APPLICATION QUESTIONS AND PROBLEMS

Section 14.1
*19. Duchenne muscular dystrophy is caused by a mutation in a gene that
comprises 2.5 million nucleotides and specifies a protein called dystrophin.
However, less than 1% of the gene actually encodes the amino acids in the
dystrophin protein. On the basis of what you now know about gene
structure and RNA processing in eukaryotic cells, provide a possible
explanation for the large size of the dystrophin gene.
The large size of the dystrophin gene is likely due to the presence of many
intervening sequences or introns within the coding region of the gene. Excision
of the introns through RNA splicing yields the mature mRNA that encodes the
dystrophin protein.
Section 14.2
20. How do the mRNAs of bacterial cells and the pre-mRNAs of
eukaryotic cells differ? How do the mature mRNAs of bacterial and
eukaryotic cells differ?
Bacterial mRNA is translated immediately upon being transcribed. Eukaryotic
pre-mRNA must be processed. Bacterial mRNA and eukaryotic pre-mRNA have
similarities in structure. Each has a 5′ untranslated region as well as a 3′
untranslated region. Both also have protein-coding regions. However, the
protein-coding region of the pre-mRNA is disrupted by introns. The eukaryotic
pre-mRNA must be processed to produce the mature mRNA. Eukaryotic mRNA
has a 5′ cap and a poly(A) tail, unlike bacterial mRNAs. Bacterial mRNA also
contains the Shine-Dalgarno consensus sequence. Eukaryotic mRNA does not
have the equivalent.
*21. Draw a typical eukaryotic gene and the pre-mRNA and mRNA
derived from it. Assume that the gene contains three exons. Identify the
following items and, for each item, give a brief description of its function:
a. 5′ untranslated region
b. Promoter
c. AAUAAA consensus sequence
d. Transcription start site
e. 3′ untranslated region
f. Introns
g. Exons
h. Poly(A) tail
i. 5′ cap
22. How would the deletion of the Shine–Dalgarno sequence affect a
bacterial mRNA?
In bacteria, the small ribosomal subunit binds to the Shine-Dalgarno sequence
to initiate translation. If the Shine-Dalgarno sequence is deleted, then
translation initiation cannot take place, preventing protein synthesis.
*23. How would the deletion of the following sequences or features most
likely affect a eukaryotic pre-mRNA?
a. AAUAAA consensus sequence
The deletion of the AAUAA consensus sequence would prevent binding of the
cleavage and polydenylation factor (CPSF), thus resulting in no cleavage or
polyadenylation of the pre-mRNA. This would affect the stability and
translation of the mRNA.
b. 5′ cap
The deletion of the 5′ cap would most likely prevent splicing of the intron that
is nearest to the 5′ cap. Ultimately, elimination of the cap will affect the
stability of the pre-mRNA as well as its ability to be translated.
c. Poly(A) tail
Polyadenylation increases the stability of the mRNA. If eliminated from the
pre-mRNA, then the mRNA would be degraded quickly by nucleases in the
cytoplasm
24. Suppose that a mutation occurs in an intron of a gene encoding a
protein. What will the most likely effect of the mutation be on the amino
acid sequence of that protein? Explain your answer.
Because introns are removed prior to translation, an intron mutation would
have little effect on a protein's amino acid sequence unless the mutation
occurred within the 5′ splice site, the 3′ splice site, or the branch point. If
mutations within these sequences altered splicing, then the mature mRNA
would be altered, thus altering the amino acid sequence of the protein. The
result could be a protein with additional amino acid sequence. Or, possibly,
the altered splicing could introduce a stop codon that stops translation
prematurely. If a mutation in the intron induced a frameshift, the reading
frame and the amino acid sequence would be altered.
25. A geneticist induces a mutation in a line of cells growing in the
laboratory. The mutation occurs in one of the genes that encodes proteins
that participate in the cleavage and polyadenylation of eukaryotic mRNA.
What will the immediate effect of this mutation be on RNA molecules in
the cultured cells?
The protein is needed as part of the process for cleavage of the 3′ UTR and for
polyadenylation. A nonfunctional protein would result in mRNA lacking a
poly(A) tail, and the mRNA would be degraded more quickly in the cytoplasm
by nucleases.
*26. A geneticist mutates the gene for proteins that bind to the poly(A) tail
in a line of cells growing in the laboratory. What will the immediate effect
of this mutation be in the cultured cells?
The stability of the mRNA is dependent on the proteins that bind to the poly(A)
tail. If the proteins are unable to bind to the tail, then the mRNA that contain
poly(A) tails will be degraded at a much more rapid rate within the cells.
27. A geneticist isolates a gene that contains five exons. He then isolates the
mature mRNA produced by this gene. After making the DNA single
stranded, he mixes the single-stranded DNA and RNA. Some of the
singlestranded DNA hybridizes (pairs) with the complementary mRNA.
Draw a picture of what the DNA–RNA hybrids will look like under the
electron microscope.
28. A geneticist discovers that two different proteins are encoded by the
same gene. One protein has 56 amino acids, and the other has 82 amino
acids. Provide a possible explanation for how the same gene can encode
both of these proteins.
a. The pre-mRNA molecules transcribed from the gene are likely processed by
alternative processing pathways. 2 possible mechanisms that could have
produced the two different proteins from the same pre-mRNA are alternative
splicing or multiple 3' cleavage sites in the pre-mRNA. The cleavage of the
pre-mRNA molecule at different 3' cleavage sites would produce alternatively
processed mRNA molecules that differ in size. Translation from each of the
alternative mRNAs would produce proteins containing different numbers of
AAs.
b. Alternative splicing of the pre-mRNA could produce different mature mRNAs
each containing a different number of exons. Again translation from each
alternatively spliced mRNA would generate proteins that differ in the number
of AAs contained.
29. Explain how each of the following processes complicates
the concept of colinearity.
a. Trans-splicing
In trans-splicing, exons from different mRNAs are spliced together during RNA
processing events. Essentially, the mature mRNA product is not produced by
DNA sequences that are contiguous or even necessarily on the same
chromosome. This results in an amino acid sequence of the translated protein
from trans-spliced mRNA being encoded by two or more different genes.
According to the principle of colinearity, we would have expected the DNA
sequence of a single gene to correspond to the amino acid sequence of the
protein.
b. Alternative splicing
Different mature mRNAs from a single gene can be produced by alternative
splicing. Different arrangements of the gene's exons can occur in the mature
mRNAs. Thus, different proteins can be encoded within the same gene as
opposed to one gene corresponding to one protein as is predicted by the
concept of colinearity.
c. RNA editing
In RNA editing, genetic information is added to the pre-mRNA after it is
transcribed. In other words, the mature mRNA will contain information that
was not part of the DNA from which it was transcribed. The result is that the
nucleotide sequence of the gene does not correspond to the amino acid
sequence of the protein—a clear violation of the concept of colinearity.
Section 14.5
30. In the early 1990s, Carolyn Napoli and her colleagues were working on
petunias, attempting to genetically engineer a variety with dark purple
petals by introducing numerous copies of a gene that codes for purple
petals (C. Napoli, C. Lemieux, and R. Jorgensen. 1990. Plant Cell 2:279–
289). Their thinking was that extra copies of the gene would cause more
purple pigment to be produced and would result in a petunia with an even
darker hue of purple. However, much to their surprise, many of the plants
carrying extra copies of the purple gene were completely white or had only
patches of color. Molecular analysis revealed that the level of the mRNA
produced by the purple gene was reduced 50-fold in the engineered plants
compared with levels of mRNA in wild-type plants. Somehow, the
introduction of extra copies of the purple gene silenced both the
introduced copies and the plant’s own purple genes. Provide a possible
explanation for how the introduction of numerous copies of the purple
gene silenced all copies of the purple gene.
Overexpression of the purple gene mRNA led to the formation of double-
stranded regions by these RNA molecules which stimulated RNA silencing
mechanisms or the RNA-Induced-Silencing Complex (RISC) leading to rapid
degradation of the mRNA molecules.