APPLICATION QUESTIONS AND PROBLEMS

Section 14.1

*21. Duchenne muscular dystrophy is caused by a mutation in a gene that comprises 2.5
million base pairs 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.

Solution:
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.

22. What would happen in the experiment illustrated in Figure 14.2 if the DNA and RNA
that are mixed together came from very different organisms, for example a worm and a
pig?

Solution:
The RNA might not be complementary to the DNA in all regions of the genome due to
DNA sequence differences that exist between pig and worm. Thus, the DNA might not
anneal with RNA because it was not complementary, in which case the introns would
not be revealed.

23. For the ovalbumin gene shown in Figure 14.3, where would the 5′ untranslated region
and 3′ untranslated regions be located in the DNA and in the RNA?

Solution:
Assuming that alternative splicing is not occurring then the 5′ UTR would be located in
exon 1 and the 3′ UTR would be located in exon 8 for both the DNA and RNA
molecules.

Section 14.2

24. 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?

Solution:
Bacterial mRNA is translated immediately upon being transcribed. Eukaryotic pre-
mRNA must be processed and exported from the nucleus. 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.

25. Are the 5′ untranslated regions (5′ UTR) of eukaryotic mRNAs encoded by sequences in
the promoter, exon, or intron of the gene? Explain your answer.

Solution:
The 5′ UTR is located in the first exon of the gene. It is not part of the promoter because
promoters for RNA polymerase II (which transcribes pre-mRNA) are not normally
transcribed, and the 5′ UTR is in the mRNA. It is not located in an intron because
introns are removed during processing of pre-mRNA, and the 5′ UTR is part of the
mRNA.

*26. 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

Solution:
The 5′ untranslated region lies upstream of the translation start site. The reyuoktaic
ribosome binds at the 5′ cap of the mRNA molecule and scans to the first
methionine codon (AUG). The region 5′ of this start codon is the 5′ UTR.

b. Promoter

Solution:
The promoter is the DNA sequence that the transcription apparatus recognizes and
binds to initiate transcription.

c. AAUAAA consensus sequence

Solution:
The AAUAAA consensus sequence lies near the 3′ end of the pre-mRNA. It
determines the location of the 3′ cleavage and poly(A) tail addition to the pre-
mRNA molecule.

d. Transcription start site

Solution:
 The transcription start site is the location of the first transcribed nucleotide of the
mRNA and is located 25 to 30 nucleotides downstream of the TATA box.

e. 3′ untranslated region

Solution:
The 3′ untranslated region is a sequence of nucleotides at the 3′ end of the mRNA
that is not translated into proteins. However, it does affect the translation of the
mRNA molecule as well as the stability of the mRNA.

f. Introns

Solution:
Introns are noncoding sequences of DNA that intervene within coding regions of a
gene.

g. Exons

Solution:
Exons are transcribed regions that are not removed in intron processing. They
include the 5′ UTR, coding regions that are translated into amino acid sequences,
and the 3′ UTR.

h. Poly(A) tail

Solution:
A poly(A) tail is added to the 3' end of the pre-mRNA. It affects mRNA stability.

i. 5′ cap

Solution:
The 5′ cap functions in the initiation of translation and mRNA stability.
27. How would the deletion of the Shine–Dalgarno sequence affect a bacterial mRNA?

Solution:
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.

28. What would be the most likely effect of moving the AAUAAA consensus sequence
shown in Figure 14.7 10 nucleotides upstream?

Solution:
The cleavage site would also be moved 10 nucleotides upstream, likely resulting in a
shorter mRNA.

29. How would the deletion of the following sequences or features most likely affect a
eukaryotic pre-mRNA?

a. AAUAAA consensus sequence

Solution:
The deletion of the AAUAAA 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

Solution:
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

Solution:
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.

30. Suppose that a mutation occurs in the middle of a large 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.

Solution:
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 such that a new exon is recognized by the spliceosome, 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
inclusion of the new exon that was previously intron could introduce a stop codon that
stops translation prematurely. If a mutation in the new exon or inclusion of the new
exon induced a frameshift, the reading frame and the amino acid sequence would be
altered from that point onward in the protein.

*31. A geneticist induces a mutation in a cell line growing in the laboratory. The mutation
occurs in a gene that encodes a protein that participates in the cleavage and
polyadenylation of eukaryotic mRNA. What will be the immediate effect of this
mutation on RNA molecules in the cultured cells?

Solution:
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.

32. A geneticist induces a mutation in a cell line growing in the laboratory. The mutation
occurs in a gene that encodes a protein that binds to the poly(A) tail of eukaryotic
mRNA. What will be the immediate effect of this mutation in the cultured cells?

Solution:
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.

33. A geneticist isolates a gene that contains eight 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 single-stranded DNA hybridizes (pairs)
with the complementary mRNA. Draw a picture of what the DNA–RNA hybrids would
look like under the electron microscope.

Solution:

DNA

RNA
34. A geneticist discovers that two different proteins are encoded by the same gene. One
protein has 56 amino acids and the other 82 amino acids. Provide a possible
explanation for how the same gene could encode both of these proteins.

Solution:
The pre-mRNA molecules transcribed from the gene are likely processed by alternative
processing pathways. Two 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 amino acids.
Alternative splicing of the pre-mRNA could produce different mature mRNAs, each
containing a different number of exons and thus the mRNAs differ in size. Again,
translation from each alternatively spliced mRNA would generate proteins that differ in
the number of amino acids contained.

35. Suppose that a 20-bp deletion occurs in the middle of exon 2 of the gene depicted in
Figure 14.12a. What will be the likely effect of this deletion in the proteins produced by
alternative splicing?

Solution:
The proteins produced by the mRNA with exons 1, 2, and 3 will likely contain more
amino acids and be larger than the protein produced by the mRNA with exons 1 and 3.

*36. Explain how each of the following processes complicates the concept of colinearity.

a. Trans-splicing

Solution:
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

Solution:
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

Solution:
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

37. RNA interference may be triggered when inverted repeats are transcribed into an RNA
molecule that then folds to form double-stranded RNA. Write out a sequence of
inverted repeats within an RNA molecule. Using a diagram, show how the RNA with
the inverted repeats can fold to form double-stranded RNA.

Solution:
5′--------AGCUGGCCA -----UGGCCAGCU--------3′

5′ A U 3′
G C
C G
U A
G C
G C
C G
C G
A U

38. n Ithe 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. 1C9e9l0l. Plant 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.
Solution:
The overexpression of the purple gene mRNA led potentially to
the formation of double-stranded regions by these RNA
molecules because of areas of homology within the mRNAs
being produced. These double-stranded molecules stimulated
RNA silencing mechanisms or the RNA-induced silencing
complex (RISC), leading to rapid degradation of the mRNA
molecules. Trehseult would be a reduction in translation of
the protein needed for the production of purple petals and
the phenotypic loss of pigmentation.