the Minimal Buffer? Suggest one alternative for each of these components of
the Minimal Buffer. [30 points]
h)
Thermodynamic
analysis of protein X with a SH3 domain shows that the binding constant (Kd)
is 1
mM
and that the enthalpy of binding (DH)
is +5 kcal/mol at 25°C.
Given the relationship
DG
= RTlnKd [where R is the universal molar gas constant (2 cal/K/mol)
and T is the absolute temperature in degrees Kelvin (K)], calculate the free
energy of binding (DG)
of protein X to the SH3 domain at 25°C.
Given the relationship
DG
=
DH
- TDS
(where
DS
is the entropy of binding), calculate the entropic contribution to the free
energy of binding of protein X to the SH3 domain at 25°C.
Is the binding of protein X to the SH3 domain under enthalpic (enthalpy-driven)
or entropic (entropy-driven) control? What is the molecular basis of enthalpic
and entropic factors contributing to the free energy of binding? [30 points]
Q2.
ACF is an ATP-dependent
nucleosome remodeler that repositions nucleosomes to generate regularly-spaced
nucleosomal arrays.
A. First you reconstitute nucleosomes with salt
dialysis on
B. Nucleosomes can slide
gradually to a new position or be transferred directly to the position by
histones disengaging from the
Figure 2A) and a Cy5 acceptor on histone H2A
(red circle). You initiate the
C. Next you put the Cy3 on
the very end of the sequence as shown (green circle) which should place it near
the Cy5 on histone H2A (Figure 3A). You do the
D. Now you use this assay to determine the
kinetics of remodeling for
E. Based on the results in
Figure 4, how does
Q3.
Recent major advances in
the field of biochemistry and biotechnology include the development of big
science approaches in the areas of genomics, proteomics, and most recently,
metabolomics. Write a 5-paragraph "news and views" perspective on the emerging
field of metabolomics. Hints: (i) Develop
a theme that resonates throughout the essay. For example, the theme should
provide an energetic prospective, particularly emphasizing the significance of
awaited outcomes that will result from integration of metabolomic data with
designated cellular genomic and proteomic profiles. (ii)
Develop a well organized outline.
Elaborating on the theme, the body of the essay may include details that define
the initial goals and describe emerging technologies that enabled development of
genomics, proteomics, and metabolomics. Additional details may be included that
discuss the significance of both outcomes and limitations within each isolated
endeavor. These points should resoundingly reinforce
your theme.
The genes for proline utilization (put)
are located at 22 min on the genetic map of
S.
typhimurium LT2. The
putP gene encodes the major proline permease, a Na+/Proline
symport protein residing in the cytoplasmic membrane. The
putA gene encodes a cytoplasmic membrane-associated enzyme that
possesses both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase
activities in a single polypeptide. The proline utilization pathway is shown in
Figure 1.
Polar transposon Tn5 insertion mutations in the
putP or the
putA gene completely eliminate
activity of the mutated gene but do not inactivate or reduce expression of the
other gene. The absence of polarity effects suggests that these genes are
transcribed independently, even though they map close to one another in the
genome.
In order to determine if the put
genes are 1) in an operon,
2) are regulated at the level of
transcription, and 3) to identify
regulatory elements, operon fusions were created that fuse the
put genes to the structural genes of the
lac operon by use of the hybrid Mu
phage derivative Mu d(Amp lac). A
series of strains bearing independent ampicillin resistant (AmpR) Mu
d(Amp lac) insertions in the
put genes were identified by their
ability to grow on minimal medium containing
succinate as a carbon source
and ammonium as a nitrogen
source and their inability to grow on minimal medium containing
succinate as a carbon source
and proline as a nitrogen
source (Put -).
Approximately 40% of the Put -
AmpR transductants contained Mu d(Amp
lac) insertions in the correct
orientation to express the lac genes
from the put regulatory region. Put
- AmpR Lac+
isolates shown to have only a single Mu d(Amp
lac) insertion were characterized as
putP::Mu d(Ap lac) or
putA:,:Mu d(Ap lac) by the
following criteria: (i) ability to use the dipeptide leucyl-proline as the sole
nitrogen source, (ii) sensitivity to the toxic proline analog dehydroproline,
and (iii) mapping against known
putP and
putA mutants. Insertion of Mu d(Amp
lac) into
putP or
putA resulted in a Put
- phenotype. Since the
dipeptide leucyl-proline enters the cell through a peptide transport system,
putP::Mu d(Amp lac) fusions
which are defective in proline permease can use leucyl-proline but not L-proline
as the sole nitrogen source. In addition,
putP::Mu d(Amp lac) insertion
mutants fail to transport toxic proline analogs and thus are
resistant to growth
inhibition by dehydroproline. In contrast,
putA::Mu d(Amp lac) mutants
cannot use either leucyl-proline or L-proline as the sole nitrogen source since
they lack the required degradative enzymes. Furthermore,
putA::Mu d(Amp lac) fusion
mutants can transport proline analogs and are
more sensitive to
dehydroproline inhibition than are put
+ cells.
PART ONE (of
TWO):
Interpret the information provided in the preceding text and the data in the
following tables and develop a model for regulation of proline utilization in
S. typhiumurium. Support your
statements with specific examples from the data in the text and these tables. BE
SPECIFIC about each type of regulation, the targets and the mediating factors,
sites or physiological conditions.
In the three tables shown below, β-Galactosidase
was assayed in permeabilized cells and β-Galactosidase activity
expressed as nanomoles per minute per optical density unit (650 nm). All
values represent means of triplicate assays. Since the expression of the
lacZ gene product, β-galactosidase, reflects the transcription of the operon
to which Mu d(Ap lac) is fused, the
β-galactosidase activity expressed by
putP::lac and
putA::lac fusion mutants was determined for cells grown under a variety
of conditions. Although the absolute level of β-galactosidase expressed from
different
put::lac fusions in a single gene
varied somewhat, all of the fusions in the same gene showed the same
general pattern of regulation. F' is the
episomal F factor bearing some bacterial genes.
Tn5 is a transposon; the Tn5 alleles
shown are polar loss of function alleles. The gene
crp encodes cAMP receptor protein and
cya encodes adenylate cyclase.
In order to map the
orientation of transcription of
putP and
putA, Hfr derivatives were constructed by integrating F' ts
lac + Tn10 into each gene
via homologous recombination between lac
sequences in the F' and in the Mu d(Amp
lac) insertion in each gene (selecting for TcR at high
temperature). The origin of conjugal transfer resides within the F' and is
unidirectional. By determining the frequency of transfer of neighboring genetic
markers (pyrC
+ is
putP proximal and
pyrD + is
putA proximal), the relative
orientation of the
putP and
putA transcripts was determined. This is illustrated in Figure 2, below:
PART TWO (of
TWO):
Draw a simple map of the put operon. In your
map, place the genes pyrC,
putP,
putA,
pyrD, and any putative regulatory
regions, promoters, operators, and other elements as necessary to explain the
lac fusion data in the preceding
tables. Make your figure and model complete, but try to make it as simple as
possible!
Q5
This question relates to the recent structure of
the SAM riboswitch [Montange, RK & Batey, RT, Nature (2006) 441, 1172-1175].
The relevant pages of this paper are attached (note: the two pages are
not contiguous. Also, it is not
necessary to read the other pages or supplementary information to answer these
questions).
(a) What is an RNA "pseudoknot"?
(b) The authors refer to a "genetically
validated pseudoknot". How do you
think this was validated?
(c) How was this structure solved?
Can you evaluate the quality of this structure from the attached figures?
(d) Are all the nucleotides hydrogen bonded to
SAM highly conserved?
(e) Why are the A6-U88 and U7-A87 base-pairs
universally conserved?
(f) Can U57 be mutated to a C?
Estimate roughly, the consequences of such a mutation.
(g) Why does this riboswitch strongly prefer SAM
over S-adenosylcysteine?
(h) What is the type of loop that is present at
the end of helix P4? What structure
does it adopt?
(i) What kind of interactions are seen between
C45, A46, and A47?
(j) In your own words, how does this riboswitch
work? What is it "switching"?
COMPREHENSIVE
EXAMINATION
IN
BIOCHEMISTRY AND
MOLECULAR BIOLOGY
PAPER #2
Thursday, February 1, 2007
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)
Q6.
Upon cold shock of Chinese hamster ovary cells,
it was found that RNA helicase activity using ribosomal RNA as a substrate was
elevated 3-fold. What experiments would you undertake to help understand the
cellular mechanisms responsible for this elevation of activity?
Q7.
A kinase Q is active and present in a cell, yet
its substrate P is not phosphorylated until a specific time during the cell
cycle as analyzed by immunofluorescence or western blotting.
What could be the explanation(s) and how would you test to see if you
were right?
ANSWER ONE OF
THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q8.
The expression of protein X
, while required during the early stages of leukocyte differentiation, needs to
be shut down for final differentiation. You hypothesize that this shutdown of
expression of protein X is caused by a microRNA that appears in the cells during
differentiation. Outline an experimental approach to test your hypothesis.
Please be sure to include controls.
OR
Q9.
The fidelity of translation
is relatively high. Only one mistake is made in 10,000 peptide bond formations.
How is this high degree of accuracy accomplished? Be specific and draw
structures of intermediates.
ANSWER ONE OF
THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q10.
The average numbers of exons in the human genes are 9.8. Supposing the gene transcript with 10 exons (9 introns) is regulated fully and solely by "exon-inclusion/skipping"-type of alternative splicing, potentially how many mature mRNA can be generated from this single transcript? [Hint: Calculate smartly the number of all the possible combinations of exons, retaining the 5’ and 3’ end exons in all the spliced mRNAs.]
Q11.
You are studying chemotactic behavior at the
single cell level in cultures of a bacterial strain and you observe an
unusual behavior in one out of every 25 cells in one culture.
You don't observe any of this abnormal behavior in several other cultures
so you suspect it has a genetic basis.
You hypothesize that your observation is due to a spontaneous mutation
that has occurred in this culture and you want to isolate a pure culture of this
mutant so you can characterize it.
You isolate a number of clones and observe each one of them until you obtain a
culture that is genetically homogeneous with regard to this behavior.
a)
If you isolate 25 clones and observe each one separately, what is the
probability that at least one will exhibit this behavior in all cells.
Assume for this part of the question that the phenotype is due to a
single mutation that is genetically stable and expressed in all cells that
contain the mutation. (A table of
values of e-m for various values of m is given below for your
use, if appropriate.)
b)
With the same assumptions as in (a), how many clones do you need to
analyze to have a 95% chance of obtaining a pure clone for the mutant behavior?
c)
It is possible that the mutant behavior will not be expressed in all
cells that contain the mutation.
Suppose that a clone in which 100% of the cells are mutant shows the mutant
behavior in only 1/3 of the cells.
If you isolate 25 clones from the original mixed culture, what is the
probability that at least one will be genetically homogeneous for the mutation?
e-m for various values of m
|
m |
e-m |
m |
e-m |
m |
e-m |
m |
e-m |
m |
e-m |
|
0.1 |
0.905 |
0.8 |
0.449 |
1.5 |
0.223 |
2.4 |
0.091 |
3.8 |
0.022 |
|
0.2 |
0.819 |
0.9 |
0.407 |
1.6 |
0.202 |
2.6 |
0.074 |
4.0 |
0.018 |
|
0.3 |
0.741 |
1.0 |
0.368 |
1.7 |
0.183 |
2.8 |
0.061 |
4.2 |
0.015 |
|
0.4 |
0.670 |
1.1 |
0.333 |
1.8 |
0.165 |
3.0 |
0.050 |
4.4 |
0.012 |
|
0.5 |
0.607 |
1.2 |
0.301 |
1.9 |
0.150 |
3.2 |
0.041 |
4.6 |
0.010 |
|
0.6 |
0.549 |
1.3 |
0.273 |
2.0 |
0.135 |
3.4 |
0.033 |
4.8 |
0.008 |
|
0.7 |
0.497 |
1.4 |
0.247 |
2.2 |
0.111 |
3.6 |
0.027 |
5.0 |
0.007 |
ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q12.
You are
studying the transcriptional regulation
of a novel human gene and have cloned 1
kb of 5' flanking sequence. To identify cis elements, you subclone
the upstream sequence into a luciferase
reporter plasmid. However, you are surprised that
in transient transfection studies
using human cells, regulation of
reporter activity
is not consistent with the
previously observed patterns of
regulation of the endogenous gene. Thus, physiological and pharmacological
stimuli, which induce endogenous gene expression, do not stimulate reporter
activity. Assuming that there was not a technical "screw up" in your
subcloning, discuss the possible reasons
for this divergence of data. Also, how
might you experimentally discriminate between these various possibilities?
OR
Q13.
Expression vectors for
expression of a recombinant protein in E. coli often make use of fusions
with E. coli Lac operon promoter because this allows inducible expression
of the recombinant protein and helps to reduce possible toxic effects of high
level expression of the recombinant protein while the culture is being expanded.
Expression of recombinant protein is often improved by further
modifications introduced into the host and expression vector.
From your knowledge of the regulation of the Lac operon, explain what is
accomplished by each of the following modifications and why each modification is
beneficial for plasmid growth and high-level expression of the correct
recombinant protein.
a)
The upstream portion of the Lac promoter is replaced by a portion of the
promoter for the tryptophan operon or a specific point mutation (lac UV5)
is introduced into the
–10 sequence element of the Lac promoter.
b)
The i gene is replaced by a mutant lac iq that
has increased affinity for the Lac operator.
(END OF PAPER #2)
COMPREHENSIVE
EXAMINATION
IN
BIOCHEMISTRY AND
MOLECULAR BIOLOGY
PAPER #3
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)
Q14.
Various techniques can be used to determine the
molecular mass of a protein. However, it is sometimes found that the mass
determined by gel filtration is very different than that determined by sucrose
gradient centrifugation. Provide an explanation for this apparent discrepancy
and suggest a method for mass determination that would not be subject to this
problem.
Q15.
In the sedimentation
equilibrium method, the molecular weight of a macromolecule is given as:
2RT ln (c2/c1)
M = -----------------------
(1-vρ) ω2 (r22 – r12)
Explain the meanings of the terms: v, ρ and ω in this equation. What experimental parameters must be measured to calculate molecular weight? Under what conditions must they be measured ?
ANSWER ONE OF
THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q16.
What distinguishes
Resonance Raman scattering from regular Raman scattering ?
What types of information about protein molecules can be obtained using
Resonance Raman scattering ?
Q17.
Explain how a protein
structure can be determined by the method of “Molecular Replacement”. What
measurements must be made ? What is meant by the “Rotation problem” and how can
it be solved ? What is the “translation Problem” and how is this solved ?
ANSWER ONE OF
THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q18.
With only a limited
repertoire of amino acid side chain chemical groups, enzymes catalyze a myriad
of biological reactions employing such side chain groups as either nucleophiles,
electrophiles, general-base catalysts, and general-acid catalysts. The key to
their adapted role or function lies in their state of protonation, and the state
of protonation is determined by the pKa of the given group
relative to the pH of the reaction environment.
Q19.
Protein P is a homotetramer
(a4)
and is known to bind a small molecule ligand L. Briefly describe two different
physical methods, which can be used to detect the amount of L that binds to P as
a function of the concentration of L. Give an equation and define the parameters
that can be used to analyze the binding data. From this equation, diagram three
binding analysis plots, properly labeling the x-axis and y-axis and showing the
curve or line that would result if there was (i) no cooperativity, (ii) positive
cooperativity, and (iii) negative cooperativity. Finally, indicate the parameter
that quantifies the degree of cooperativity and give the exact value of this
parameter that indicates (i) no cooperativity and values relative to this value
that indicate either (ii) positive cooperativity or (iii) negative cooperativity.
ANSWER ONE OF
THE FOLLOWING TWO QUESTIONS (30 MINUTES)
Q20.
DNA binding proteins often
bind specific sequences, while most RNA binding proteins are non-specific.
What features of DNA and/or protein motifs make DNA binding proteins more
sequence specific than proteins that bind RNA.
Use at least one DNA binding and one RNA binding motif to illustrate your
answer.
Q21.
There are many examples of
RNA catalysts, but almost none of DNA acting as an enzyme.
What features of RNA allow it to catalyze reactions?
How do RNA catalysts compare with protein catalysts?
(END OF EXAM)