Chapter 8: From Gene to Protein- RNA processing (Book: Chapter 14)

16. At the beginning of the chapter, we considered Duchenne muscular dystrophy

and the dystrophin gene. We learned that the gene causing Duchenne muscular
dystrophy encompasses more than 2 million nucleotides, but less than 1% of the
gene encodes the protein dystrophin. 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.

Actually, the size of gene do not define the function of a gene. There are multiple nucleotides in
a gene but only some specific nucleotide sequences that code for the a particular type of protein
i.e. is here dystrophin. Not all the gene encode for the protein hence the size of gene doesn't
define its function and if mutation occurs at that particular sequence from where gene was
coding dystrophin it will lead to disease. The processing of gene takes place mainly at 2 steps
1. Transcription (DNA to RNA)
2. Translation (RNA to Proteins)

17. How do the mRNA of bacterial cells and the pre-mRNA of eukaryotic cells
differ? How do the mature mRNAs of bacterial and eukaryotic cells differ?

Messenger RNA (mRNA) is a single stranded RNA involved in the synthesis of protein. The
difference between mRNA in bacteria and pre-mRNA in eukaryotic cells are:

 In bacteria mRNA is immediately transcribed after being transcribed whereas, in

eukaryotic cell, pre-mRNA is processed as well as exported from the nucleus.
 mRNAs of bacterial cells have presence of exons whereas pre-mRNA of eukaryotic
cells have both exons and introns that get spliced during the process of splicing to
yield mature mRNAs.
 The functional mRNA in prokaryotes have no post transcriptional processing whereas
eukaryotic pre-mRNA do not have such modifications (happens post splicing forming
a mature mRNA).
 The mature mRNA in prokaryotes that get translated into functional proteins whereas
pre-mRNA needs an additional step of splicing processing and modification
(polyadenylation and capping) to be further translated into a functional protein.

Messenger RNA (mRNA) is a matured RNA which contains coding sequence of a gene but pre-
mRNA is the primary transcript and contains both coding and non coding genes.
The differences in matured RNA in bacteria and eukaryotic cells are:

 . The mRNA of bacteria are polygenic in nature. The term polygenic means single
mRNA is transcribed by the several structural genes of an operon. They contain many
sites for initiation and termination codons. A single mRNA can code for several
 different protein molecules whereas in eukaryotic cells, mRNA have only one site for
initiation and termination of protein synthesis. Therefore, eukaryotic mRNA is
monocistronic in nature.
 Life span of the prokaryotic mRNA is very short. In E.coli, the average half life of
mRNA is about two minutes. Eukaryote mRNAs have much longer life span than
prokaryotic mRNAs as they are more stable.
 In most of the bacterial cells translation of the mRNA begins while the mRNA is still
being transcribed from the DNA molecule indicates a process of coupled transcription
and translation. In eukaryotes ,the mRNA produced from DNA template is first
transported into the cytoplasm where it forms ribosome complexes and then the
synthesis of proteins take place. Thus translation process begging only after
transcription of mRNA is completed.
 Prokaryotic mRNA undergoes very little post transcriptional changes an
modifications. Also, there is very short time interval between transcription and
translational process. In eukaryotes the transcribed mRNA undergoes major post
transcriptional modifications such as splicing.

 Messenger RNA (mRNA) is present in both bacteria and eukaryotic cells but they are
different in both species. In bacteria mRNA is present whereas in eukaryotic cells pre-
mRNA is present. Their functions are different in bacteria and eukaryotic species.

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 (e) 3’ untranslated region
(b) Promoter (f) Introns
(c) AAUAAA consensus sequence (g) Exons
(d) Transcription start site (h) Poly(A) tail
(i) 5’ cap
Promoter:- promoter is a region where RNA polymerase attached and recognise the start codon
where the transcription started.
Transcription start site:-- for this site transcription will be started. And help to produce pre
mRNA.

Intron:-- it is the functional part of the mRNA which encoded by some codon that produces
specific amino acids which requires for our body by translation.

Exon:-- it is the non functional part of the mRNA which is cut off when the mature mRNA form.

3' untranslated region:--the three prime untranslated region (3′-UTR) is the section of messenger
RNA (mRNA) that immediately follows the translation termination codon. The 3′-UTR often
contains regulatory regions that post-transcriptionally influence gene expression. It helps in
mRNA stability and translation efficiency.

5' untranslated region:-- The 5′ untranslated region (5′ UTR) (also known as a leader sequence,
transcript leader, or leader RNA) is the region of an mRNA that is directly upstream from the
initiation codon. It helps in the regulation of translation.

5' cap:-- it is a the first nucleotide in the transcription contain a modified guanine nucleotide. It
helps to protect the mature mRNA from any injuries.it also helps to attached the ribosome with
the mRNA and start the translation process.

Poly A tail:-- at the 3' end region of the mRNA that contain a poly A tail which composed of
many adenosine nucleotide. Poly A tail helps to stabilize the mRNA during the translation
process and also helps to protect the mRNA from the degradation.

19. 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.

20. How would the deletion of the following sequences or features most likely affect
a eukaryotic pre-mRNA?
(a) AAUAAA consensus sequence
Translation is a process where mRNA is translated into the protein. In eukaryotes mRNA
translation occur in the cytoplasm.
AAUAAA deletion:- AAUAA consesus sequence deletion stop the binding of the cleavage and
the polydenylation so, no clevage or polydenylation of the pre mRNA. It would be only affect
the translation of mRNA and stability.
when we deleted the AAUAAA consensus sequence then we can see there is change occur in the
sequence of pre mRNA. So, the pre mRNA would bot cleaved at the cleavage site.

AAUAAA consensus sequence:- The pre mRNA is not cleaved at the cleavages site due to
AAUAAA deltion.

(b) 5’ cap
5'cap deletion:- 5' cap deletion mostly stop the splicing of the introns which is nearest to the 5'
cap. Eventually cap elimination will affect the pre mRNA stability and also effect the ability to
be translated.
5' cap:- introns are not removed from the pre mRNA due to 5' deletion.

(c) Poly(A) tail

Poly(A) tail deletion:- if poly(A) tail is deleted from the pre mRNA the mRNA degraded fastly
through the nucleases in the cytoplasm because polydenylation increased the mRNA stability and
if tail is deleted mRNA doesn't transported to the cytoplasm.
Poly(A) tail:- the mRNA is not transported to the cytoplasm due to poly (A) tail deletion.

21. What would be the most likely effect on the amino acid sequence of a protein of a
mutation that occurred in an intron of the gene encoding the protein? Explain your
answer.

DNA molecule acts as genetic material and stores genetic information that is used to code for
amino acid sequence in proteins. One segment of DNA that codes for one protein is known as a
gene. It is to be noted that eukaryotic genes consists of two types of regions; the exons that
contain coding regions and entrance that have non-coding regions.

When the gene is transcribed, messenger RNA molecule is made and is processed before its
translation to protein. In the processing of messenger RNA, the introns are removed and exons
are joined together to give rise to a mature messenger RNA. Therefore the introns are non coding
regions and are removed from RNA molecules and hence have no contribution in amino acid
sequence of final protein. Therefore if a mutation occurs in a large intron, most probably the
intern will be removed and there will be no effect on the amino acid sequence of final
protein.
It is to be noted that the removal of introns also require special sequences present at the ends of
introns. Their mutation can lead to alternative splicing and can give rise to a different protein but
I am not considering this case because the question says the mutation is in the middle of intron.
Also, considering the overall size of intron and the sequence of removal, most probably a
random mutation would not affect the sequence and introj will be removed anyway having no
effect on protein.

22. A geneticist induces a mutation in the gene that codes for cleavage and
polyadenylation specificity factor (CPSF) in a line of cells growing in the laboratory.
What would be the immediate effect of this mutation on RNA molecules in the
cultured cells?

Since the gene encodes a protein that participates in the Polyadenylation process becomes
mutated, Polyadenylation on those cultured cells will not be successful, resulting in the
immediate effect mentioned above. Polyadenylation is an important step in the process of post-
transcriptional modification of nascent mRNA molecules. Cleavage at polyadenylation site
followed by polyadenylation at 3'end is required to form poly(A).

 Polyadenylation on those cultured cells will not be successful, resulting in the immediate
effect mentioned above.

The exportation of mRNA is very important for mRNA stability, nucleocytoplasmic export, and
translation. If the exportation does not succeed, they become targets for degradation by
exosomes. Due to the mutation, fully mature mRNA molecules will not be processed in the
cultured cells. As a result, mRNA becomes unstable and can be degraded by enzymes. Its
transport efficiency from the nucleus to the cytoplasm and translation efficiency will also be
reduced.
 Its transport efficiency from the nucleus to the cytoplasm and translation efficiency will
also be reduced.

Polyadenylation will not take place in mRNA molecules in those cultured cells as the gene
encodes a protein that participates in that process, resulting in the above mentioned immediate
effect.

23. A geneticist mutates the gene for proteins that bind to the poly(A) tail in a line of
cells growing in the laboratory. What would be the immediate effect of this mutation
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.

24. An in vitro (within a test tube) splicing system has been developed that contains
all the components (snRNAs, proteins, splicing factors) necessary for the splicing of
nuclear pre-mRNA genes. When a piece of RNA containing an intron and two exons
is added to the system, the intron is removed as a lariat and the exons are spliced
together. What intermediate products of the splicing reaction would accumulate if
the following components were omitted from the splicing system? Explain your
reasoning.
(a) U1 (c) U6 (e) U4
(b) U2 (d) U5

If the U1 component is omitted from the splicing system, then the splicing reaction will not be able to
proceed as normal because U1 is required for the recognition of the 5' splice site. Without U1, the splicing
reaction will not be able to recognize where to start splicing the RNA, and the intron will not be removed.

If the U2 component is omitted from the splicing system, then the splicing reaction will not be able to
proceed as normal because U2 is required for the recognition of the branch point. Without U2, the
splicing reaction will not be able to recognize where to insert the lariat, and the intron will not be
removed.

If the U4 component is omitted from the splicing system, then the splicing reaction will not be able to
proceed as normal because U4 is required for the formation of the spliceosome, which is the complex of
proteins and snRNPs (small nuclear ribonucleoproteins) that carries out the splicing reaction. Without U4,
the spliceosome will not be able to form, and the intron will not be removed.

If the U5 component is omitted from the splicing system, then the splicing reaction will not be able to
proceed as normal because U5 is required for the release of the newly spliced RNA from the spliceosome.
Without U5, the spliced RNA will not be released from the spliceosome, and the intron will not be
removed.

If the U6 component is omitted from the splicing system, then the splicing reaction will not be able to
proceed as normal because U6 is required for the recognition of the 3' splice site. Without U6, the splicing
reaction will not be able to recognize where to end splicing the RNA, and the intron will not be removed.

In conclusion, the splicing reaction requires all of the components (U1, U2, U4, U5, and U6) in
order to proceed normally. If any of these components are omitted from the splicing system, the
splicing reaction will not be able to proceed as normal and the intron will not be removed. This is
because each of these components plays a specific role in the recognition of splice sites,
formation of the spliceosome, and release of the spliced RNA.
25. The splicing system introduced in Problem 24 is used to splice an RNA molecule
containing two exons and one intron. This time, however, the U2 snRNA used in the
splicing reaction contains several mutations in the sequence that pairs with the U6
snRNA. What would be the effect of these mutations on the splicing process?

The U2 snRNA plays a crucial role in the splicing process by forming a base-pairing interaction with the
U6 snRNA at the branch point of the intron. This base-pairing interaction helps to stabilize the
spliceosome and facilitate the splicing reaction. If the U2 snRNA contains several mutations in the
sequence that pairs with the U6 snRNA, it is likely that the base-pairing interaction will be disrupted.

As a result of these mutations, the splicing process may be impaired or inhibited. The spliceosome may
not form properly, or the splicing reaction may not proceed efficiently. It is also possible that the splicing
reaction may not occur at all, leading to the accumulation of unspliced RNA molecules.
In general, mutations in the sequence of the U2 snRNA that affect its ability to form a base-pairing
interaction with the U6 snRNA will have a negative impact on the splicing process. These mutations may
interfere with the proper formation and function of the spliceosome, leading to impaired or inhibited
splicing.

In conclusion, mutations in the sequence of the U2 snRNA that affect its ability to form a base-pairing
interaction with the U6 snRNA will have a negative impact on the splicing process. These mutations may
interfere with the proper formation and function of the spliceosome, leading to impaired or inhibited
splicing. As a result, the splicing process may be impaired or inhibited, and the accumulation of unspliced
RNA molecules may occur.

26. 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 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.
27. The chemical reagent psoralen can be used to elucidate nucleic acid structure.
This chemical attaches itself to nucleic acids and, on exposure to UV light, forms
covalent bonds between closely associated nucleotide sequences. Such cross-links
provide information about the proximity of RNA molecules to one another in
complex structures.
Psoralen cross-linking has been used to examine the structure of the spliceosome. In
one study, the following cross-linked structures were obtained during splicing. U1,
U2, U5, and U6 became cross-linked to pre-mRNA. U2 was cross-linked to U6 and
to pre-mRNA. The U1, U5, and U6 cross-links with pre-mRNA were mapped to
sequences near the 5- splice site, whereas the U2 snRNA cross-links with pre-mRNA
were mapped to the branch site. After splicing, U2, U5, and U6 were cross-linked to
the excised lariat.
Explain these results in regard to what is known about the structure of the
spliceosome and how it functions in RNA splicing. (Based on D. A. Wassarman, and
J. A. Steitz, 1992, Interactions of small nuclear RNAs with precursor messenger
RNA during in vitro splicing, Science 257:1918–1925.)

The snRNAs U1, U2, U5, and U6 play important roles in the splicing process. (explain)

The spliceosome is the complex ribonucleoprotein machine that catalyzes the removal of introns from
pre-mRNA during RNA splicing. The spliceosome is composed of small nuclear ribonucleoproteins or
snRNPs which are composed of small nuclear RNAs (snRNAs) and protein components.

After splicing, the excised lariat is formed by the joining of the 5'-splice site and the 3'-splice site. The
cross-links between U2, U5, and U6 and the excised lariat likely reflect the role of these snRNAs in
stabilizing the lariat structure. (explain)
The results obtained in the study using psoralen cross-linking suggest that the snRNAs U1, U2, U5, and
U6 are closely associated with the pre-mRNA during splicing. The cross-links between these snRNAs and
pre-mRNA likely reflect interactions between these molecules that are necessary for the splicing reaction
to occur. The cross-link between U2 and U6 and the cross-link between U2 and pre-mRNA likely reflect
the role of U2 in forming the spliceosome's active site, where the splicing reaction takes place. The cross-
links between U1, U5, and U6 and pre-mRNA that were mapped to sequences near the 5'-splice site may
reflect the role of these snRNAs in recognizing and interacting with the splice site in the pre-mRNA.

Overall, these results support the current understanding of the structure and function of the spliceosome in
RNA splicing and provide further insight into the specific interactions between snRNAs and pre-mRNA
that are necessary for the splicing reaction to occur.