TRANSCRIPTION

BY PROF DR SHAHEENA
 LEARNING OBJECTIVES
 Basic concept of Transcription
 Identify the key enzyme required for
transcription
 How transcription is controlled
 What is post transcriptional modification
FLOW OF GENETIC INFORMATION

• The genetic information flows from DNA

to RNA and then to the protein synthesizing
machinery.
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 Definition:
 It is the process of copying of genetic information
contained in DNA. in the form of RNA by a multi
enzyme system.
Features:
i. It is highly selective process.
ii. It results in the formation of three kind of RNA
i.e. mRNA, tRNA and rRNA.
iii.The mRNA formed with particular sequence of
N-Bases decided by genetic information on
DNA.
iv.By the combined work of mRNA, tRNA and
rRNA, a specific protein is synthesized with in
the cell.
 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;

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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.

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 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.
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 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.

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 TEMPLATE STRAND

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.

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SENSE STRANDS:-

It is the coding (non template) strand

of DNA. It has the same sequence of N-Base as the
RNA transcribed, except of thymine and sugar.

ANTI SENSE STRAND:

It is the template strand of DNA,
which is not utilized in the process of transcription.
PROMOTOR REGION:
It is the region of “nucleotide
Sequence” at the beginning of a stretch of DNA that is
to be transcribed.
1)PROKARYOTIC PROMOTOR
REGION:
i)Pribnow Box:-
It is a stretch of 6 or 7 nucleotide
situated about 10 nucleotide to left of initial base of
DNA strand coding for mRNA molecule.
 ii) T TGACA (-35 Sequence)
It is the second part of promoter
region situated 35 nucleotide to the left of initial base of
DNA strand coding for mRNA.
2.EUKARYOTIC PROMOTOR REG
i) TATA or Hogness Box
It is the stretch of nucleotide
bases situated 25 bases to the left of the initial base of
DNA strand coding for mRNA molecule.
 ii) CAAT Box:
It is situated about 70-80 nucleotide to
the left of the initial base of DNA strand coding for
mRNA.
Transcribed region;it is stretch of DNA that
is to be transcribed into RNA
TERMINATIOR
REGION:
It is the region of nucleotide
sequence at the terminal portion of the segment of DNA
that is to be transcribed. This region can be recognized
by RNA polymerase itself or by rho (p) factor .
 RNA POLYMERASE:-
RNA polymerase is a multi enzyme
system that can recognizes the promoter region at the
beginning of the length of DNA which is to be
transcribed. It then made complementary copy of DNA
template and finally recognizes the terminator region.

RNA is synthesized from its 5’end to 3’end anti

parallel to its DNA template.
 A transcription unit extends from the promoter to
terminator region and the product of process of
transcription by RNA polymerase is termed as “Primary
transcript.”
The RNA polymerase has following two parts.
 i. CORE ENZYME
The RNA polymerase core enzyme
consists of two alpha (2α), one beta (β), one beta
prime (β') and one omega (ω) and it is responsible for
5’ 3’ RNA polymerase activity.

σ- Unit CORE ENZYME

Holoenzyme

α α

β Β’
 ii.σ(Sigma) Subunit:
It enables the RNA to recognize the promoter region on
DNA.
The σ subunit plus core enzyme make up the
holoenzyme.
STEPS IN TRANSCRIPTION:
The process of synthesis of RNA (transcription) can be
described well in following steps:-

i. Binding of RNA polymerase to the DNA double

helix.
ii. Unwinding (of DNA template)
iii.Initiation and Elongation of RNA chain.
iv.Termination of RNA chain.
i) BINDING OF RNA POLYMERASE
WITH DNA HELIX

The process of RNA synthesis involve 1st the

binding of the RNA holopolymerase molecule to the
template at promoter site. Binding is carried out by
sigma factor with the promoter region.
 ii) UNWINDING OF DNA:
The unwinding of DNA molecule takes place
from promoter region by RNA polymerase. It is only
for the distance of about 15-17 base pairs. By
unwinding template become accessible for incoming
ribonucleosides triphosphates. It is a transition stage
from closed promoter complex to the open promoter
complex.
iii) INITIATION AND
ELONGATION OF RNA CHAIN
 The initiation of RNA synthesis takes place at the 5’
end of adenine or guanine triphosphate residue, and
primer is not needed.
 Nucleoside triphosphate is used and pyrophosphate
is released each time during the addition of
nucleotide.
 Direction of elongation is 5’ 3’antiparallel to
the DNA template.
 The localized melted region of DNA with RNA
polymerase and nascent RNA is termed as
“TRANSCRIPTION BUBBLES”.
 After the addition of About 10 nucleotide “sigma”
subunit is removed from the holoenzyme and
combined with the next core enzyme and repeat the
process.
The base sequence is complementary to that of the
primer RNA strand, with the following specification.

DNA STRAND RNA STRAND

G C
C G
T A
A U
iv) TERMINATION OF CHAIN
ELONGATION:
The termination of the process of
“Transcription” is signaled by a sequence in the
template strand of DNA molecule.

Termination can occur in following two ways:

 a)ρ-independent termination:
It is carried out by RNA polymerase (sometimes
only) by recognizing the terminator region on DNA. It
is achieved through:-
Formation of stable hair pin turn, which slows
down the progress of RNA polymerase.
The hair pin turn of the RNA is complementary
to a region of DNA template near terminator
region. (Plaindromes)
Near the base of the stem of hair pin turn, G and C
is richly found (this stabilizes the structure of hair
pin)
Following the hair pin turn there is string of Us.
 The bonding between U and A is weak, which help
separation of newly synthesized RNA from DNA
template.
 a)ρ- dependent Termination:
It is bring about by involvement of an addition
protein called ρ (rho) factor which has an RNA
dependent ATPase activity.
 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. 3
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 EUKARYOTIC TRANSCRIPTION
• In eukaryotes,
transcription occurs in
the cell's nucleus,
mRNA then moves to
the cytoplasm for
translation.

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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.
 THE PROMOTERS OF GENES TRANSCRIBED BY RNA
POLYMERASE II CONSIST OF A CORE PROMOTER AND A
REGULATORY PROMOTER

BRE DPE
Inr
(basal promoter)

or
Upstream promoter element

Not all the consensus sequences are found in all promoters

 Eukaryotic RNA polymerases alone are not able
to discriminate between promoter sequences and
other regions of DNA
 The TATA box is bound by 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
PREINITIATION COMPLEX
TFIIA

General Transcription Factors TFIID

TFIIB

TFIIE

TFIIF

preinitiation complex,
Combine with RNA polymerase II to form a TFIIH
which is competent to initiate transcription as soon as
nucleotides are available

TFIID is a complex protein containng a TATA-box-binding protein (TBP)

and a 8-10 TBP-associated factors (TAFs)

TBP is a universal transcriptional factor required by all three classes of genes

 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 4relative to
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the initiation site.
ASSEMBLY OF THE
RNA POLYMERASE
II PRE-INITIATION
COMPLEX

The first step in assembly of the pre-initiation complex

is recognition of the TATA box and possibly the
Inr sequence by the TATA-binding protein (TBP),
in conjunction with the TBP-associated factors (TAFs).

Y-S-P-T-S-P-S
26x up to 52x

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II,
must be phosphorylated before the polymerase can leave the promoter, and start transcription
 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
 FUNCTIONS OF HUMAN GTFS
(GENERAL TRANSCRIPTION FACTORS)
GTF Function

TFIID (TBP Recognition of the TATA box and possibly Inr sequence; forms a
component) platform for TFIIB binding

TFIID (TAFs) Recognition of the core promoter; regulation of TBP binding

TFIIA Stabilizes TBP and TAF binding

TFIIB Intermediate in recruitment of RNA polymerase II; influences

selection of the start point for transcription

TFIIF Recruitment of RNA polymerase II

TFIIE Intermediate in recruitment of TFIIH; modulates the various activities

of TFIIH
TFIIH Helicase activity responsible for the transition from the closed to
open promoter complex; possibly influences promoter clearance
by phosphorylation of the C-terminal domain (cinase activity)
of the largest subunit of RNA polymerase II

GTF- dictate the starting point and direction of transcription (low level of transcription)
 EUKARYOTIC TRANSCRIPTION
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.
 EUKARYOTIC TRANSCRIPTION
Termination of transcription
• The signals for the termination of transcription by eukaryotic
RNA polymerase II are very poorly understood.
 Antibiotics can inhibit protein synthesis by targeting
either the 30S subunit, examples of which include
spectinomycin, tetracycline, and the aminoglycosides
kanamycin and streptomycin, or to the 50S subunit,
examples of which include clindamycin,chloramphenicol,
linezolid, and the macrolides erythromycin
 Rifampicin binds to the exit channel of the RNA
polymerase by making specific contacts that involve the
β-subunit of RNA polymerase. Rifampicin blocks the exit
channel thereby inhibiting transcription
 POST TRNASCRIPTION
MODIFICATION (OF RNA)
 The newly synthesis RNA is called as “primary
transcript.” It is a linear copy of transcriptional unit.
 Primary tRNA and rRNA of both prokaryotes and
Eukaryotes are post transcriptionally modified (By
ribonuclease)
 Prokaryotic mRNA is generally identical to its
primary transcript.
 Eukaryotic mRNA is extensively modified after the
synthesis.
 1)RIBOSOMAL RNA:

 Newly synthesized RNA of both prokaryotes and

eukaryotes is called preribosomal RNA (45S)
 The long pre rRNA of prokaryotes is cleaved into
23S, 16S and 5S ribosomal RNA by ribonuclease.
 Similarly by the same enzyme eukaryotic pre rRNA is
cleaved into 28S, 18S and 5.8S ribosomal RNAs.
 The 5S ribosomal RNA of eukaryote is synthesized by
RNA polymerase III and is modified separately.
 RIBOSOMAL RNA GENE

DNA

RNA POLYMERASE

28S 5.8S 18S 23S 5S 16S

RIBONUCLEASE RIBONUCLEASE

28S 5.8S 18S 23S 5S 16S

EUKARYOTES PROKARYOTES
 2) TRANSFER RNA

 Both eukaryotic and prokaryotic RNAs are trimmed

(shortened) after transcription.
 Addition of -CCA sequence at 3’ terminal by
nucleotidyl transferase.
 Modification of specific bases also takes place to
produce unusual bases like pseudouracil or N4 Acetyl
cytosine etc.
3) MESSENGER RNA
(EUKARYOTIC ONLY)
 The primary transcript of mRNA is called as
heterogeneous nuclear RNA (hn RNA)
 It is synthesized by RNA polymerase II
 It undergoes following modification.
 a) 5’ “Capping”:
 it is the 1st modification takes place on hn RNA.
 It is the addition of 7-methylguanosine on 5’ end of
mRNA through triphosphate linkage.
 This addition carried out by nuclear enzyme called
guanine-7-methyl transferase.
 Methylation of guanine takes place in cytosol by
guanine-7- methyl transferase.
S-adenosyl methionine is the source of methyl group

 The cap is added through 5’ 5’ triphosphate

linkage.
 By capping :
i) mRNA is stabilized.
ii) mRNA is easily
translated
 b) Addition of poly-A tail:-
 Most of the eukaryotic mRNA have a chain of 40-250
adenine nucleotide at 3’ end.
 This poly A tail is not the result of transcription from
DNA.
 This addition is carried out by nuclear enzyme poly-A
polymerase.
i) Stablization of mRNA
ii) Their exit from Nucleus.
 After entering into the cytosol, the poly A tail of
mRNA is gradually shortened
Removal of introns:
 Intron is the region of RNA which do not code for
protein synthesis.
 Exon is the region actually coding.
 For maturation of mRNA, removal of intron is
necessary.
 Small nuclear RNAs (Sn RNAs) in association with
special proteins called small nuclear
ribonucleoprotiens (Sn RNPs) facilitates the splicing
of some Exon segments by forming base pairs with
each end of the introns.
 Introns are removed from the primary transcript in the
nucleus, exons (coding sequences) are ligated to form
the mRNA molecule
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SPLICING OF M-RNA
The steps of splicing are as follows-
• i) Role of small nuclear RNA (sn RNA)  and
Spliceosome
• The molecular machine that accomplishes the task of
splicing is known as the spliceosome. Spliceosomes
consist of the primary transcript, five small nuclear RNAs
(U1, U2, U4, U5, and U6) and more than 60 proteins.
• Collectively, these form a small ribonucleoprotein
(snRNP) complex, sometimes called a "snurp"
(snRNPs)

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SPLICING OF M-RNA
 Snurps are thought to position the RNA segments
for the necessary splicing reactions.
 These facilitate the splicing of exon segments by
forming base pairs with the consensus sequence
at each end of the intron.

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SPLICING OF M-RNA

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SPLICING OF M-RNA
• The newly- feed 3'OH of the
upstream exon 1 then forms a
phosphodiester bond with the
5'end of the downstream exon 2.
• The excised intron is released as
a "lariat" structure, which is
degraded
• After removal of all the introns,
the mature m RNA molecules
leave the nucleus by passing in
to the cytosol through pores in
to the nuclear membrane.
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