DNA Transcription ()

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 Flow of genetic information

• The genetic information flows from DNA

to mRNA and then to the protein
synthesizing machinery.
Biochemistry For Medics- Lecture Notes 2
 DNA Transcription-
Introduction
•The synthesis of an RNA molecule from DNA
is called Transcription.
•All eukaryotic cells have five major classes of
RNA: ribosomal RNA (rRNA), messenger RNA
(mRNA), transfer RNA (tRNA), small nuclear
RNA and microRNA (snRNA and miRNA).
•The first three are involved in protein
synthesis, while the small RNAs are involved
in mRNA splicing and regulation of gene
expression.
 Similarities between Replication
and Transcription
The processes of DNA and RNA
synthesis are similar in that
they involve-
(1)the general steps of initiation ,
elongation, and termination
with 5' to 3' polarity;
(2)large, multicomponent
initiation complexes; and
(3)adherence to Watson-Crick
base-pairing rules.
Biochemistry For Medics- Lecture Notes 4
Differences between Replication
and Transcription
(1)Ribonucleotides are used in RNA synthesis
rather than deoxy ribonucleotides;
(2)U replaces T as the complementary base pair for
A in RNA;
(3) A primer is not involved in RNA synthesis;
(4)Only a portion of the genome is transcribed or
copied into RNA, whereas the entire genome
must be copied during DNA replication; and
(5)There is no proofreading function during RNA
transcription.

Biochemistry For Medics- Lecture Notes 5

 Template strand

•The strand that is transcribed or copied 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.
Biochemistry For Medics- Lecture Notes 6
 Template strand
• The information in the template strand is
read out in the 3' to 5' direction
• The sequence of ribonucleotides in the RNA
molecule is complementary to the sequence of
deoxy ribonucleotides in template strand of
the double-stranded DNA molecule
• In the coding strand (complementary strand)
the sequence is same as that of the sequence
of nucleotides in the primary transcript.

Biochemistry For Medics- Lecture Notes 7

 Template strand (contd.)

With the exception of T for U changes,

coding strand corresponds exactly to the
sequence of the RNA primary transcript,
which encodes the (protein) product of
the gene.
Biochemistry For Medics- Lecture Notes 8
 Template strand (contd.)
• In the case of a double-stranded DNA
molecule containing many genes, the template
strand for each gene will not necessarily be
the same strand of the DNA double helix.
• Thus, a given strand of a double-stranded DNA
molecule will serve as the template strand for
some genes and the coding strand of other
genes.

Biochemistry For Medics- Lecture Notes 9

 Transcription unit
• A transcription unit is defined as that region of DNA
that includes the signals for transcription initiation,
elongation, and termination.
• DNA-dependent RNA polymerase is the enzyme
responsible for the polymerization of ribonucleotides
into a sequence complementary to the template
strand of the gene.
• The enzyme attaches at a specific site—the
promoter—on the template strand.
• This is followed by initiation of RNA synthesis at
the starting point, and the process continues until a
termination sequence is reached.

Biochemistry For Medics- Lecture Notes 10

Transcription unit and Primary
transcript

Biochemistry For Medics- Lecture Notes 11

 Primary transcript
• The RNA product, which is synthesized in the
5' to 3' direction, is the primary transcript.
• In prokaryotes, this can represent the product of
several contiguous genes
• In mammalian cells, it usually represents
the product of a single gene
• The 5' terminals of the primary RNA
transcript and the mature cytoplasmic RNA
are identical.
• The starting point of transcription corresponds
to the 5' nucleotide of the mRNA.
Biochemistry For Medics- Lecture Notes 12
 Primary transcript
• This is designated position +1, as is the
corresponding nucleotide in the DNA
• The numbers increase as the sequence
proceeds downstream.
• The nucleotide in the promoter adjacent to the
transcription initiation site is designated -1,
• These negative numbers increase as the sequence
proceeds upstream, away from the initiation site.
• This provides a conventional way of defining the
location of regulatory elements in the promoter.

Biochemistry For Medics- Lecture Notes 13

Transcription unit

Biochemistry For Medics- Lecture Notes 14

 Bacterial DNA-Dependent RNA
Polymerase
The DNA-dependent RNA
polymerase (RNAP) of the
bacterium Escherichia coli exists
as an approximately 400 kDa
core complex consisting of-
•two identical α subunits,
•similar but not identical β and β
' subunits, and
•an ω subunit and a
•A sigma subunit (σ)
•Beta is thought to be the
catalytic subunit.
Biochemistry For Medics- Lecture Notes 15
 Bacterial DNA-Dependent RNA
Polymerase
• RNAP, a metalloenzyme, also contains two zinc
molecules.
• The core RNA polymerase associates with a
specific protein factor (the sigma σ factor) that
helps the core enzyme recognize and bind to the
specific deoxynucleotide sequence of the
promoter region to form the preinitiation complex
(PIC)
• Bacteria contain multiple factors, each of
which acts as a regulatory protein.
Biochemistry For Medics- Lecture Notes 16
Mammalian DNA-Dependent RNA
Polymerases
Mammalian cells possess three distinct
nuclear DNA-Dependent RNA
Polymerases
• RNA polymerase I is for the synthesis of
r RNA
• RNA polymerase II is for the synthesis of
m RNA and miRNA
• RNA polymerase III is for the synthesis
of tRNA/5S rRNA, snRNA
Biochemistry For Medics- Lecture Notes 17
 Prokaryotic transcription
Steps of RNA Synthesis-
The process of transcription of a typical gene
of
E. Coli can be divided in to three phases-
i) Initiation
ii) Elongation
iii) Termination

Biochemistry For Medics- Lecture Notes 18

Overview of Prokaryotic DNA
Transcription

Biochemistry For Medics- Lecture Notes 19

 i) Initiation of Transcription
• Initiation of transcription involves the binding of the
RNA polymerase holoenzyme to the promoter region
on the DNA to form a preinitiation complex, or
PIC
• Characteristic "Consensus" nucleotide sequence of
the prokaryotic promoter region are highly
conserved.

Biochemistry For Medics- Lecture Notes 20

 Structure of bacterial prokaryotic
promoter region
Pribnow box
• This is a stretch of 6 nucleotides (
5'- TATAAT-3') centered about 8-
10 nucleotides to the left of the
transcription start site.
-35 Sequence
• A second consensus
nucleotide sequence ( 5'-
TTGACA-3'), is centered about
35 bases to the left of the
transcription start site.
Biochemistry For Medics- Lecture Notes 21
 i) Initiation of Transcription
(contd.)
• Binding of RNA-polymerase (RNAP) to the
promoter region is followed by a conformational
change of the RNAP, and the first nucleotide
(almost always a purine) then associates with the
initiation site on the subunit of the enzyme.
• In the presence of the appropriate
nucleotide, RNAP catalyzes the formation of a
phosphodiester bond, and the nascent chain is
now attached to the polymerization site on
the subunit of RNAP.

Biochemistry For Medics- Lecture Notes 22

 i) Initiation of Transcription
(contd.)
• In both prokaryotes and eukaryotes, a
purine ribonucleotide is usually the first to
be polymerized into the RNA molecule.
• After 10–20 nucleotides have been
polymerized, RNAP undergoes a second
conformational change leading to promoter
clearance.
• Once this transition occurs, RNAP
physically moves away from the promoter,
transcribing down the transcription unit,
leading to the next phase of the process,
elongation. Biochemistry For Medics- Lecture Notes 23
i) Initiation of Transcription
(contd.)

Biochemistry For Medics- Lecture Notes 24

 II) Elongation step of
Transcription
• As 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 template strand.
• The extent of this transcription bubble (i.e., DNA
unwinding) is constant throughout and is
about 20 base pairs per polymerase molecule.

Biochemistry For Medics- Lecture Notes 25

 II) Elongation step of
Transcription
• RNA polymerase has
associated with it an
"unwindase" activity that
opens the DNA helix.
• Topo isomerase both
precedes and follows the
progressing RNAP to
prevent the formation of
super helical
complexes.
• Base pairing rule is followed
Biochemistry For Medics- Lecture Notes 26
during the incorporation of
ribonucleotides
II) Elongation step of
Transcription

Biochemistry For Medics- Lecture Notes 27

 III) Termination of transcription

Termination of the synthesis of the RNA molecule

in bacteria is of two types-
a) Rho (ρ) dependent termination-
•The termination process is signaled by a
sequence in the template strand of the DNA
molecule—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
Biochemistry For Medics- Lecture Notes 28
complex.
Biochemistry For Medics- Lecture Notes 29
 III) Termination of transcription
(contd.)
b) Rho independent termination
• This process requires the presence of intrachain
self complementary sequences in the newly
formed primary transcript so that it can acquire a
stable hair pin turn that slows down the
progress of the RNA polymerase and causes it
to pause temporarily.
• Near the stem of the hairpin, a sequence occurs
that is rich in G and C.
• This stabilizes the secondary structure of the hair
pin.
Biochemistry For Medics- Lecture Notes 30
 III) Termination of transcription
(contd.)
•Beyond the hair
pin, the RNA
transcript contains
a strings of Us,
the bonding of Us
to the
corresponding As
is weak.
•This facilitates
the dissociation of
the primary
transcript from
DNA.
Biochemistry For Medics- Lecture Notes 31
 III) Termination of transcription
(contd.)

• After termination of synthesis of the RNA

molecule, the enzyme separates from the DNA
template.
• With the assistance of another factor, the core
enzyme then recognizes a promoter at which
the synthesis of a new RNA molecule
commences.

Biochemistry For Medics- Lecture Notes 32

 Eukaryotic transcription
• The general process of transcription can
be applied to both prokaryotic cells and
eukaryotic cells.
• The basic biochemistry for each is the
same; however, the specific mechanisms
and regulation of transcription differ
between prokaryotes and eukaryotes.
• Transcription of eukaryotic genes is far more
a complicated process than prokaryotes.

Biochemistry For Medics- Lecture Notes 33

 Prokaryotic versus Eukaryotic
Transcription
1) Location
• In prokaryotes (bacteria), transcription
occurs in the cytoplasm.
• Translation of the mRNA into proteins
also occurs in the cytoplasm

Biochemistry For Medics- Lecture Notes 34

 Prokaryotic versus Eukaryotic
Transcription
• In eukaryotes,
transcription occurs
in the cell's nucleus,
mRNA then moves to
the cytoplasm for
translation.

Biochemistry For Medics- Lecture Notes 35

 Prokaryotic versus Eukaryotic
Transcription
2) Genome size
• The genome size is much larger in
eukaryotes,
• Greater specificity is needed for
the transcription of eukaryotic
genes.

Biochemistry For Medics- Lecture Notes 36

 Prokaryotic versus Eukaryotic
Transcription
3) Chromatin Structure
• DNA in prokaryotes is much more
accessible to RNA polymerase
than DNA in eukaryotes.
• Eukaryotic DNA is wrapped
around proteins called histones to
form structures called nucleosomes
• Eukaryotic DNA is packed to
form chromatin .
• While RNA polymerase
interacts directly with
prokaryotic DNA, other
proteins mediate the interaction
between RNA 37
 Prokaryotic versus Eukaryotic
Transcription
4) RNA polymerases
• There are three distinct classes of RNA polymerases
in eukaryotic cells. All are large enzymes with
multiple subunits. Each class of RNA polymerase
recognizes particular types of genes.
• RNA polymerase I- Synthesizes the precursor of
the large ribosomal RNAs (28S, 18S and 5.8S).
• RNA polymerase II - Synthesizes the precursors of
messenger RNA and small nuclear
RNAs(snRNAs).
• RNA polymerase III- Synthesizes small
RNA, including t RNAs, small 5S RNA and
some snRNAs. 38
 Prokaryotic versus Eukaryotic
Transcription
5) Promoter regions
• Eukaryotic promoters are more complex.
• Two types of sequence elements are promoter-proximal
and distal regulatory elements.
• There are two elements in promoter proximal ,One of
these defines where transcription is to
commence along the DNA, and the other contributes
to the mechanisms that control how frequently this
event is to occur.
• Most mammalian genes have a TATA box that
is usually located 25–30 bp upstream from the
transcription start site.

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 Prokaryotic versus Eukaryotic
Transcription
• The consensus sequence for a TATA box is
TATAAA, though numerous variations have been
characterized.
• Sequences farther upstream from the start site determine
how frequently the transcription event occurs.
• Typical of these DNA elements are the GC and CAAT
boxes, so named because of the DNA sequences involved.
• Each of these boxes binds a specific protein.
• Distal regulatory elements enhance or decrease the rate of
transcription.
• They include the enhancer/ silencer regions and other
regulatory elements.

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Prokaryotic versus Eukaryotic
Transcription

Biochemistry For Medics- Lecture Notes 41

 Prokaryotic versus Eukaryotic
Transcription
6) Promoter identification
• In contrast to the situation in prokaryotes,
eukaryotic RNA polymerases alone are not able to
discriminate between promoter sequences and
other regions of DNA
• The TATA box is bound by 34 kDa TATA
binding protein (TBP), which in turn binds
several other proteins called TBP-associated
factors (TAFs).
• This complex of TBP and TAFs is referred to as
TFIID
Biochemistry For Medics- Lecture Notes 42
 Prokaryotic versus Eukaryotic
Transcription

• Formation of the basal transcription complex

begins when TFIID binds to the TATA box.
• It directs the assembly of several other components
by protein-DNA and protein-protein interactions. T
• The entire complex spans DNA from position -30 to
+30 relative to the initiation site.
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 Prokaryotic versus Eukaryotic
Transcription
• Binding of TFIID to the TATA box sequence
is thought to represent the first step in the
formation of the transcription complex on the
promoter.
• Another set of proteins—co activators—
help regulate the rate of transcription
initiation by interacting with transcription
activators that bind to upstream DNA
elements
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 Prokaryotic versus Eukaryotic
Transcription
7) Enhancers and Repressors
• A third class of sequence elements can
either increase or decrease the rate of
transcription initiation of eukaryotic genes
• These elements are called either enhancers
or repressors (or silencers), depending on
which effect they have.

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 Prokaryotic versus Eukaryotic
Transcription
• They have been found in a variety of locations both
upstream and downstream of the transcription start site
and even within the transcribed portions of some
genes.
• In contrast to proximal and upstream promoter
elements, enhancers and silencers can exert their
effects when located hundreds or even thousands of
bases away from transcription units located on the
same chromosome.
• Hormone response elements (for steroids, T3, retinoic
acid, peptides, etc) act as—or in conjunction with—
enhancers or silencers
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 Prokaryotic versus Eukaryotic
Transcription
7) Termination of transcription
• The signals for the termination of transcription
by eukaryotic RNA polymerase II are very poorly
understood.
8) Processing of primary transcript
• mRNA produced as a result of transcription is not
modified in prokaryotic cells. Eukaryotic cells
modify mRNA by RNA splicing, 5' end capping, and
addition of a polyA tail.
• Most eukaryotic RNAs are synthesized as precursors
that contain excess sequences which are removed
prior to the generation of mature, functional RNA.

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