Chapter 14: RNA Molecules and RNA Processing

Problem Solutions:

COMPREHENSION QUESTIONS

1. What is the concept of colinearity? In what way is this concept fulfilled in bacterial and eukaryotic cells?

Solution:

Colinearity is the concept that the sequence of codons in the DNA of a gene has a direct correspondence with
the sequence of amino acids in the protein. If we examine the location of three codons for three amino acids in a
protein, the order of the codons in the gene is always the same as the order of the amino acids in the protein.
The presence of large regions of noncoding DNA in introns means that DNA sequences that code for adjacent
amino acids may be separated by many base pairs of DNA, but introns to not alter the order of codons in the
gene.

2. What are some characteristics of introns?

Solution:

Introns are intervening sequences that typically do not encode proteins. Eukaryotic genes commonly contain
introns. However, introns are rare in bacterial genes. The number of introns found in an organism’s genome is
typically related to complexity – more complex organisms possess more introns.

3. What are the three principal elements in mRNA sequences in bacterial cells?

Solution:

(1) The 5′ untranslated region, which contains the Shine–Dalgarno sequence

(2) The protein-encoding region, which begins with the start codon and ends with the stop codon
(3) The 3’ untranslated region

4. In bacteria, what is the function of the Shine–Dalgarno consensus sequence?

Solution:

The Shine–Dalgarno consensus sequence functions as the ribosome-binding site on the mRNA molecule.

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5. Summarize the different types of processing that can take place in pre-mRNA.

Solution:

Several modifications to pre-mRNA take place to produce mature mRNA in eukaryotes.

(1) Addition of the 5′ 7-methylguanosine cap to the 5′ end of the pre-mRNA

(2) Cleavage at a site downstream of the AAUAAA consensus sequence at the 3′ end of the pre-mRNA
(3) Addition of the poly(A) tail to the 3′ end of the mRNA immediately following cleavage
(4) Removal of the introns (splicing)

6. a. In eukaryotes, what is the 5′ cap?

Solution:

The 5′ end of eukaryotic mRNA is modified by the addition of the 7-methyl guanine 5′ cap. The cap consists of
an extra guanine nucleotide linked 5′ to 5′ to the mRNA molecule. This nucleotide is methylated at position 7 of
the base. The ribose sugars of adjacent bases may be methylated at the 2′ –OH.

b. What is the function of the 5′ cap?

Solution:

CAP binding proteins recognize the 5′ cap and stimulate binding of the ribosome to the 5′ cap and to the mRNA
molecule. The 5′ cap may also increase mRNA stability in the cytoplasm. Finally, the 5′ cap is needed for
efficient splicing of the intron that is nearest the 5′ end of the pre-mRNA molecule.

7. How is the poly(A) tail added to pre-mRNA? What is the purpose of the poly(A) tail?

Solution:

Cleavage of the mRNA occurs 11-30 nucleotides downstream of the polyadenylation consensus sequence
(AAUAAA). 50-250 adenines are then added to the 3’ end of the RNA molecule.

The presence of the poly(A) tail increases the stability of the mRNA molecule through the interaction of
proteins at the poly(A) tail. The poly(A) tail also assists with the binding of the ribosome to the mRNA.

8. What is a spliceosome and what does it do?

Solution:

The spliceosome consists of small RNA molecules and a large number of proteins. Splicing of pre-mRNA
nuclear introns occurs through the action of the spliceosome which recognizes consensus sequences at the 5’
splice site, the 3’ splice site and the branch point.

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9. Describe two types of alternative processing pathways. How do they lead to the production of multiple
proteins from a single gene?

Solution:

Alternative processing of pre-mRNA can take the form of either alternative splicing of pre-mRNA introns or the
alternative cleavage of 3′ cleavage sites in a pre-mRNA molecule containing two or more cleavage sites for
polyadenylation. Alternative splicing results in different exons of the pre-mRNA being ligated to form mature
mRNA. Each mRNA formed by an alternative splicing process will yield a different protein. In pre-mRNA
molecules with multiple 3′ cleavage sites, cleavage at the different sites will generate mRNA molecules that
differ in size. Each alternatively cleaved RNA potentially could code for a different protein depending on the
location of the alternative 3′ cleavage site or produce mRNAs with different 3’UTRs. Each 3’UTR could confer
different stability on the mRNA thus controlling how much protein is produced. A single pre-mRNA transcript
can undergo both alternative processing steps, thus potentially producing multiple proteins.

10. What is RNA editing? Explain the role of guide RNAs in RNA editing.

Solution:

RNA editing alters the sequence of a RNA molecule after transcription either by the insertion, deletion, or
modification of nucleotides within the transcript. The guide RNAs (gRNAs) provide templates for the alteration
of nucleotides in RNA molecules undergoing editing and are complementary to regions within the pre-edited
RNA molecule. At these complementary regions, the gRNAs base pair to the pre-edited RNA molecule. This
binding determines the location of the alteration of nucleotides.

11. Describe the basic structure of ribosomes in bacterial and eukaryotic cells.

Solution:

Ribosomes in both eukaryotes and bacteria consist of a complex of protein and RNA molecules. A functional
ribosome is composed of a large and a small subunit. The bacterial 70S ribosome consists of a 30S small
subunit and a 50S large subunit. Within the small subunit are a single 16S RNA molecule and proteins. The 23S
and the 5S RNA molecules, along with proteins, are found in the large bacterial subunit. The eukaryotic 80S
ribosome is comprised of a 60S large subunit and a 40S small subunit. Three RNA molecules, the 28S RNA, the
5.8S RNA, and the 5S RNA, are located in the large subunit as well as proteins. The eukaryotic small subunit
contains only a single 18S RNA molecule and proteins.

12. What role do CRISPR-Cas systems naturally play in bacteria?

Solution:

The CRISPR-Cas system essentially serves as an adaptive RNA defense system or immune system for bacterial
cells and protects cells against foreign DNA genomes from plasmids or infecting bacteriophages. The spacers
between the palindromic repeat sequences consist of short nucleotide sequences captured from previous
invading DNA molecules and can help to provide adaptive protection by targeting homologous invading foreign
DNA molecules for cleavage by the Cas proteins.

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13. Outline the three stages of CRISPR-Cas action.

Solution:

The action of the CRISPR-Cas system occurs through the three distinct stages of acquisition, expression, and
interference. In the acquisition stage, short DNA sequences from the destroyed foreign DNA molecules are
inserted into a spacer region that is flanked by the palindromic repeat sequences within the CRISPR array.
During the expression stage, a long precursor RNA is transcribed from the CRISPR array. The Cas proteins
cleave and process the precursor RNA into shorter RNA fragments called crRNAs. Each of these crRNAs
contains one of the spacer regions. These crRNAs then combine with the Cas proteins to form the effector
complexes. In the interference stage, the effector complexes bind to the homologous invading foreign DNA
molecules allowing for their cleavage by the Cas proteins.

14. 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.

15. 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.

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16. Explain how each of the following processes complicates the concept of colinearity.

a. 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.

b. 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.

APPLICATION QUESTIONS

17. 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 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.

18. 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 eukaryotic ribosome binds at the 5′ cap
of the mRNA molecule and scans to the first methionine codon (AUG) in a Kozak consensus. The region 5′ of
this start codon is the 5′ UTR.

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b. Promoter

Solution:

The promoter is the DNA sequence that the transcription apparatus recognizes and binds to initiate
transcription. It is 5’ or upstream to the start site for transcription (+1).

c. Polyadenylation consensus sequence

Solution:

The polyadenylation 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 a
protein. 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 and translation.

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i. 5′ cap

Solution:

The 5′ cap functions in the initiation of translation and mRNA stability.

19. How would the deletion of the Shine–Dalgarno sequence affect a bacterial mRNA?

Solution:

In bacteria, the ribosome 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.

20. What would be the most likely effect of moving the polyadenylation signal (AAUAAA) consensus sequence
shown in 10 nucleotides upstream?

Solution:

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

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

a. polyadenylation signal (AAUAAA) consensus sequence

Solution:

The deletion of the polyadenylation signal consensus sequence would result in no cleavage or polyadenylation
of the pre-mRNA. This would affect the stability and translation of the mRNA.

b. 5′ cap

Solution:

The loss of the 5′ cap would most likely prevent splicing of the intron that is nearest to the 5′ cap. In addition,
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 and is required for translation. If eliminated from the pre-
mRNA, then the mRNA would be degraded quickly by exonucleases in the cytoplasm.

22. 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
and prevented removal of the intron from the pre-mRNA.

23. 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 not be translated and
would be degraded more quickly in the cytoplasm by nucleases.

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24. 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 and translation 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 and not translated.

25. 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 could 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.