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Homework 10
Biology 350
due April 7 at 1:00 p.m. in 1005 Haworth
1. Define the following and explain how they work and affect the cell or
organism? Give examples.
RNAi: RNA interference is a post-transcriptional method of regulating gene
expression. A short (several hundred bps) dsRNA gets into the cell, is cleaved
by a DICER enzyme into 21-22 bp fragments. The cut-up RNAs bind to RISC
complex as ssRNAs and initiate destruction of all cellular RNAs that share
homology to the dsRNA. RNAi has been incredibly useful to researchers
because it can be used to reduce the expression of genes that are tough to
mutate.
TFIID is a complex of proteins within the basal/general transcriptional
machinery that turns on genes. It includes the TATA-binding factor, which
binds to the TATA box, located at -35 of many eukaryotic promoters.
Riboswitches are small molecules which stabilize one of two alternate stemloop conformations found in the leaders (5’UTRs) of some eukaryotic
transcripts. In one conformation, the gene will form a terminator structure and
RNA polymerase will fall off the template; in the alternate conformation, the
gene will not form a terminator structure and the entire gene will be
transcribed.
Alternative splicing: This is a post-transcriptional method of regulating which
gene product is made from a primary transcript. It produces different mRNAs
from the same primary transcript. E.g., perhaps exon 2 needs to be included in
the final protein in some cells, but not in others. Alternative splicing allows
the exon to be included in some mature mRNAs but excluded from others.
Thus different versions of the protein are made in different cells.
2. What is the difference between an enhancer and a promoter? How does each
contribute to the regulation of gene expression?
An enhancer binds transcriptional activators and brings them to the DNA.
The activation domain of the transcriptional activator will attract the basal
transcription machinery—including RNA Polymerase—and help them find
the promoter. They increase the amount of transcription from a promoter.
Promoters bind general transcription factors (aka basal transcription
machinery)—including TFIID (which includes TATA-binding factor). A
promoter without an enhancer will have very low levels of transcription
because its chance of attracting RNA polymerase is low.
Homework 10
Biology 350
due April 7 at 1:00 p.m. in 1005 Haworth
Enhancers can work from upstream or downstream of the promoter, from far
or near, in either orientation. Promoters (or PolII transcribed genes) are not so
flexible. They set the start site and direction of transcription. If you flip a
promoter in the wrong orientation it would not transcribe the proper
sequences.
3. What is meant by the term epigenetic regulation?
Epigenetic regulation is a way of modifying the DNA without changing its
sequence in a way that is inheritable from one generation to the next. It is
often accomplished by methylation of DNA, which affects chromatin structure
and imprinting.
4. A girl expresses a mutant phenotype because she has received a mutation in a
paternally imprinted gene. From which parent did the girl inherit the mutant
allele? Explain your answer
Imprinting is a way of silencing a gene in a gender-specific way. A paternallyimprinted gene is one that the child inherited from the father but that—
because of methylation or chromatin structure—is not expressed. (similarly a
maternally-imprinted gene would be one that is inherited from the mom, but
that is silent). The maternal allele will be the only allele expressed in the child
(whether male or female).
The allele the girl inherited from her father is silent. Therefore, the mutant
phenotype must come from the allele that she inherited from her mother.
5. In which yeast genes should you be able to isolate galactose-constitutive
mutations? What characteristics of the normal protein would the mutation
disrupt?
The GAL4 protein is the transcriptional activator that regulates GAL gene
expression (i.e., transcription from GAL1, 7, and 10). GAL4 activity is
repressed by GAL80, which binds to GAL4’s activation domain and obscures
it, such that it cannot attract RNA Polymerase.
Mutations in GAL4 that prevent GAL80 from binding to it could lead to
constitutive expression. Similarly mutations that delete GAL80 or prevent it
from binding to GAL4 would also lead to constitutive expression of the GAL
genes.
Homework 10
Biology 350
due April 7 at 1:00 p.m. in 1005 Haworth
6. Does each of the following types of gene regulation occur in eukaryotes only,
prokaryotes only, or in both?
Differential splicing—only in eukaryotes
Positive regulation—in both
Chromatin remodeling—in eukaryotes only.
Attenuation—attenuation requires coupled transcription/translation, which is
only found in prokaryotes. However, riboswitches can cause transcription to
terminate in eukaryotes if the RNA is not needed.
Negative regulation—genes can be turned off by repressors in both eukaryotes
and prokaryotes.
7. You have isolated a gene expressed in differentiated neurons in mice. You
then fused the upstream DNA and beginning of the gene to lacZ (which will act
as a reporter for gene activity; the arrow indicate the cut sites for the restriction
enzymes that you used). Different fragments (shown as dark lines in the
following figure) were cloned next to a lacZ gene that lacked its own promoter.
The clones were introduced into neurons to monitor expression. From the
results that follow, which region contains the promoter and which contains an
enhancer? Explain your answer.
lacZ
Fragments fused to lacZ
LacZ expression
0
0
0
5
5
80
5
80
Homework 10
Biology 350
due April 7 at 1:00 p.m. in 1005 Haworth
The fusion genes that show no expression do not have the promoter. You cannot
have any transcription without a promoter. You can have low levels of
transcription if you have a promoter without an enhancer. The ones that are
only expressing “5” amount of lacZ have only the promoter. To get high levels
of transcription, there needs to be both an enhancer and a promoter. You see
that in the two fusion genes making 80 amount of lacZ. The enhancer probably
lies within the common region of the two possible enhancer fragments (in circle)