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BIO 118 Exam I Name___________________________________

May 30, 2000

Please limit your answer to the space provided! Please do not use red pen!

1. The heat shock proteins (HSPs) were first identified as a group of proteins whose concentration in the
cell dramatically increases soon after 'heat shock', a sudden exposure to higher than normal temperatures
(e.g. from 37°C to 42°C). The proteins presumably help protect the cell from detrimental effects of heat
shock.

1a. (8 pts) Based on your knowledge of protein structure, suggest a reason why a small increase in
temperature might be detrimental to a cell. You need not identify specific proteins.

1b. (4 pts) Heat shock proteins protect cells by binding nonspecifically to a variety of other proteins. The
HSPs bind to the amino acids side chains that are only exposed after heat shock. List the names of two
amino acids that are likely to be bound by a HSP. What characteristic of these amino acids makes them
candidates for being bound by an HSP?

1.

2.

characteristic:

1c. (4 pts) Histones, on the other hand, bind nonspecifically to DNA. List the names of two amino acids
that are likely to stabilize the binding of histones to DNA. What characteristic of these amino acids makes
them candidates for binding to DNA?

1.

2.

characteristic:
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1. (cont’d) The increase in heat shock proteins (HSPs) after heat shock is the consequence of increased
expression of the HSP genes. This is the result of activation of a ' heat shock transcription factor' (HSTF).
The HSTF is normally bound to a heat shock protein called HSP90. Under conditions of heat shock,
HSP90 will also bind a variety of other proteins, effectively reducing the amount of HSP90 available to
bind HSTF. It is believed that this interaction between HSTF and HSP90 regulates the activity of HSTF.
To test this model, you use the normal human HSTF protein (#1) and a series of truncated
proteins in which you’ve deleted portions from the C-terminus to produce 3 shortened proteins with the
same N-terminus (#2-4). You test each protein , in the presence and absence of HSP90, for binding
activity to a DNA fragment containing the HSTF binding site (Y = binds to DNA; N = no binding to DNA):

HSTF: wild type (#1) & truncated mutants (#2 - 4) DNA Binding Activity
1 50 100 150 200 250 300 350 400 + HSP90 – HSP90
#1 N--------------------------------------------------------------------------------------C N Y
#2 N----------------------------------------------------------------C Y Y
#3 N----------------------------------------C Y Y
#4 N-------------------C N N

1d. (10 pts) Based on the results above, draw a clearly labeled diagram of wild type HSTF (#1) which can
explain how it’s activity is regulated by HSP90. Your diagram should clearly indicate: (a) any domains
and binding sites on HSTF; (b) the locations of the N- & C-termini; and (d) any necessary conformational
changes. You should also diagram the truncated forms (#2-4) to explain the experimental results.

1e. (3 pts) HSP90 is called that because it has a molecular weight of about 90,000 daltons, equivalent to
approximately 850 amino acids. How does the size of this protein compare to a typical protein in E. coli?
(Answer in a few words, please!)
page 3 Name___________________________________

1. (cont’d) Looking more closely at the results in the experiment above, you find that truncation #3 (1-200)
binds DNA with significantly lower affinity than truncation #2 (1-300) or wild type. In subsequent
experiments you find that the critical sequence for this change is from amino acid 232-251:

--Leu Ser Asp His Leu Asp Ala Met Asp Ser Asn Leu Asp Asn Leu Gln Thr Met Leu--

1f. (6 pts) Predict the likely secondary structure of this sequence. What is the basis of your prediction?
(show your conclusions in a diagram)

1g. (8 pts) You suspect that the decrease in DNA binding affinity with the deletion of amino acids 232-251
in truncation #3 (see q#1f) may be related to a change in the 4° structure (= subunit interactions) of the
protein. Additional evidence comes from the sequence of the HSTF binding site on the bound DNA:

5’----GAAACTTC----3’
||||||||
3’----CTTTGAAG----5’

Based on these two pieces of evidence, predict the 4° structure (monomer?, homodimer?, heterodimer?,
tetramer?, etc.) of HSTF. Explain how the protein & DNA sequences above support your conclusion.
page 4 Name___________________________________

1h. (6 pts) Each HSTF polypeptide binds to DNA using the surface of a single alpha helix. What kind of
molecular interactions account for the specificity of binding to the specific sequences of bases above? Be as
specific as possible!

1i. (4 pts) Estimate the number of binding sites in the human genome for each HSTF polypeptide. Assume
that the genome is random and that each polypeptide binds individually. Show your work – round off as appropriate.

1j. (8 pts) Transcription factors, like HSTF, activate the expression of genetic information stored in DNA.
List two properties of DNA , as it is found in the cell, that distinguish it from RNA. Briefly explain how
those properties are advantageous to organisms with respect to the storage of genetic information.

1.

2.
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1k. (8 pts) The binding of HSTF in the absence of HSP0 to fragments of DNA in the test tube is found to
be ~10X faster than the binding to DNA in a human cell, even under activating conditions of heat shock.
You find that the slower binding in the cell can be mimicked by the addition of histones to the test tube.
Propose a model for how the addition of histones would inhibit HSTF binding in the test tube. Which
histones would you need to add? (Be as specific as possible – name any relevant structures. Diagram, please!)

1l. (6 pts) You decide to test the model of how histones might inhibit HSTF binding (see q #1j) by adding
varying amounts of histones. You then digest the DNA with a micrococcal nuclease, an enzyme that cuts
both strands simultaneously. You digest extensively (for a long time) and then extract the DNA and
analyze it by gel electrophoresis. Draw a clearly labeled diagram of the results you expect, based on the
model you proposed above (q#1k).

DNA alone DNA + Histones DNA + Histones


(low concentration) (high concentration)

I have better things to do than cheat – so I didn’t. Signed ________________________________________

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