Download BME205-Tutorial 6 Solutions2015-06-15 15

Systems Biology BME 205 (Winter 2013)
Tutorial 6 Problem Set
J. Rocheleau, P. Gilbert
1. How does binding of the lac repressor to the lac operator block transcription initiation?
a. lac repressor binding blocks RNA polymerase from interacting with DNA at the start
site
b. lac repressor binding induces a DNase that cleaves the DNA at the transcription start
site
c. lac repressor binding causes a conformational change in RNA polymerase
d. lac repressor binding induces a protease that degrades the sigma subunit of RNA
polymerase
2. Most eukaryotic genes are controlled at the level of
a.
b.
c.
d.
transcription initiation.
transcription elongation.
transcription termination.
transcription initiation.
3. Which of the following statements regarding the E. coli two-component PhoR/PhoB
regulatory system is (are) true?
a.
b.
c.
d.
PhoB has kinase activity.
PhoB is a cytosolic protein.
Phosphorylated PhoB is an inactive transcriptional activator.
None of the above.
4. An enhancer
a.
b.
c.
d.
is a DNA element that stimulates transcription of eukaryotic promoters.
binds to RNA polymerase and stimulates transcription.
acts as a binding site for RNA polymerase.
interacts with repressor proteins to enhance transcriptional repression.
5. The TATA box
a.
b.
c.
d.
serves as a promoter sequence for gene transcribed by RNA polymerase III.
is located approximately 100 base pairs upstream of the start site for mRNAs.
is present in all eukaryotic genes.
acts to position RNA polymerase II for transcription initiation.
6. All the following elements can function as eukaryotic promoters except
a. a TATA box.
b. an initiator element.
c. CpG islands.
d. an enhancer.
7. Refer to figure below, can lactose be metabolized under the conditions shown? Explain.
P – Lac promoter
O – Lac operon
I – inhibitor
No, because the inhibitor is bound to the lac operator and the RNA polymerase cannot
move past is to transcribe the mRNA.
8. Describe two levels of regulation of cell activity.
1. At the gene level where proteins (which control cell activity) are synthesized (usually at a
much slower rate).
2. At the protein level where enzymatic activity and cell surface receptors enact control over
cell activity (usually at a much faster rate).
9. RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by
short double-stranded RNA molecules called small interfereing RNAs (siRNA). In
mammalian cells, transfection of 21-22 nucleotide siRNAs leads to degradation of
mRNA molecules that contain the same sequence as the siRNA. In the following
experiment, siRNA and knockout mice are used to investigate two related cell surface
proteins designated p24 and p25 that are suspected to be cellular receptors for the uptake
of a newly isolated virus.
a. To test the efficacy of RNAi in cells, siRNAs specific to cell surface proteins p24
(siRNA-p24) and p25 (siRNA-p25) are transfected individually into cultured
mouse cells. RNA is extracted from these transfected cells and the mRNA for
proteins p24 and p25 are detected on Northern blots using labeled p24 cDNA or
p25 cDNA as probes. The control for this experiment is a mock transfection with
no siRNA. What do you conclude from this Northern blot about the specificity of
the siRNAs for their target mRNAs?
The Northern blot reveals the steady state mRNA levels for p24 (in the center)
and p25 (on the right). For p24, only the transfection of siRNA-p24 reduces the
amount of p24 mRNA whereas the control transfection without siRNA and
transfection with siRNA-p25 did not affect p24 mRNA levels. Similarly, for p25,
only siRNA-p25 reduced p25 RNA levels. These results demonstrate that the
siRNAs can cause specific degradation of their target RNAs.
b. Next, the ability of siRNAs to inhibit viral replication is investigated. Cells are
transfected with siRNA-p24 or siRNA-p25 or with siRNA to an essential viral
protein. A day later, transfected cells are infected with the virus. After a further
incubation period, the cells are collected and lysed. The number of viruses
produced by each culture is shown below. The control is a mock transfection with
no siRNA. What do you conclude about the role of p24 and p25 in the uptake of
the virus? Why might the siRNA to the viral protein be more effective than
siRNA to the receptors in reducing the number of viruses?
Cell Treatment
Control
siRNA-p24
siRNA-p25
siRNA-p24 and siRNA-p25
siRNA to viral protein
Number of Viruses/ml
1 x 107
3 x 106
2 x 106
1 x 104
1 x 102
In the cultured cells, transfection of either siRNA-p24 or siRNA-p25 yielded a
viral titer that was slightly slower than the control transfection. This result
indicates that reduction of either p24 mRNA or p25 mRNA (and presumably the
proteins encoded by them) only minimally affects the ability of the virus to infect
the cells. However, transfection of both siRNA-p24 and siRNA-p25 did result in a
significant reduction in viral tier. In combination with the previous results, these
results indicate that either p24 or p25 mRNA would lead to a decrease in p24 and
p25 protein, resulting in inhibition of viral infection and replication. Transfection
of siRNA to a viral protein resulted in an even greater reduction of virus titer
compared to transfection with siRNA to both p24 and p25. One possible
explanation is that transfection with either siRNA-p24 or siRNA-p25 leads to a
reduction but not a complete elimination of cell surface receptors for the virus,
whereas siRNA to a viral protein can inhibit viral replication/assembly directly. A
second possibility has to do with the half-life of p24 and p25 proteins. A
reduction of p24 and p25 mRNA may not lead to an immediate reduction in
p24/p25 protein due to the stability of the protein. The protein level is determined
by the half-life of the protein and the length of time after transfection of siRNA.
For example, if the half-lives of p24 and p25 proteins are greater than a day, then
during the one day incubation after transfection with siRNA, there should still be
more than half of the protein still present.