SIR 2018

(Molecular Genetics)
Dr. Nur Ardiyana Rejab
ACADEMIC SESSION 1, 2023/2024
Tutorial 1, 2
NAME: MAI SUHIER M.Y.
MATRIC ID: S2124397

Tutorial 1

1. Explain why regulation of transcription frequently involves the promoter and protein
interactions with the promoter.

Transcription begins with RNA polymerase binding to the promoter, inducing structural
changes for initiation. The promoter serves as a regulatory nexus, interacting with proteins to
precisely control gene expression. This direct modulation of initiation, whether inhibiting or
enhancing at the promoter, allows cells to finely tune transcription. Responsive to
environmental signals, the promoter enables dynamic adjustments, ensuring a nuanced
orchestration of gene activity.

2. What steps in the eukaryotic transcription cycle are stimulated by phosphorylation of the
carboxyl terminal (CTD) of the large subunit of RNA polymerase II and beyond?

Phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a crucial role in
regulating transcription. Initially unphosphorylated, Pol II is recruited into the preinitiation
complex (PIC) and undergoes phosphorylation during the transition from initiation to
elongation. CTD phosphorylation has dual roles: inhibiting initiation before PIC assembly
and stimulating promoter escape and elongation post-assembly. Predominantly occurring on
serine 2 and serine 5, phosphorylation at these sites influences the distribution of Rpb1, with
serine 2-phosphorylated Rpb1 enriched distally from the promoter and serine 5-
phosphorylated Rpb1 enriched at promoter-proximal regions. Hyperphosphorylation of the
CTD is linked to essential mRNA synthesis events, including the recruitment of modification
enzymes and pre-mRNA splicing factors.

3. What purposes do capping and poly-A tail addition serve for eukaryotic mRNAs?

The capping and poly-A tail addition processes in eukaryotic mRNA serve to protect the
transcript and help it get exported from the nucleus and translated on the ribosomes (protein-
making "machines") found in the cytosol.

• Poly-A tails and mRNA export: The poly-A tails contribute to the export of the mRNA
into the cytoplasm, ensuring that it reaches the site of protein synthesis for translation.
• 5' cap and protection: The 5′ cap protects RNA from being degraded by an RNase,
safeguarding the mRNA molecule during its journey through the cellular environment.
• Poly-A tails and stability: Additionally, the poly-A tails help in stability, preventing rapid
degradation of the mRNA in the cytoplasm and ensuring a longer functional lifespan.

these modifications play crucial roles in safeguarding the mRNA transcript, facilitating its
export from the nucleus, and contributing to its stability during translation on ribosomes in
the cytosol."

4. If the 5' splice site sequence changed from 5 '- GUAAGU-3' to 5'-GUAUGU-3', predict the
effect of the sequence change on U1 binding and U6 snRNP binding in an in vitro protein–
RNA binding assay.
SIR 2018
(Molecular Genetics)
Dr. Nur Ardiyana Rejab
ACADEMIC SESSION 1, 2023/2024
Tutorial 1, 2
NAME: MAI SUHIER M.Y.
MATRIC ID: S2124397

The mutation is expected to lead to a reduction in the binding of U1 to the 5' splice site,
although it is unlikely to completely disrupt this interaction. Conversely, the mutation is
anticipated to strengthen the binding of U6 snRNP to the 5' splice site. This mutation results
from a purine-to-pyrimidine transition, specifically an A to U change in the sequence. As a
silent mutation, it replaces the codon AGU with UGU, encoding for serine and cysteine,
respectively. Both amino acids are hydrophilic. U1 to U6 are small nuclear ribonuclear
proteins (SnRNPs) crucial for pre-mRNA splicing. While the amino acid change at the U1
position is not expected to disrupt binding, it may decrease U1 snRNP binding. Conversely,
the mutation is likely to enhance U6 snRNP binding due to the hydrophilic amino acid
providing a binding site at the U6 snRNP with the 5' splice site.

5. In a biochemical experiment, you compare the products from splicing reactions carried out in
vitro using three different substrates. In each case the substrate is a construct containing a
single intron surrounded by two exons, and in all cases the construct is the same overall size.
But in one case, the intron is a group I intron, in another a group II intron, and in the third an
intron removed by the spliceosome. Each construct is labelled in a manner that allows it to be
detected after gel electrophoresis, and each is tested in two reactions—one, conditions that
support self- splicing, and two, in the presence of nuclear extract as well. Match the intron
type with the appropriate results (A, B, or C) in the gel shown below. Note that, for
simplification, only the final products of the splicing reaction are seen, but before degradation
of the introns.

Group I self-splicing intron -- Sample C: Group I introns can self-splice. Therefore, under
conditions that support self-splicing, Sample C (Group I) is expected to undergo self-splicing.

Group II self-splicing intron -- Sample B: Group II introns are also capable of self-splicing.
Therefore, under conditions that support self-splicing, Sample B (Group II) is expected to undergo
self-splicing.

Intron that requires a spliceosome -- Sample A: If the intron in Sample A requires a spliceosome, it
means that it cannot self-splice and needs the cellular machinery for splicing. Therefore, under
conditions that support self-splicing, Sample A would likely show limited or no splicing.
SIR 2018
(Molecular Genetics)
Dr. Nur Ardiyana Rejab
ACADEMIC SESSION 1, 2023/2024
Tutorial 1, 2
NAME: MAI SUHIER M.Y.
MATRIC ID: S2124397

Tutorial 2

1. Figure 1 shows the last replicon at one end of a human chromatid: the ends of the DNA
strands are shown on the right side of the figure, while the dotted line on the left side
indicates that the DNA molecule continues in that direction. The origin of replication closest
to the end of the DNA molecule is indicated by ori. The following questions are related to the
region between this ori and the end of the DNA. Solve the following MCQs!

Figure 1
The end of the DNA of a human chromatid.

Four-choice Association
(In this type of question, a set of lettered headings is followed by a list of numbered words or
phrases. Select:

• (a)if the word or phrase is associated with A only;

• (b)if the word or phrase is associated with B only;
• (c)if the word or phrase is associated with A and B;
• (d)if the word or phrase is associated with neither A nor B.)
o A Strand A
o B Strand B
o C Both of them
o D Neither of them
▪ 1___B__ Replication of this strand involves a single primer molecule.
▪ 2___A__ Replication of this strand involves several primer molecules.
▪ 3___D__ Replication of this strand does not need primer.
▪ 4___A__ Okazaki fragments are synthesized on this strand.
▪ 5___B__ This strand is the leading strand template.
▪ 6___A_ This strand is the lagging strand template.
▪ 7___A__ The regular DNA replication machinery of the cell can not
prevent the shortening of this strand during successive replication
cycles.

2. The role of telomeres and telomerase in cell proliferation was tested in the following
experiment. Telomerase-negative human embryonic kidney cells were infected with an
“empty” retroviral vector (lanes 1–2 in Fig. 2A.) or with a vector coding for the human
telomerase protein (lanes 3–7). Southern blot analysis was performed using a restriction
SIR 2018
(Molecular Genetics)
Dr. Nur Ardiyana Rejab
ACADEMIC SESSION 1, 2023/2024
Tutorial 1, 2
NAME: MAI SUHIER M.Y.
MATRIC ID: S2124397

endonuclease that does not cut into the telomeric DNA; the blot was hybridized with a
telomere-specific probe (Fig. 2A). Growth of the two cultures was also measured (Fig. 2B).
What conclusions can be drawn from the experiment?

Figure 2
Expression of the human telomerase protein in human embryonic kidney cells. (A). Southern blot
analysis of genomic DNA using a telomere-specific probe. (B). Growth curves of cells (PD,
population doubling)

(The following statements are related to the information presented in the description of the
experiment. Based on the information given, select:

• A if the statement is supported by the information given;

• B if the statement is contradicted by the information given;

• C if the statement is neither supported nor contradicted by the information given.)

o 8___A__ Telomeres in a cell population without telomerase have a uniform size.

o 9___B__ Telomeres in a cell population expressing telomerase have a uniform size.

o 10__C__ Human embryonic kidney cells express telomerase RNA.

o 11__B___ Telomere shortening is a factor in cellular aging.

o 12__B__ The retrovirus vector used in this study immortalizes the cells.

Q2:
Telomerase, a ribonucleoprotein comprising both RNA and protein components, serves the
crucial function of counteracting the inherent shortening of chromosomes during DNA
SIR 2018
(Molecular Genetics)
Dr. Nur Ardiyana Rejab
ACADEMIC SESSION 1, 2023/2024
Tutorial 1, 2
NAME: MAI SUHIER M.Y.
MATRIC ID: S2124397

replication in eukaryotic cells. By adding TTAGGG sequences to the chromosomal ends,

telomerase effectively elongates chromosomes and mitigates the effects of regular
fragmentation.

Southern blotting, a technique employed to identify specific DNA samples within a given
sample, is utilized to analyze the telomere distribution in DNA samples. In Figure (A), the
lengths of telomeres are depicted, revealing a uniform distribution that implies the addition of
similar sequences in the same polarity.

Contrastingly, Figure (B) illustrates a distinct scenario. In the presence of telomerase enzyme,
cell growth exhibits a rapid increase, and there is a noticeable absence of chromosome
shortening or senescence. This is evident from the straightforward linear graph. In the absence
of telomerase, however, chromosome shortening ensues, leading to cellular death. The depicted
contrast in the growth patterns underscores the pivotal role of telomerase in maintaining
chromosome integrity and cellular vitality.