BT 205: Cell & Molecular Biology

-Prof. Siddhartha Sankar Ghosh

Lec-10

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Q What are the three events of post
transcription?

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Post Transcriptional Modifications

Splicing of mRNA

5/ Capping

3/ Polyadenylation

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Q What are the steps of RNA Splicing?

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 Steps Involved
 Formation of the primary
transcript and its processing
during maturation of mRNA
in a eukaryotic cell.
 The 5′ cap is added before
synthesis of the primary
transcript is complete.
 A noncoding end sequence
(intron) following the last
exon is shown in orange.
 Splicing can occur either
before or after the cleavage
and polyadenylation steps.

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Q. What is 5’ Capping?

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 1. The 5′ cap

In eukaryotes, the 5′ cap (cap-0), found on

the 5′ end of an mRNA molecule, consists of
a guanine nucleotide connected to mRNA
via an unusual 5′ to 5′ triphosphate linkage.
This guanosine is methylated on the 7
position directly after capping in vivo by
methyltransferase. It is referred to as a 7-
methylguanylate cap, abbreviated m7G.

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Q. What are four functions of 5’ Cap?

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The 5′ cap has four main functions:

1.Regulation of nuclear export

2.Prevention of degradation by exonucleases
3.Promotion of translation
4.Promotion of 5′ proximal intron excision

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 The 5′ cap of mRNA
(a) 7-Methylguanosine (m7G) is joined to the 5′ end of almost all eukaryotic mRNAs in
an unusual 5′,5′- triphosphate linkage.
Methyl groups (light red) are often found at the 2′ position of the first and second
nucleotides. RNAs in yeast cells lack the 2′-methyl groups.
The 2′- methyl group on the second nucleotide is generally found only in RNAs from
vertebrate cells.
(b) Synthesis of the cap is carried out by enzymes tethered to the CTD of Pol II. The cap
remains tethered to the CTD through an association with the cap-binding complex
(CBC).

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 2. Polyadenylation:

It is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA

(mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words,
it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of
the process that produces mature mRNA for translation.

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Q. What is the use of 3’ Polyadenylation commercially?

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Oligo-dT Cellulose Columns. Oligo dT isolation is a very useful method for isolating
sequences with a poly A tag.

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3. Splicing of mRNA

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Q. How many classes of RNA Splicing are present?

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3. Splicing of mRNA

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Spliceosomal introns generally have the dinucleotide
sequence GU at the 5′ end and AG at the 3′ end, and
these sequences mark the sites where splicing occurs.

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 In eukaryotes, most introns undergo splicing by the same lariat-forming mechanism.
 Splicing takes place within a large protein complex called a spliceosome.
 A spliceosome is made up of multiple specialized RNA-protein complexes called small
nuclear ribonucleoproteins (snRNPs, often pronounced snurps).
 Each snRNP contains one of a class of eukaryotic RNAs, 100 to 200 nucleotides long,
known as small nuclear RNAs (snRNAs).
 Five snRNAs (U1, U2, U4, U5, U6) involved in splicing reactions are generally found
in abundance in eukaryotic nuclei.
 Spliceosomes are thus among the most complex macromolecular machines in any
eukaryotic cell.

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 Splicing mechanism in mRNA primary transcripts
 RNA pairing interactions in the formation of spliceosome complexes. The U1 snRNA
has a sequence near its 5′ end that is complementary to the splice site at the 5′ end of
the intron. Base pairing of U1 to this region of the primary transcript helps define the
5′ splice site during spliceosome assembly.
 U2 is paired to the intron at a position encompassing the A residue (shaded light red)
that becomes the nucleophile during the splicing reaction.
 Base pairing of U2 snRNA causes a bulge that displaces and helps to activate the
adenylate, the 2′ OH of which will form the lariat structure through a 2′,5′-
phosphodiester bond.
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 Assembly of spliceosomes

 All steps are reversible, but are shown proceeding in the forward direction for
simplicity.
 The U1 and U2 snRNPs bind, then the remaining snRNPs (the U4-U6 complex and
U5) bind to form an inactive spliceosome.
 Internal rearrangements convert this species to an active spliceosome in which U1
and U4 have been expelled and U6 is paired with both the 5′ splice site and U2. This
is followed by the catalytic steps, which parallel those of the splicing of group II
introns. .

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Alternative Splicing

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Reverse Transcription

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Reverse Transcription

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Drug: AZT

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• A reverse transcriptase (RT) is an enzyme used to
generate complementary DNA (cDNA) from an RNA
template, a process termed reverse transcription.
• HIV infection – AZT used analog of dTTP
• AZT taken up by susceptible T lymphocytes
• Reverse Transcriptase has higher affinity for AZT
triphosphate than dTTP unlike mammalian
polymerases
• AZT triphosphate lacks 3’-OH group terminating viral
replication.

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 Mechanism of Action

 AZT is taken up by T lymphocytes, immune system cells that are particularly vulnerable
to HIV infection, and converted to AZT triphosphate. (AZT triphosphate taken directly
would be ineffective because it cannot cross the plasma membrane.)
 HIV’s reverse transcriptase has a higher affinity for AZT triphosphate than for dTTP, and
binding of AZT triphosphate to this enzyme competitively inhibits dTTP binding.
 When AZT is added to the 3′ end of the growing DNA strand, lack of a 3′ hydroxyl means
that the DNA strand is terminated prematurely and viral DNA synthesis grinds to a halt.

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Inhibitors of Transcription

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 Rifampicin (rifamycin)

• It binds to the beta subunit of prokaryotic RNA polymerase

• It is an inhibitor of prokaryotic transcription initiation
• It binds only to bacterial RNA polymerases but not to eukaryotic
RNA polymerases
• It is a powerful drug for the treatment of bacterial infections.
• It is an antibiotic used to treat tuberculosis and leprosy

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Rifampicin (rifamycin)

Ref: https://journals.asm.org/doi/10.1128/ecosalplus.esp-0017-2019
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Actinomycin D:

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 Actinomycin D:

• It is an antibiotic that shows antibacterial and antitumor activity.

• Antibiotic, inhibits transcription in bacteria and eukaryotes
• The planar portion of this molecule inserts (intercalates) into the
double helical DNA between successive GC base pairs, deforming the
DNA
• Inhibits RNA elongation by restricting RNA polymerase along the
template
• Concentration dependent action
• Acridine also functions in a similar fashion
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 Alpha amanitin:
• It is a eukaryotic inhibitor isolated from Amanita
phalloides which inhibits RNA II polymerase by blocking
the initiation and elongation process.
• A cyclic peptide of 8 amino acids
• Selective inhibitor of RNA Pol II & Pol III
• α-Amanitin interacts with the bridge helix in Pol II. This
interaction interferes with the translocation of RNA and
DNA needed to empty the site for the next round of RNA
synthesis.
• No effect on affinity for NTPs & phosphodiester bond can
be formed.
• Non-Competitive binding slows down rate of elongation

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Q. A. Show synthesis of beta galactosidase, permease for the
followings in presence and absence of IPTG and give brief
reasons:

B. Draw the graph of Z production vs time.

a) I+Z+Y+/I-Z- Y+
b) I+Z+Y+/I-Z- Y+
c) I+Z-Y+/I-Z+ Y-
b) O+Z-Y+/O-Z+Y-
c) O+Z+Y+/OcZ+Y-
d) O-Z-Y+/OcZ+Y+
e) O+Z+Y+/OcZ+Y-
f) O+Z-Y+/O+Z+Y-

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THANK YOU

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