Maria D.

Teklemariam
Department of Medical Biochemistry
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DNA packaging into chromosomes
DNA molecules are very large that require special
packaging to enable them to reside within cells.
• All DNA is packaged as structures called nucleosomes
which are the basic organizational units of chromatin.
• Chromatins are the complexes between eukaryotic DNA
and Proteins
• The DNA in the chromatin is very tightly associated with
proteins called histones.
Histones, which are small basic proteins containing large
amounts of arginine and lysine.
Histones are the major structural proteins of
chromosomes.

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 DNA packaging contd…
The organization of eukaryotic DNA into chromatin is
essential for regulation of transcription
There are different types of chromatin
 Euchromatin- chromatin that is diffuse
 heterochromatin- chromatin that is condensed
There are two types of heterochromatin:
 Constitutive heterochromatin
 Facultative heterochromatin

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 DNA packaging contd…
The genes in heterochromatin are inactive, whereas those
in euchromatin are active and can produce mRNA.
There are five major classes of histones these are H1, H2A,
H2B, H3, and H4.
H2A, H2B, H3 and H4 are known as the core histones,
while H1 is known as the linker histones.

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 DNA packaging contd…
In eukaryotes, activation of a gene requires changes in the
state of chromatin (chromatin remodeling) that are
facilitated by acetylation of histones and methylation of
bases.
These changes in DNA determine which genes are available
for transcription.

Figure 1. Model for the structure of the nucleosome, in which

DNA is wrapped around.

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 DNA packaging contd…
Chromatin has the appearance of beads on a string.
The beads (nucleosome cores) with DNA protruding from
each end are known as nucleosomes.
Two molecules of each of four histone classes (H2A, H2B,
H3, and H4) form the center of the core around which
approximately 140 base pairs of DNA.
The DNA wrapped around the nucleosome core is
continuous and joins one nucleosome core to the next.
The DNA joining the cores is complexed with the fifth
type of histone, H1.

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 DNA packaging contd…
Other types of proteins are also associated with DNA in
the nucleus.
These proteins are “non-histone chromosomal proteins.”
The cells of different tissues contain different amounts
and types of these proteins, which include
 enzymes that act on DNA replication and repair
 factors that regulate transcription (proteins involved in
RNA synthesis, processing and transport)

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DNA packaging contd…

Figure 2. Histones H2A, H2B, H3 and H4 are known as the core histones,
while histones H1 is known as the linker histones.

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DNA packaging contd…
 The histones interact with each other in very specific ways.
 H3 and H4 form a tetramer containing two molecules of
each (H3-H4)2, while H2A and H2B form dimers (H2A-
H2B).
 These histone oligomers associate to form the histone
octamer of the composition (H3-H4)2-(H2A-H2B)2.

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 DNA packaging contd…
These four core histones are subject to at least five types
of covalent modification:
 acetylation,
 methylation,
 phosphorylation,
 ADP ribosylation,
 sumoylation and
 covalent linkage to ubiquitin. (H2A only)

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 DNA packaging contd…
Histone acetylation and deacetylation.
Histone acetylase and other enzymatic activities are
involved in regulation of gene transcription.
Acetylation is known to occur on -amino group of lysine
residues in the amino terminal tails of histone molecules by
Histone acetyltransferase (HATs)
It reduces the positive charge of these tails and decreases
the binding affinity of histone for the negatively charged
DNA.
Histone deacetylation (HDAC) would have the opposite
effect.

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 DNA packaging contd…

Figure 3. Histone acetylation and deacetylation and their effects

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 DNA packaging contd…

Methylation of DNA
Cytosine residues in DNA can be methylated to produce 5-
methylcytosine.
The methylated cytosines are located in GC-rich
sequences (CpG-islands), which are often near or in the
promoter region of a gene.
In certain instances, genes that are methylated are less
readily transcribed than those that are not methylated.

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 DNA packaging contd…
Histone methyltransferases (HMTs) are enzymes that
catalyze the methylation of lysine or arginine residues of
histone proteins.
Methylated histones can either repress or activate
transcription
Methylation of a pair of lysine amino acids at the 4th and
9th positions of the N-terminus of H3 have different effect.
Methylation of lysine-9 forms a binding site for the
Heterochromatin protein-1 (HP1) protein which induces
chromatin packaging and silences gene expression
Methylation of lysine-4 has the opposite effect and
promotes an open chromatin structure.

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DNA packaging contd…

Possible Roles of Modified Histones

 Acetylation of histones H3 and H4 is associated with the
activation or inactivation of gene transcription.
Acetylation of core histones is associated with
chromosomal assembly during DNA replication.
Phosphorylation of histone H1 is associated with the
condensation of chromosomes during the replication
cycle.
ADP-ribosylation of histones catalyzed by ADP-ribose
transferases and is associated with DNA repair, cell
proliferation and differentiation

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DNA packaging contd…
Possible Roles of Modified Histones
Monoubiquitylation is associated with gene activation,
repression, and heterochromatic gene silencing.
Sumoylation of histones (SUMO; small ubiquitin-related
modifier) is associated with transcription repression.

Maria 17
 DNA packaging contd…
Remodeling of chromatin, is also carried out by large
protein complexes SWItch/Sucrose Non-Fermentable
(SWI/SNF) complex.
Sw1/Snf complexes have ATPase and helicase activity.
The Sw1/Snf complex is thought to transiently dissociate
DNA from the surface of nucleosomes, permitting
nucleosomes to “slide” along the DNA and promoting the
unfolding of condensed higher-order chromatin
structures.
facilitate the binding of transcription factors to DNA in
chromatin

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DNA packaging contd…
SWI/SNF complexes are also required for the repression of
some genes, because
 they help expose histone tails to deacetylases or
 they assist in the folding of chromatin into condensed,
higher-order structures.
These complexes are thought to recruit histone acetylases
that modify chromatin to make genes accessible to
transcription factors.
Mutations affecting the Swi/Snf chromatin-remodeling
complex, are associated with a variety of tumors.

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DNA packaging contd…

Figure 4. Chromatin access:

factor that mainly belong
to the SWI/SNF family
medicate DNA accessibility
by nucleosome
rearrangement
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 Replication contd…
Nucleic acids are required for the storage and expression of
genetic information.
DNA replication is the process by which the genome's DNA
is copied in cells.
Replication of DNA must be complete and carried out with
high fidelity to maintain genetic stability.
The two strands of the DNA double helix are separated;
each can serve as a template for the replication of a new
complementary strand, producing two daughter molecules.
This process is called semi conservative replication.
The enzymes involved in DNA replication process are
template directed polymerases.

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Replication contd…
Polymerases synthesize the complementary sequence of
each strand.
New DNA strands are
synthesized by using the
existing (parent) strands as
templates.
The formation of new, daughter
strands that are complementary
to the parent strands.

Figure 5. DNA Double Helix

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 Replication contd…

Figure 6. Semi-conservative DNA replication

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Replication contd…
Synthesis of DNA in Eukaryotes requires
1. DNA polymerase

DNA Polymerases Require a Primer to Initiate Replication.

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 Replication contd…
Table 1. Class of proteins involved in replications

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Replication contd…
DNA polymerases cannot initiate chain synthesis it requires
a short, RNA strand, called a primer, to begin chain growth.

Figure 7. DNA replication

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Replication contd…
DNA polymerase catalyzes the stepwise addition of a
deoxyribonucleotide (dAMP, dGTP, dTMP, …) to the 3ʹ-
OH end of a polynucleotide chain.
• Helicases and topoisomerases unwind the parental
strands, and single-strand binding proteins prevent them
from reannealing.
 Type I topoisomerases cleave just one strand of DNA,
 Type II enzymes cleave both strands.

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Replication contd…

Figure 8. DNA replication in Eukaryotes

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Replication contd…
Replication of the circular,
double-stranded DNA in
Prokaryotes is almost the same
in eukaryotes.

Figure 8. DNA replication of a

bacterial genome.
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Replication contd…
Replication of the Ends of Chromosomes
Eukaryotic chromosomes are linear, and the ends of the
chromosomes are called telomeres.
Telomeres consist of a repeating sequence of bases
(TTAGGG for humans).
The enzyme telomerase contains both proteins and RNA
and acts as an RNA dependent DNA polymerase.
(human telomerase reverse transcriptase gene (hTERT)).
The polymerase activity of telomerase then uses the
existing 3- hydroxyl group of the overhang as a primer,
and its own RNA as a template, and synthesizes new DNA
that
lengthens the 3- end of the DNA strand.
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Replication contd…
The 3 -overhang can also form a complicated structure
with
telomere binding proteins to protect the ends of the
chromosomes from damage and nuclease attack.

Figure 9. Telomerase action

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 Mutation ; DNA repair
Genomes are dynamic entities that change over time as a
result of the cumulative effects of mutation and
recombination.
Mutation is a heritable change in the nucleotide sequence
of a short region of a genome.
Mutations occur at a low frequency as the result of
copying errors introduced by DNA polymerases
Recombination is the exchange of genetic material
between chromosomes.
process responsible for crossing-over and exchange of
DNA segments between homologous chromosomes during
meiosis of eukaryotic cells

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 Mutation
DNA damage can be caused by;-
 spontaneous cleavage of chemical bonds in DNA,
 by environmental agents such as ultraviolet and ionizing
radiation
 by reaction with genotoxic chemicals that are by-products
of normal cellular metabolism or occur in the
environment. Eg. aflatoxin B1 is the most potent liver
carcinogen
several “proofreading” mechanisms that act sequentially to
correct any initial mispairings
Many mutations are point mutation that replace one
nucleotide with another; others involve insertion or
deletion of one or a few nucleotides.
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DNA repair
Cells possess efficient DNA repairing systems.
All cells possess DNA-repairing enzymes that attempt to
minimize the number of mutations that occur.
The different DNA repairing system :-
1. Direct repair:-
act directly on damaged nucleotides, converting each
one back to its original structure.
2. excision repair
involves excision of a segment of the polynucleotide
containing a damaged site, followed by resynthesis of
the correct nucleotide sequence by a DNA polymerase.

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 DNA repair contd….
3. Mismatch repair
corrects errors of replication, again by excising a stretch
of single-stranded DNA containing the aberrant
nucleotide
and then repairing the resulting gap.
4. Recombination repair:-
is used to repair double-strand breaks.

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 DNA Repair contd…

Figure 10. DNA damaging factors, DNA lesion types and possible
repair mechanisms
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 DNA Repair contd…
Table 2. Mechanisms of DNA Repair

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DNA repair
Excision endonuclease, also known as excinuclease or UV-
specific endonuclease
Xeroderma pigmentosum results from failed DNA repair.
Patients with this disorder are highly sensitive to light,
exhibit premature skin aging, and are prone to malignant
skin tumors.

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 DNA repair

Figure 11. The general pathway of Nucleotide excision repair

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DNA Repair contd…
Once cells lose their ability to effectively repair damaged
DNA, there are three possible responses

 The cell may become senescent, i.e., irreversibly dormant.

stopping mitosis.
 The cell may become apoptotic. Sufficient DNA damage
may trigger an apoptotic signaling cascade, forcing the
cell into programmed cell death.
 The cell may become malignant.

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Mitochondrial DNA
Major Features of the Structure and Function of Human
Mitochondrial DNA
1. Is circular, double-stranded, and composed of heavy (H)
and light
2. Contains 16,569 bp
3. Encodes 13 protein subunits of the respiratory chain (of
a
total of about 67).
4. High mutation rate (5 to 10 times that of nuclear DNA)

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Mitochondrial DNA

Figure 12. Human Mitochondrial DNA structure

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Prokaryotic and Eukaryotic Genetic transcription

• RNA is a working copy of DNA. The copying process,

which uses one of the two strands of DNA as a template is
called transcription.
• RNA is the only macromolecule known to have a role both
in the storage and transmission of information and in
catalysis.
• Transcription requires the action of enzymes called RNA
polymerases.

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Genetic transcription contd….
• There are four major types of RNA:-
1. mRNA- mRNA is transcribed from DNA and contains the
genetic blueprint to make proteins that encode the
amino acids sequence of one or more polypeptides
specified by a gene
2. tRNA- serve as adapters molecule
3. rRNA - rRNA forms ribosomes, which are essential in
protein synthesis.
4. Small RNAs- small nuclear RNA (snRNA) Small
Interfering RNAs (siRNAs) and micro RNA (miRNA)….

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 Eukaryotic genetic transcription contd….

• RNA polymerase (RNA pol) is a complex enzyme (8 to 14

subunits) that synthesizes RNA in the 5′ to 3′ direction.
• Eukaryotic cells contain three distinct nuclear RNA pol
that transcribe different classes of genes.
RNA pol II transcribes Protein-coding genes to yield
mRNAs.
RNA pol I and III transcribe ribosomal RNAs (rRNAs) and
transfer RNAs (tRNAs).
RNA pol I is specifically devoted to transcription of the
three largest species of rRNAs (28S, 18S, and 5.8S).

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 Eukaryotic genetic transcription contd….

 RNA pol III transcribes the genes for tRNAs, for the 5S
rRNA, snRNAs and scRNAs.
• RNA Pol II is responsible for the synthesis of mRNA
from protein-coding genes.
• Many different proteins are involved in the activation
of transcription.
• Transcription in the eukaryotic system requires
specific Proteins called transcription factors required
for RNA pol
II to initiate transcription.

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Eukaryotic genetic transcription contd…
• Expression of eukaryotic protein-coding genes is regulated
by multiple protein-binding DNA sequences referred to as
transcription control regions.
These include:-
 promoters
 TATA box
 CAAT box
 CpG Islands
 silencer sequence, and others

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Eukaryotic genetic transcription contd…
Transcription factor is a protein that binds to a specific
site on DNA and regulates the rate of transcription of a
gene.
• These Proteins bind to specific regulatory sequences and
modulate the activity of RNA pol.
The expression of eukaryotic genes is controlled primarily
at the level of initiation of transcription.
The sequence of an RNA is complementary to the
sequence of DNA in one strand of the double-stranded
DNA.

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 Eukaryotic genetic transcription contd….
The strand that is transcribed into an RNA molecule is
referred to as the template strand of the DNA.
The other DNA strand, the non-template strand, is
frequently referred to as the coding strand of that gene.
Transcription has initiation, elongation, and termination
steps and the newly synthesized RNA is called the primary
transcript
DNA-Dependent RNA Polymerase initiates Transcription
at a Distinct site called the Promoter

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 Eukaryotic genetic transcription contd….
Eukaryotic genes encoding proteins have promoter sites
with a TATAAA consensus sequence, called a TATA box or
a Hogness box.
The TATA box is usually located 25–30 bp upstream from
the transcription start site in mammalian genes.
Promoters also have a CAAT box with a GGNCAATCT
consensus sequence centered at about -75.
The starting point of transcription corresponds to the 5
nucleotide of the mRNA and is designated as +1 position.
The base in the coding strand of the gene serving as the
start point for transcription is numbered +1.

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 Eukaryotic genetic transcription contd….
Untranscribed sequences to the left of the start point,
known as the 5-flanking region of the gene and numbered
as –1, –2, –3, etc., starting with the nucleotide (–1)
immediately to the left of the start point (1).
By analogy, the sequences to the left of the start point are
said to be upstream from the start point and those to the
right are said to be downstream.
This designation provides a conventional way of defining
the location of regulatory elements in the promoter.

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Transcription contd….
Five general transcription factors (TF) (or basal TF) are required
for initiation of transcription termed TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, and TFIIH.

The human TATA box is bound by the 34 kDa TATA-binding

protein (TBP), which is multisubunit complexes.

 The non-TBP subunits of TFIID are proteins called TBP-

associated factors (TAFs).

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 Transcription contd….

Figure 13. Promoter site for transcription of eukaryote and

prokaryotes.
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 Transcription contd….
Binding of the TBP-TAF, TFIID complex to the TATA box
sequence is thought to represent a first step in the
formation of the transcription complex on the promoter.

Figure 14. The eukaryotic basal transcription complex

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 Transcription contd….
Some mRNA-encoding genes lack a consensus TATA box.
In this case, additional cis elements, an initiator sequence
(Inr) and downstream promoter element (DPE), direct the
RNA polymerase II transcription machinery.
The Inr element spans the start site (from −3 to +5).
The DPE has the consensus sequence localized about 25
bp downstream of the +1 start site.
The Inr, DPE sequences are also bound by the TAF
subunits of TFIID.

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 Transcription contd….

Figure 15. Schematic diagram showing the transcription control regions in

a hypothetical mRNA producing, eukaryotic gene transcribed

by RNA polymerase II.
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 Transcription contd….
Sequences just upstream from the transcription start site
also determine how frequently a transcription event
occurs.
Typical of these elements are the cis-acting the GC rich
sequence and CAAT boxes in the region between –40 and
–110.
Eukaryotic genes also contain promoter-proximal
elements in the region of –100 to - 200.
These boxes bind to transacting transcription factors Sp1
and CTF (CAAT box binding transcription factor).
Sp1 binds to the GC rich motif and CTF to the CAAT box;
through their distinct DNA binding domains (DBDs).

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 Transcription contd….
There are other class of sequence elements that can either
increase or decrease the rate of transcription initiation of
eukaryotic genes. These elements are:-
 Enhancers
 Repressors (or silencers)
They have been found in a variety of locations both
upstream and downstream of the transcription start site
and even within the transcribed protein coding portions of
some genes.

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 Transcription contd….
Enhancers and silencers (repressors) can exert their effects
when located thousands of bases away from transcription
start site.
Enhancer binding transactivator factors have been shown
to interact with other transcription proteins like:-
 Chromatin-modifying coactivators,
 Mediator, (mediate signals from DNA-binding TF directly
to RNA Pol II)
 Components of the basal transcription machinery.
transactivator factor-enhancer DNA binding events result
in an increase in the binding of the basal transcription
machinery to the promoter.

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Transcription contd….
Some regions are controlled by complex DNA elements
called insulators.
It blocks enhancer-promoter interactions.
Regulation of transcription also requires that regulatory
proteins bind with high affinity and specificity to the
correct region of DNA.
 Three unique motifs—
 the helix-turn- helix,
 the zinc finger, and
 the leucine zipper—account for many of these specific
protein-DNA interactions.

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Transcription contd….

conserved structural motif in

DNA binding domain of the
transcription factors.

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Transcription contd….

Figure 16. Mechanism of Transcription

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Transcription contd….

Figure 17. Schematic view of a eukaryotic gene and steps required

to produce an mRNA
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Transcription contd….
After RNA poly II has finished the region of the
transcription unit encoding the 3′ end of the transcript,
RNA endonucleases cleave the primary transcript at a
position about 15 bases 3′ of the consensus sequence
AAUAAA that serves in eukaryotic transcripts as a
cleavage and polyadenylation signal

Figure 18. Relationship between the coding strand of DNA, the

DNA template strand, the mRNA transcript
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Transcription contd….
Function, structure and synthesis of RNAs
tRNA- serve as adapters for the translation of the
information in the sequence of nucleotides of the mRNA
into specific amino acids.
It has a cloverleaf shape
They vary in length from 74 to 95 nucleotides.
tRNAs compose roughly 20% of total cellular RNA.
All tRNA molecules contain four main arms that contain
modified bases by posttranscription.
•The acceptor arm terminates in the nucleotides 3’-CCA.

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Genetic transcription contd….
These three nucleotides are added posttranscriptionally by a
specific nucleotidyl transferase enzyme.
T-loop (TC) contains both ribothymidine (T) and
pseudouridine ().
Near the 5’ end the D arm is there where D is
dihydrouridine.
The anticodon, loop contains the trinucleotide anticodon
that base pairs with the codon on mRNA.
A fourth loop, known as the variable loop because it varies in
size.
Some tRNA precursors contain introns that are removed by
endonucleases.

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Genetic transcription contd….

The pre-tRNA is
subsequently cleaved
at the 5- and 3-ends
by RNase P.

Figure 19. tRNA structure

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Genetic transcription contd….

Figure 20. Overview of tRNA synthesis

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Genetic transcription contd….

3. rRNA - rRNA forms ribosomes, which are essential in

protein synthesis.
 All of the ribosomal RNA molecules except the 5S rRNA,
are transcribed from a single precursor 45S rRNA.
 This large 45S transcript is cleaved to produce the 18S,
28S, and 5.8S rRNAs.
 one portion of the 45S rRNA precursor becomes the 18S
rRNA that, complexed with proteins, forms the small
40S ribosomal subunit.

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Genetic transcription contd….
Another segment of the precursor folds back on itself and
is cleaved, forming 28S rRNA, hydrogen-bonded to the
5.8S rRNA.
Proteins complex with the 28S and 5.8S rRNAs to form
the 60S.
In the cytoplasm, the 40S and 60S ribosomal subunits
interact with mRNA forming the 80S ribosomes on which
protein synthesis occurs.

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Genetic transcription contd….

Figure 21. rRNA and ribosome synthesis

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Genetic transcription contd….
4. Small RNAs- small nuclear RNA (snRNA) Small
Interfering RNAs (siRNAs) and micro RNA (miRNA)……
snRNA involved in rRNA and mRNA processing and gene
regulation.
of the several snRNAs, U1, U2, U4, U5, and U6 are
involved in intron removal and the processing of mRNA
precursors into mRNA.
The U7 snRNA is involved in production of the correct
3′ ends of histone mRNA - which lacks a poly(A) tail.

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Genetic transcription contd….
miRNA
 a class of small RNAs found in most eukaryotes.
 are typically 21–25 nucleotides in length.
 are derived from large primary transcripts through
specific nucleolytic processing
 Majority of miRNA are transcribed by RNA pol II into
primary transcripts termed pri-miRNAs.
• In the nucleus, the DROSHA and DGCR8 microprocessor
complex cleaves pri-miRNA to mature miRNA
• The pri-miRNAs are 5′-capped and 3′-polyadenylated
 miRNA cause inhibition of gene expression by decreasing
specific protein production.

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Transcription contd….
Reverse Transcriptase
Reverse transcriptase is an enzyme that uses a single-
stranded RNA template and makes a DNA copy.
Retroviruses (RNA viruses) contain a reverse transcriptase,
which copies the viral RNA genome. (HIV, Covid 19,
Hepatitis, …... ).
A double-stranded cDNA is produced, which can become
integrated into the human genome
This enzyme catalyzes the production of a DNA copy from
an RNA template.
The RNA of a DNA-RNA hybrid is degraded, and the
single DNA strand is used as a template to make double-
stranded DNA.
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Transcription contd….

Figure. 22. Action of

reverse transcriptase.

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Transcription contd….

Figure 23. Integration of retrovirus into the host chromosomes

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 Prokaryotic genetic transcription contd….
Transcription and translation are coupled in prokaryotic
cells.
Prokaryotic mRNAs are subjected to little processing prior
to carrying out their intended function in protein
synthesis.
Bacterial cells have a single RNA polymerase that
transcribes DNA to generate all of the different types of
RNA (mRNA, rRNA, and tRNA).

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 Prokaryotic genetic transcription contd….
The DNA-dependent RNA Pol of the bacterium E coli exists as
an approximately 400 kDa core complex consisting of five
subunit:-
 two identical α subunits,
 similar but not identical β and β′ subunits, and
 an ω subunit.
The β subunit binds Mg2+ ions and composes of the catalytic
subunit.
Another protein called a  (sigma) factor binds the core
enzyme and directs binding of RNA pol to specific promoter
regions of the DNA template.

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 Prokaryotic contd….

A number of different  factors are there that recognize

the promoter regions of different genes, the major one is
70.

A number of protein-producing genes may be linked

together and controlled by a single promoter called an
operon.
Bacterial promoters are approximately 40 nucleotides (40
bp) in length.

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 Prokaryotic genetic transcription contd….

Figure 24. RNA pol catalyzes the polymerization of

ribonucleotides

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 Prokaryotic contd….
There are two short, conserved consensus sequence
elements in the promotor region.
 Approximately -35-bp upstream of the transcription start
site there is a consensus sequence of eight nucleotide pairs
(consensus: 5′-TGTTGACA-3′) involved in binding of RNA
pol.
 proximal to the transcription start site about -10
nucleotides upstream—is a six-nucleotide-pair A+T-rich
sequence (consensus: 5′-TATAAT-3′)(Pribnow box). It is
recognize by the sigma factor 70

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 Prokaryotic contd….

Figure 25. Bacterial promoters with the two highly conserved

nucleotide sequence
 By convention, all nucleotides upstream of the transcription initiation
site (at +1) are numbered in a negative sense and are referred to as 5′-
flanking sequences.
 All nucleotides downstream of the transcription start site is in a positive
sense
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Prokaryotic contd….
The process of RNA synthesis in bacteria involves first the
binding of the RNA polymerase holoenzyme molecule to
the template at the promoter site to form a preinitiation
complex, or PIC.
A  factor of RNA polymerase binds to the promoter
region of DNA and causes the two DNA strands to unwind
and separate within a region approximately 10 to 20
nucleotides
TATA box is centered about –10 and is recognized by the
sigma factor 70
The sigma factor is released when the growing RNA chain
is approximately 10 nucleotides long.

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Prokaryotic contd….
The enzyme polymerizes the ribonucleotides in the
specific Sequence.
Pyrophosphate (PPi) is released following each cycle of
polymerization.
purine ribonucleotide is usually the first to be polymerized
into the RNA molecule.
After 10–20 nucleotides have been polymerized, RNA Pol
undergoes a second conformational change leading to
promoter clearance.
Once this transition occurs, RNA Pol physically moves
away from the promoter.

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Prokaryotic contd….
The elongation complex containing the core RNA
polymerase progresses along the DNA molecule.
DNA unwinding must occur in order to provide access for
the appropriate base pairing to the nucleotides of the
coding strand.
Elongation of the RNA molecule from the 5′ to its 3′ end
continues cyclically, antiparallel to its template.
RNA polymerase has an intrinsic helicase activity that
opens the DNA helix.
The elongation reactions continue until the RNA
polymerase encounters a transcription termination signal.

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Prokaryotic contd….

• Termination:- a signal that is recognized by a

termination protein, the rho (ρ) factor.
• Rho is an ATP-dependent RNA-stimulated helicase that
disrupts the nascent RNA-DNA complex.
Rho-dependent transcription termination signals have a
distinct consensus sequence, 40 nucleotide pairs in length
and contain a hyphenated or interrupted inverted repeat
followed by a series of AT base pairs.
Transcription continues into the AT region with the aid of
the ρ protein the RNA polymerase stops, dissociates from
the DNA template, and releases the nascent transcript.

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Prokaryotic contd….

Figure 26. Formation of a hairpin loop in the transcript, preceding a

number of U residues :- termination signal

RNA hairpin causes RNA polymerase to pause

transcription.
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Prokaryotic contd….

Figure 27. The structural genes of an operon are transcribed as one long
polycistronic mRNA
 mRNA is usually generated from an operon as a polycistronic
mRNA contains multiple sets of start and stop codons that allow
a number of different proteins to be produced from this single
transcript.
 A cistron is a region of DNA that encodes a single polypeptide
chain.
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Posttraranscriptional modification
Eukaryotic RNA primary transcripts undergo extensive
processing, whether it be as mRNA, miRNAs, rRNA, 5S
RNA, or tRNA.
Processing occurs primarily within the nucleus.
Eukaryotic pre-mRNA transcripts contain regions known
as exons and intervening sequences (introns).
Exons appear in the mature mRNA; introns are removed
from the transcript and are not found in the mature
mRNA

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Posttranscription Contd…
Regulation of transcription by RNA processing
1. Alternative splicing
Alternating splicing of mRNA is a regulatory mechanism
that can affect quantitative control of gene expression and
functional diversification of protein.
Transcription of a single gene can be initiated from a
variety of alternative promoters and can result in a variety
of isoforms.
These isoforms can be tissue specific, developmental stage
specific, sub-cellular localization specific and sex specific.

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Posttraranscription contd….
The coding portions (Exons) of most Eukaryotic genes are
Interrupted by introns.
Exons ultimately translated into the amino acid sequence
of a protein molecule.
The intron RNA sequences are cleaved out of the
transcript.
The exons of the transcript are appropriately spliced
together in the nucleus before the resulting mRNA
molecule appears in the cytoplasm for translation.
Alternative splicing provides different mRNAs.

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Posttraranscription contd….
A special multicomponent complex, the spliceosome, is
involved in converting the primary transcript into mRNA.
Spliceosomes consist five small nuclear RNAs (snRNAs)
(U1, U2, U4, U5, and U6) and more than 60 proteins, many
of which contain conserved “RNP” and “Signal
recognition” protein motifs.
Introns are removed as a lariat-like structure in which the
5’ G of the intron is joined in an unusual 2,5-
phosphodiester bond to an adenosine near the 3’ end of
the intron.

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Posttraranscription contd….
The consensus sequences at the intron/exon boundaries of
the pre-mRNA are AGGU (AGGT in the DNA).
Almost all introns begin with a 5’ GU and end with a 3’ AG

Figure 28. Splice junctions in hnRNA intron sequences

shown in blue dashed boxes
snRNA bind to the intron, causing it to form a loop
(Lariate).
U1 within the snRNP complex binds first by base pairing
to the 5′ exon-intron boundary.
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Posttraranscription contd….

U2 binds within the intron in a region containing an

adenine nucleotide residue.
Another group of snRNAs, U4, U5, and U6, binds to the
complex, and the loop is formed.
The phosphate attached to the G residue at the 5-end of
the intron forms a 2–5 linkage with the 2-hydroxyl group
of the adenine nucleotide residue.

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Posttraranscription contd….
Cleavage occurs at the end of the first exon, between the
AG residues at the 3’ end of the exon and the GU residues
at the 5 end of the intron.
A second cleavage occurs at the 3-end of the intron after
the AG sequence.
The exons are joined together. The intron, shaped like a
lariat, is released and degraded to nucleotides.

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 Posttraranscriptional contd….

Figure 29. Alternative Splicing process

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 Contd…

Figure 30. Model of

spliceosome-
mediated splicing
of pre-mRNA
.

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Posttranscription contd….
1. Capping of mRNA at the 5’ end
Eukaryotic mRNAs at the 5′ terminal is “capped” by a 7-
methylguanosine triphosphate.
The 5′ cap of the RNA transcript is required both for
efficient translation initiation and protection of the 5′ end
of mRNA from attack by 5′ → 3′ exonucleases.
7-methylguanylate that is connected to the terminal
nucleotide of the RNA by an unusual 5,5 triphosphate
linkage

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Posttraranscription contd….

Figure 31. Methylated

cap of Eukaryotic
mRNA
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Posttranscription contd….
RNA editing changes mRNA after transcription
RNA editng is a form of post-transcriptional processing
which can involve enzyme-mediated insertion or deletion
of nucleotides or substitution of single nucleotides.
Individual mRNAs may undergo multiple C → U or U → C
editing events.
Example :- C to U editing of human APOB lipoprotein
mRNA
The cell-type-specific expression of the two isoforms of
apoB results from editing of apoB pre-mRNA
In the liver the APOB gene encodes a 14.1 kb mRNA
transcript and a 4536 amino acid product, ApoB100.

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Posttraranscription contd….
In the intestine the same gene encodes a 7 kb mRNA
which contains a premature stop codon and encodes a
product, ApoB48 with 2152 amino acids.

Figure 32. RNA editing of ApoB pre-mRNA

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Inhibitors of RNA Pol contd….
Rifampin is thought to inhibit bacterial DNA-dependent
RNA polymerase, by forming a stable drug-enzyme
complex selectively killing the bacteria that cause the
infection.
The toxin  amanitin is capable of causing irreversible
inhibition of mammalian RNA polymerases.
 amanitin a peptide toxin from the mushroom Amanita
phalloides,

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Inhibitors of RNA Pol contd….
 α-Amanitin blocks the translocation of RNA polymerase
during phosphodiester bond formation.
 Actinomycin D forms a very stable complex with DNA
preventing the unwinding of the DNA double-helix, thus
inhibiting the DNA-dependent RNA polymerase activity.
 Remedsivir – inhibitor of viral RNA pol.
 Entecavir - viral reverse transcriptase inhibitor

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 Thank you

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