Unit 1 transcription

By – Dr. Mafatlal M. Kher
Unit: 1 Transcription

DNA
Transcription
RNA
Translation
PROTEIN
Central
Dogma
of
Life
2

Transcription
Synthesis of
RNA from DNA
3

Why DNA Transcription is
important?
Because the information that is contained
in DNA can not be transferred without
transcription.
4

Functions of Different Types of RNA
RNA Type Size Function
Messenger RNA (5%) Variable in size Informational RNA: Carries genetic information
provided by DNA
Transfer RNA (15%) Small Adapter/Soluble: Transport amino acids to site of
protein synthesis.
Ribosomal RNA (80%) Variable in size Catalytic: Combines with proteins to form
ribosomes, the site of protein synthesis.
Small Nuclear RNA Small Process initial mRNA to its mature form in
eukaryotes.
Small interfering RNA Small Affects gene expression; used by scientist to knock
out a gene to be studied.
Micro RNA Small Affects gene expression; important for growth and
development.
5

1866
1909
1911
1941
1949
1965
GJ Mendel
A unit factor that controls specific
phenotypic trait
Wilhelm Johannsen
Coining the terms gene, phenotype and genotype
Yanofsky
One gene (cistron) – one polypeptide
hypothesis
TH Morgan
Gene theory is the idea that genes are the
basic units in which characteristics are passed
from one generation to the next
George Beadle and Edward Tatum
The one gene–one enzyme hypothesis is the idea
that genes act through the production of
enzymes, with each gene responsible for
producing a single enzyme that in turn affects a
single step in a metabolic pathway.
Pauling and Ingram
Established the role of genes in protein
synthesis
The concept of Gene
8

 In a typical nucleus, some region of chromatin are loosely packed (and
stains light) and referred to as “euchrmoatin”.
 The chromatin that is more densely packed and stains dark is called
heterochromatin, specifically euchromatin is said to be
transcriptionally active and heterochromatin is transcriptionally
inactive.
Transcription at chromosome level
9

 A gene is defined as the functional unit of inheritance.
 Cistron is defined as functional unit of gene, it is a segment of RNA
coding for a polypeptide.
 The structural gene in a transcription unit is monocistronic (mostly
in eukaryotes) and polycistronic mostly in prokaryotes or in bacteria.
 Monocistronic gene synthesises one type of polypeptide or protein.
Polycistronic gene synthesises different types of polypeptides or
proteins.
The concept of Gene
10

Exon
Intron
Cistron
DNA v/s RNA v/s Genetic Code
11

1. The term “Exon” is derived from expressed region
Walter Gilbert coined the term.
2. Both are related to gene.
3. Found in Prokaryotes and Eukaryotes
4. These are DNA bases, which translated into mRNA.
5. Exons are code of proteins (Coding).
6. Exons are very much conserved which means that
their sequence does not change rapidly over time or
in-between the species.
1. The term “Intron” is derived from intragenic region,
a region inside a gene. It also referred as intervening
sequences Walter Gilbert coined the term.
2. Both are related to gene.
3. Only present in Eukaryotes and Archaea.
4. These are also DNA bases, found in between exon.
5. Not at all implicated with protein coding
(Noncoding). Therefore it is also referred as “Junk
DNA”: In reality, we don't entirely understand how
intron sections work. They are probably composed of
old code, sections of DNA that are no longer used.
6. Introns are less conserved which means that their
sequence changes very frequently over time.
Exons v/s Introns
Exon Intron
12

 Bacteria don’t have introns to maximise the capacity to store genetic
information in a confined space that is their tiny cell.
 Mitochondria and Chloroplast are thought to be a symbiotic bacteria in
Eukaryotic cells, and hence their DNA don’t contain introns.
Why introns are absent in Prokaryotes/Bacteria?
13

 Introns help create variation in the mRNA molecules produced from a
gene and thus the resulting proteins.
 Non-coding RNA may get produced from introns.
 Introns may have once encoded proteins but these functions were lost
over the course of evolution.
Importance of introns in Eukaryotes?
14

 These are coding region of DNA, i.e. an exon
has several cistron.
 These appear in mature or processed RNA.
 It was discovered by Richard Roberts.
 It is the DNA segment that directs the synthesis
of a peptide sequence.
 It is an alternative term for gene.
 Cistron was discovered by Seymour Benzer.
 A cistron is an alternative term for "gene". The
term cistron arises from the identification of
gene function in a cis-trans test; distinct
positions (or loci) within a genome are cistronic.
 Cistron (or gene) is a segment of DNA
consisting of a stretch of base sequences
those codes for one polypeptide, one
transfer RNA (tRNA) molecule. or one
ribosomal RNA (rRNA).
Exon, Cistron/Gene & Genetic code
Exon/ Coding portion Cistron/ Gene Genetic Code
 The genetic code is a dictionary that gives
the correspondence between a sequence
of nucleotide bases & a sequence of
amino acids.
 Each individual word in the code is
composed of 3 nucleotide bases. These
genetic words are called codons.
 The codons are usually presented in the
messenger RNA language of adenine (A),
guanine (G), cytosine (C), & uracil (U).
Four nucleotide bases are used to
produce the three base codons. Therefore
there are 64 different combinations
possible. Nucleotide sequence is always
read from 5'end to the 3'end.
 For example, 5'-CAU-3' codes for
histidine, 5'-AUG-3' codes for methionine.
There are stop codons - UAG, UGA, &
UAA - dont code for any amino acids but
rather are termination codons.
 Usage of genetic code is rather consistent
throughout all living organisms
15

 Poly means “many, ” and cistron means “genes.” An mRNA is said as polycistronic
mRNA when it codes for two or many proteins; it contains more than one genes
codes on it.
 Polycistronic mRNA contains many codons of cistrons.
 When it is transcribed in the cells, it has many codons that initiate this process and
many codons that terminate them.
 The coding region that initiates the translation mostly consists of a linear structure or
sequence of codons.
 These codons make it polyfunctional for the cell.
 Moreover, these RNA have multiple ORFs (open reading frames) each of which
correspond to the single gene transcript.
 These codons or ORFs are then translated into a polypeptide according to the code.
 This mRNA is mostly present in the prokaryotes like bacteria etc.
 Many prokaryotic RNAs are completely functional and do not require any changes
(post-transcriptional changes).
What is Polycistronic mRNA?
16

 Mono means “one” and cistron means “genes.”
 An mRNA is said as monocistronic mRNA when it codes for only single
proteins; it contains only one genes code on it.
 When it is transcribed in the cell, it has only one codon that initiates this
process and one codon that terminates them.
 Moreover, these RNA has single ORF (open reading frame) each of which
corresponds to a single or specific gene transcript.
 These codons or ORFs are then translated into a polypeptide according to the
code.
 Monocistronic mRNA is present in eukaryotes like human cells.
 Many eukaryotic RNAs are non-functional and require many kinds of changes
(post-transcriptional changes). These changes may be like splicing, splicing,
removal of introns, etc.
What is monocistronic mRNA?
17

Gene
Type Polycistronic mRNA Monocistronic mRNA
Messenger Polycistronic mRNA is that messenger
RNA which encodes for two or more
proteins.
Monocistronic mRNA is that messenger
RNA which encodes for only one or
specific protein or polypeptide.
Codons Polycistronic mRNA contains many
codons of cistrons.
Monocistronic mRNA contains single
codon of cistron.
ORF Polycistronic mRNA have multiple ORFs
(open reading frames).
Monocistronic mRNA have single ORF
(open reading frame).
Available in Polycistronic mRNA is present mostly in
prokaryotes like bacteria etc.
Monocistronic mRNA is present in
eukaryotes like human, plants
Post-
transcriptional
changes
Polycistronic mRNA do not require post-
transcriptional changes.
Monocistronic mRNA requires post-
transcriptional changes.
18

 In molecular genetics, an open reading frame (ORF) is the part of a
reading frame that has the ability to be translated.
 An ORF is a continuous stretch of codons that begins with a start codon
(usually AUG) and ends at a stop codon (usually UAA, UAG or UGA).
 The transcription termination site is located after the ORF, beyond the
translation stop codon.
 If transcription were to cease before the stop codon, an incomplete
protein would be made during translation.
 In eukaryotic genes with multiple exons, introns are removed and exons
are then joined together after transcription to yield the final mRNA for
protein translation.
Open reading frame
19

Down stream
Upstream
Promoter
ORF/ RNA-Coding region
DNA
5’
3’
3’
5’
• Template strand
• Antisense
• Minus
• Anticoding
• Noncoding
• Transcribed
• Non template
strand
• Coding
• Sense
• Plus
Transcription start site Transcription termination site
Terminator
5’ 3’
RNA Transcript
Transcription Unit
Structural Gene
21

Down stream
Upstream
Promoter RNA-Coding region
DNA
5’
3’
3’
5’
• Template strand
• Antisense
• Minus
• Anticoding
• Noncoding
• Transcribed
• Non template
strand
• Coding
• Sense
• Plus
Transcription start site Transcription termination site
Terminator
5’ 3’
RNA Transcript
Promoter
Structural Gene
22

 Non-coding DNA sequence
 Cis-regulatory elements (CREs) are regions of non-coding DNA which
regulate the transcription of neighbouring genes. The Latin prefix cis means
"on this side", i.e. on the same molecule of DNA as the gene(s) to be
transcribed.
 CREs typically regulate gene transcription by binding to transcription factors.
 A single transcription factor may bind to many CREs, and hence control the
expression of many genes (pleiotropy).
 CREs are vital components of genetic regulatory networks, which in turn
control morphogenesis, the development of anatomy, and other aspects of
embryonic development, studied in evolutionary developmental biology.
Promoter
23

Cis-regulatory element:
 Promoters: Promoters are CREs consisting of relatively short sequences of DNA which include
the site where transcription is initiated. In eukaryotes, promoters usually have the following four
components: the TATA box, a TFIIB recognition site, an initiator, and the downstream core
promoter element. It has been found that a single gene can contain multiple promoter sites. In
order to initiate transcription of the downstream gene, a host of DNA-binding proteins called
transcription factors (TFs) must bind sequentially to this region. Only once this region has been
bound with the appropriate set of TFs, and in the proper order, can RNA polymerase bind and
begin transcribing the gene.
 Enhancers: Enhancers are CREs that influence (enhance) the transcription of genes on the same
molecule of DNA and can be found upstream, downstream, within the introns, or even relatively far
away from the gene they regulate. Multiple enhancers can act in a coordinated fashion to regulate
transcription of one gene.
 Silencers: Silencers are CREs that can bind transcription regulation factors (proteins) called
repressors, thereby preventing transcription of a gene. The term "silencer" can also refer to a region in
the 3' untranslated region of messenger RNA, that binds proteins which suppress translation of that
mRNA molecule, but this usage is distinct from its use in describing a CRE.
 Operators: Operators are CREs especially in prokaryotes that exist within operons.
Knowledge Bank
24

Trans-regulatory element:
 Trans-regulatory elements are genes which may modify (or regulate) the expression of distant
genes.
 More specifically, trans-regulatory elements are DNA sequences that encode trans-acting factors
(often proteins such as transcription factors).
 Trans-regulatory elements work through an intermolecular interaction between two different
molecules and so are said to be "acting in trans". For example (1) a transcribed and translated
transcription factor protein derived from the trans-regulatory element; and a (2) DNA regulatory
element that is adjacent to the regulated gene. This is in contrast to cis-regulatory elements that
work through an intramolecular interaction between different parts of the same molecule: (1) a
gene; and (2) an adjacent regulatory element for that gene in the same DNA molecule.
 Examples of trans-acting factors include the genes for:
 Subunits of RNA polymerase
 Proteins that bind to RNA polymerase to stabilize the initiation complex
 Proteins that bind to all promoters of specific sequences, but not to RNA polymerase (TFIID
factors)
 Proteins that bind to a few promoters and are required for transcription initiation (positive
regulators of gene expression)
Knowledge Bank
25

+1
DNA
5’
3’
3’
5’
Pribnow box (TATAAT or -10
sequence): The first place where
base pairs separate during
prokaryotic transcription to allow
access to the template strand. The
AT-richness is important to allow
this separation, since adenine and
thymine are easier to break apart
(not only due to fewer hydrogen
bonds, but also due to weaker base
stacking effects (AT: Double bond)
Upstream Downstream
From the site of transcription
Transcription start site
Prokaryotic: Promoter
TATAAT -10
Prokaryotic RNA Polymerase (Holo
enzyme)
TTGACG -35
AACTGC -35 ATATTA -10
Pribnow Box
σ Factor
26

RNA Polymerase I or II or III or IV or V
+1
DNA
5’
3’
3’
5’
TATA box (also called the Goldberg-Hogness box: The first place where base pairs separate during eukaryotic transcription
to allow access to the template strand. The AT-richness is important to allow this separation, since adenine and thymine are
easier to break apart (not only due to fewer hydrogen bonds, but also due to weaker base stacking effects (AT: Double bond).
Transcription is initiated at the TATA box in TATA-containing genes. The TATA box is the binding site of the TATA-binding protein
(TBP) and other transcription factors in some eukaryotic genes. Gene transcription by RNA polymerase II depends on the
regulation of the core promoter by long-range regulatory elements such as enhancers and silencers. Without proper regulation
of transcription, eukaryotic organisms would not be able to properly respond to their environment.
Transcription start site
Downstream
From the site of transcription
Eukaryotic: Promoter
W can be A or T
GGGCGG -110
CCCGCC -110
GC Box
27
GCCCAATCT 60-100
CGGGTTAGA 60-100
CAT/ CAAT Box
TATAWAW -25--35
ATATWTW -25--35
TATA Box
Upstream
Transcription
factor

 GC box, also known as a GSG box.
 Distinct pattern of nucleotides found in the
promoter region of some eukaryotic genes.
 The GC elements are bound by transcription
factors and have similar functions to
enhancers.
 The GC box is commonly the binding site for
Zinc finger proteins.
 CCAAT box (also sometimes abbreviated a
CAAT box or CAT box) is a distinct pattern of
nucleotides with GGCCAATCT consensus
sequence.
 The CAAT box signals the binding site for the
RNA transcription factor, and is typically
accompanied by a conserved consensus
sequence. It is an invariant DNA sequence at
about minus 70 base pairs from the origin of
transcription in many eukaryotic promoters.
 Belong to the regulatory promoter and core
promoter.
 Protein specific binding is required for the
CCAAT box activation. These proteins are
known as CCAAT box binding proteins/CCAAT
box binding factors.
Eukaryotic promoter region
GC BOX CAAT BOX TATA BOX
 TATA box (also called the Goldberg-
Hogness box: The TATA box is the binding
site of the TATA-binding protein (TBP) and
other transcription factors in some
eukaryotic genes. The first place where
base pairs separate during eukaryotic
transcription to allow access to the
template strand.
 The AT-richness is important to allow this
separation, since adenine and thymine
are easier to break apart (not only due to
fewer hydrogen bonds, but also due to
weaker base stacking effects (AT: Double
bond).
 Based on the sequence and mechanism
of TATA box initiation, mutations such as
insertions, deletions, and point mutations
can result in phenotypic changes. Which
can then turn into a disease phenotype
like: gastric cancer, Huntington's disease,
β-thalassemia, Gilbert's syndrome, and
HIV-1. The TATA-binding protein (TBP)
could also be targeted by viruses as a
means of viral transcription
A zinc finger is a small protein structural motif
that is characterized by the coordination of one
or more zinc ions (Zn2+) in order to stabilize
the fold.
• Various protein engineering techniques can
be used to alter the DNA-binding specificity
of zinc fingers and tandem repeats of such
engineered zinc fingers can be used to
target desired genomic DNA sequences
• Zinc finger nucleases: Engineered zinc
finger arrays are often fused to a DNA
cleavage domain (usually the cleavage
domain of FokI) to generate zinc finger
nucleases. Such zinc finger-FokI fusions
have become useful reagents for
manipulating genomes of many organisms 28

AUG UAG
5’
3’
3’
5’
5’ 3’
Start
codon
Stop codon
UAA UGA
ATG TAG TAA TGA
TAC ATC ATT ACT
DNA
Codon mRNA
Non template strand
Template strand
Transcription termination
Transcription
Initiation
Promoter Other sequence
Other sequence
Other sequence
Transcription: Start codon and stop codon
Methionine
Start and stop codons are important because they tell the cell machinery where to
begin and end translation, the process of making a protein.
UAC
5’
Anticodon tRNA
Amino acid
30

Knowledge Bank: Promoter
1. Eukaryotic promoters are the regulatory sequences that
initiate the transcription of eukaryotic organisms.
2. Eukaryotic promoter consists of (TATA box), CAAT box, GC
box and initiator elements.
3. Core promoter: A binding site for RNA polymerase
1. RNA Polymerase I: 18S, 5.8S and 28S ribosomal RNAs
2. RNA Polymerase II: mRNA; small nuclear RNAs and
microRNA
3. RNA Polymerase III (Eukaryotes): tRNA; 5s ribosomal RNAs
and other small RNAs
4. Proximal promoter - the proximal sequence upstream of
the gene that tends to contain primary regulatory elements
Approximately -250 Specific transcription factor binding
sites
5. Distal promoter – the distal sequence upstream of the
gene that may contain additional regulatory elements, often
with a weaker influence than the proximal promoter
Anything further upstream (but not an enhancer or other
regulatory region whose influence is positional/orientation
independent) Specific transcription factor binding sites.
1. Prokaryotic promoters are the regulatory sequences that
initiates the transcription of prokaryotic genes.
2. Prokaryotic promoter consists of upstream elements, -10
element (Pribnow box) and -35 elements.
3. Core promoter: Binding site for RNA Polymerase
holoenzyme.
4. RNA Polymerase holoenzyme: consists of five subunits:
1. Two alpha (α) subunits of 36 kDa,
2. One beta (β) subunit of 150 kDa,
3. One beta prime subunit (β′) of 155 kDa,
4. A small omega (ω) subunit.
A sigma (σ) factor binds to the core, forming the holoenzyme. After
transcription starts, the factor can unbind and let the core enzyme
proceed with its work.
The core RNA polymerase complex forms a "crab claw" or "clamp-
jaw" structure with an internal channel running along the full length.
Eukaryotic Prokaryotic
31

 RNA polymerases (RNAP) have been found in all species, but the number and composition of
these proteins vary across taxa. For instance, bacteria contain a single type of RNA polymerase,
while eukaryotes (multicellular organisms and yeasts) contain three to five distinct types. In spite
of these differences, there are striking similarities among transcriptional mechanisms.
 Using the enzyme helicase, RNA Polymerase locally opens the double-stranded DNA so that one
strand of the exposed nucleotides can be used as a template for the synthesis of RNA, a process
called transcription.
 A transcription factor and its associated transcription mediator complex must be attached to a
DNA binding site called a promoter region before RNAP can initiate the DNA unwinding at that
position.
 RNAP not only initiates RNA transcription, it also guides the nucleotides into position, facilitates
attachment and elongation, has intrinsic proofreading and replacement capabilities, and
termination recognition capability.
 In eukaryotes, RNAP can build chains as long as 2.4 million nucleotides.
RNA polymerase (RNAP): Some important facts
33

 In E. coli, as with other prokaryotes, there is only one true RNA polymerase (not including the specialty RNA
polymerase, primase, which makes short RNA primers for DNA replication).
 The polymerase is a multi-subunit holoenzyme comprised primarily of two α subunits, a β subunit, a β’
subunit, an ω subunit, and a σ subunit.
 The α subunits are primarily structural, assembling the holoenzyme and associated regulatory factors.
 The β subunit contains the polymerase activity that catalyzes the synthesis of RNA, while the β’ subunit is used
to nonspecifically bind to DNA.
 The ω subunit is involved in assembly of the holoenzyme and may also play a role in maintaining the structural
integrity of the RNA polymerase.
 Finally, there is the σ subunit, which does not stay closely associated with the core enzyme (αββ’ω) except when
helping to initiate transcription, and is used to recognize the promoter by simultaneously decreasing the affinity of
RNAP to DNA in general, but increasing the affinity of RNAP for specific DNA promoter sequences.
 Why decrease the affinity for non-specific DNA? When the RNAP is not in use, it does not just float about in
the nucleoplasm: it is bound quite tightly along the DNA. When the sigma is bound, the decreased affinity allows
the RNAP holoenzyme to move along the DNA and scan for promoter sequences.
 There are multiple isoforms of the σ subunit (such as the sigma-70), each of which recognizes different
promoter sequences. All isoforms perform the same basic function of properly locating the RNAP to the start of a
gene, and all isoforms only stay attached to the holoenzyme for that one transient purpose, after which they are
released (usually after transcribing about ten nucleotides).
RNA polymerase: Prokaryotic
34

RNA Polymerase (E.coli)
35

β
β'
ω
σ
αI
αII
β
β'
ω
σ
αI
αII
+ =
Core Enzyme
σ factor
Holoenzyme
36

β
β'
ω
σ
αI
αII
The σ subunit: Recognition of
the promoter region. Sigma
reduces the affinity of RNAP for
nonspecific DNA while
increasing specificity for
promoters, allowing transcription
to initiate at correct sites.
The β subunit is the second-largest subunit,
Chain initiation and elongation, The β
subunit contains the rest of the active center
responsible for RNA synthesis. Catalysis of
the polymerisation process.
The β' subunit is the largest subunit,
DNA Binding, The β' subunit contains
part of the active center responsible for
RNA synthesis and contains some of the
determinants for non-sequence-specific
interactions with DNA and nascent RNA.
It binds and open the DNA template. It
is split into two subunits in Cyanobacteria
and chloroplasts
The α subunit is the third-largest
subunit and is present in two copies
per molecule. Determination of
DNA to be transcribed
The ω subunit facilitates assembly of
RNAP and stabilizes assembled
RNAP, Restore denatured RNA P to
its functional form 37

RNA Polymerase
binding to promoter
Transcription
initiation
38

RNA Polymerase & their
location
RNA Type Function of RNA
RNA Pol I
(Nucleolus)
Ribosomal RNA (80%) Catalytic: Combines with proteins to form ribosomes, the
site of protein synthesis.
RNA Pol II
(Nucleoplasm)
Messenger RNA (5%) Informational RNA: Carries genetic information provided
by DNA
RNA Pol III
(Nucleoplasm)
Transfer RNA (15%) Adapter/Soluble: Transport amino acids to site of protein
synthesis.
RNA Pol IV
(Nucleoplasm)
si RNA in Plants A class of double-stranded RNA non-coding RNA
molecules, work as operator (post transcriptional gene
silencing).
RNA Pol V
(Nucleoplasm)
Si RNA in plants Pol V is involved in siRNA-directed DNA methylation
pathway which leads to heterochromatic silencing.
39
RNA polymerase: Eukaryotic

 Transcription is the process by which a strand of DNA is copied (transcribed) to mRNA, which
carries the information needed for protein synthesis; only one of the two DNA strands is
transcribed into an RNA.
 The enzyme RNA polymerase catalyzes the process of transcription The enzyme is known as
DNA-dependent RNA polymerase.
 RNA chains contain nucleotides with the base uracil instead of thymine and that uracil pairs
with adenine.
 Regulatory proteins determine whether a particular gene is available to be transcribed by RNA
polymerase
 In double-stranded DNA, the strand to be copied is known as the coding strand. The other
strand, which contains the complementary base sequence, is the template strand that will be
used to form the RNA transcription.
Transcription: Synthesis RNA from DNA.
41

Elongation
Transcription
Key steps:
42

43
RNA polymerase uses ribonuceloside
triphosphate (rNTP) to synthesize
mRNA strands (rATP, rUTP, rCTP, and
rGTP) in the 5'->3' direction.

Why only one strand of DNA is copied during transcription??
First, if both strands act as a template:
They would code for RNA molecule with different sequences (Remember
complementarity does not mean identical), and in turn, if they code for proteins, the
sequence of amino acids in the proteins would be different.
Hence, one segment of the DNA would be coding for two different proteins, and this
would complicate the genetic information transfer machinery.
Second, if both strands act as a template:
 The two RNA molecules produced simultaneously would be complementary to each
other.
 Hence would form a double stranded RNA. This would prevent RNA from being
translated into protein and the exercise of transcription would become a useless.
45

 RNA polymerase binds to promoter region of the DNA and the process of transcription begins.
 RNA polymerase moves along DNA helix and unwinds it.
 One of the two strands of DNA serves as a template for RNA synthesis.
 This results in the formation of complementary RNA strand (It is formed at a rate of about 40 to 50
nucleotides per second).
 RNA synthesis comes to a stop when RNA polymerase reaches the terminator sequence.
 The transcription enzyme, i.e., RNA polymerase is only of one type in prokaryotes and can
transcribe all types of RNAs.
 RNA polymerase is a holoenzyme that is represented as (α2ββ’ω)σ
 The enzyme without σ subunit is referred to as core enzyme.
 The sigma "factor" is usually released when the RNA chain reaches 8-9 bases.
Transcription: Mechanism (Prokaryotes)
47

 During initiation, RNA polymerase recognizes a specific site on the DNA,
upstream from the gene that will be transcribed, called a promoter site
and then unwinds the DNA locally.
 A promoter is a DNA sequence onto which the transcription machinery
binds and initiates transcription.
 First, the holoenzyme contacts the-35 sequence and then binds to the
full promoter.
 Once this interaction is made, the subunits of the core enzyme bind to
the site. DNA is still in standard double helix form, a state called the
closed promoter complex.
 The A–T-rich -10 region facilitates unwinding of the DNA template;
several phosphodiester bonds are made. The untwisted form of the
promoter is called the open promoter complex.
 When initiation succeeds, sigma is no longer necessary, and the enzyme
makes the transition to the elongation ternary complex of core
polymerase-DNA-nascent RNA.
Prokaryotic transcription: Initiation
48

 Once initiation succeeds and the elongation stage is established
 The RNA polymerase begins to move along the DNA and the sigma factor is
released
 The core enzyme alone is able to complete the transcription of the gene
 The RNA polymerase contacts about 40 bp of the DNA with approximately 25
bp in the transcription bubble.
 Within the untwisted region, about 9 RNA nucleotides are base paired to the
DNA in a temporary RNA–DNA hybrid; the rest of the newly synthesized RNA
exits the enzyme as a single strand
 RNA polymerase has two proofreading activities :
i) Pyrophosphorlytic editing – steps back and removes incorrect nucleotides
ii) Hydrolytic editing – Backtracks to cleave error containing sequence
Prokaryotic transcription: Elongation
49

Prokaryotic transcription: Elongation
50

 Once a gene is transcribed, the prokaryotic polymerase needs to be instructed
to dissociate from the DNA template and liberate the newly made mRNA.
 Depending on the gene being transcribed, there are two kinds of termination
signals.
 One is protein-based (Rho) and the other is RNA-based (Rho independent).
Prokaryotic transcription: Termination
51

 It is controlled by the rho protein, which tracks along behind the polymerase on
the growing mRNA chain.
 Rho-dependent terminators are C-rich, G-poor sequences.
 Rho attaches to recognise site on RNA.
 Rho binds to the C-rich terminator sequence present upstream of transcription
termination site.
 Rho then moves along the transcript until it reaches the RNA polymerase i.e
RNA-DNA hybrid Rho has a property of helicase.
 Rho unwinds DNA-RNA hybrid in transcription bubble. It uses energy from ATP
hydrolysis for this process.
 The new RNA strand is then released, the DNA double helix reforms RNA pol
and Rho dissociates from DNA
 Termination: RNA polymerase, Rho, and RNA are released.
Prokaryotic transcription: Rho-dependent termination
52

 It is controlled by specific sequences in the DNA template strand.
 As the polymerase nears the end of the gene being transcribed, it encounters a
region rich in C–G nucleotides.
 The mRNA folds back on itself, and the complementary C–G nucleotides bind
together.
 The result is a stable hairpin that causes the polymerase to stall as soon as it
begins to transcribe a region rich in A–T nucleotides.
 The complementary U–A region of the mRNA transcript forms only a weak
interaction with the template DNA.
 This, coupled with the stalled polymerase, induces enough instability for the
core enzyme to break away and liberate the new mRNA transcript.
Prokaryotic transcription: Rho-independent termination
53

Eukaryotic
Transcription
 Eukaryotic transcription is carried out in the nucleus of the cell and proceeds in three
sequential stages: initiation, elongation, and termination.
 Eukaryotes require transcription factors to first bind to the promoter region and then help
recruit the appropriate polymerase.
 RNA Polymerase II is the polymerase responsible for transcribing mRNA.
55

Euchromatin and Heterochromatin
56
 In a typical nucleus, some region of
chromatin are loosely packed (and stains
light) and referred to as “euchrmoatin”.
Euchromatin is said to be transcriptionally
active.
 The chromatin that is more densely
packed and stains dark is called
heterochromatin. It is transcriptionally
inactive.

 Histone protein is a family of
highly alkaline proteins present
in the nucleus of eukaryotic
cells.
 Their positive charge facilitates
the association with negatively
charged DNA.
 The main function of histone
proteins is to package and
order the DNA into structural
units called nucleosomes.
 Without histones, the unwound
DNA in chromosomes would be
very long (a length to width ratio
of more than 10 million to 1 in
human DNA).
Extra notes: “Histone protein”
58

Extra notes: None-histone Proteins
 The proteins remain in
chromatin after the histones
have been removed.
59

60
EUKARYOTIC Transcription:
Key points:
 RNA polymerase II (RNAPII) transcribes the major share of eukaryotic genes.
 During elongation, the transcription machinery needs to move histones out of the
way every time it encounters a nucleosome.
 Transcription elongation occurs in a bubble of unwound DNA, where the RNA
Polymerase uses one strand of DNA as a template to catalyse the synthesis of a
new RNA strand in the 5′ to 3′ direction.
 RNA Polymerase I and RNA Polymerase III terminate transcription in response to
specific termination sequences in either the DNA being transcribed (RNA
Polymerase I) or in the newly-synthesized RNA (RNA Polymerase III).
 RNA Polymerase II terminates transcription at random locations past the end of the
gene being transcribed. The newly-synthesized RNA is cleaved at a sequence-
specified location and released before transcription terminates.

61
 Although the enzymatic process of elongation is essentially the same in eukaryotes
and prokaryotes, the eukaryotic transcription is more complex.
 When eukaryotic cells are not dividing, their genes exist as a diffuse, but still
extensively packaged and compacted mass of DNA and proteins called chromatin.
 The DNA is tightly packaged around charged histone proteins at repeated
intervals. These DNA–histone complexes, collectively called nucleosomes.
 For polynucleotide synthesis to occur, the transcription machinery needs to move
histones out of the way every time it encounters a nucleosome.

62
 A a special protein called FACT, which stands for “facilitates chromatin
transcription.” FACT partially disassembles the nucleosome immediately ahead
(upstream) of a transcribing RNA Polymerase by removing two of the eight histones
(a single dimer of H2A and H2B histones is removed.).
 This presumably sufficiently loosens the DNA wrapped around that nucleosome so
that RNA Polymerase can transcribe through it.
 FACT re-assembles the nucleosome behind the RNA Polymerase by returning the
missing histones to it.
 RNA Polymerase will continue to elongate the newly-synthesized RNA until
transcription terminates.

63
Transcription
in
Eukaryotes

 Promoter proximal elements are important in determining how and when a gene is
expressed.
 Core promoter: +1 transcription start site, is the location at which the RNA polymerase
machinery initiates transcription.
64
EUKARYOTIC Transcription: PROMOTER REGION

 GC box, also known as a GSG box.
 The GC elements are bound by transcription
factors and have similar functions to
enhancers.
 The GC box is commonly the binding site for
Zinc finger proteins.
 CCAAT box (also sometimes abbreviated a
CAAT box or CAT box) is a distinct pattern of
nucleotides with GGCCAATCT consensus
sequence.
 The CAAT box signals the binding site for the
RNA transcription factor, and is typically
accompanied by a conserved consensus
sequence. It is an invariant DNA sequence at
about minus 70 base pairs from the origin of
transcription in many eukaryotic promoters.
 Belong to the regulatory promoter and core
promoter.
 Protein specific binding is required for the
CCAAT box activation. These proteins are
known as CCAAT box binding proteins/CCAAT
box binding factors.
EUKARYOTIC Transcription: PROMOTER REGION
GC BOX CAAT BOX TATA BOX
 TATA box (also called the Goldberg-
Hogness box: The TATA box is the binding
site of the TATA-binding protein (TBP) and
other transcription factors in some
eukaryotic genes. The first place where
base pairs separate during eukaryotic
transcription to allow access to the
template strand.
 The AT-richness is important to allow this
separation, since adenine and thymine
are easier to break apart (not only due to
fewer hydrogen bonds, but also due to
weaker base stacking effects (AT: Double
bond).
 Based on the sequence and mechanism
of TATA box initiation, mutations such as
insertions, deletions, and point mutations
can result in phenotypic changes. Which
can then turn into a disease phenotype
like: gastric cancer, Huntington's disease,
β-thalassemia, Gilbert's syndrome, and
HIV-1.
 The TATA-binding protein (TBP) could
also be targeted by viruses as a means of
viral transcription
A zinc finger is a small protein structural motif
that is characterized by the coordination of one
or more zinc ions (Zn2+) in order to stabilize
the fold.
• Various protein engineering techniques can
be used to alter the DNA-binding specificity
of zinc fingers and tandem repeats of such
engineered zinc fingers can be used to
target desired genomic DNA sequences
• Zinc finger nucleases: Engineered zinc
finger arrays are often fused to a DNA
cleavage domain (usually the cleavage
domain of FokI) to generate zinc finger
nucleases. Such zinc finger-FokI fusions
have become useful reagents for
manipulating genomes of many organisms
65

RNA Polymerase & their
location
RNA Type Function of RNA
RNA Pol I
(Nucleolus)
Ribosomal RNA (80%) Catalytic: Combines with proteins to form ribosomes, the
site of protein synthesis.
RNA Pol II
(Nucleoplasm)
Messenger RNA (5%) Informational RNA: Carries genetic information provided
by DNA
RNA Pol III
(Nucleoplasm)
Transfer RNA (15%) Adapter/Soluble: Transport amino acids to site of protein
synthesis.
RNA Pol IV
(Nucleoplasm)
si RNA in Plants A class of double-stranded RNA non-coding RNA
molecules, work as operator (post transcriptional gene
silencing).
RNA Pol V
(Nucleoplasm)
Si RNA in plants Pol V is involved in siRNA-directed DNA methylation
pathway which leads to heterochromatic silencing.
66
EUKARYOTIC Transcription: RNA Polymerases

EUKARYOTIC Transcription: Initiation
67
 Initiation is the first step of eukaryotic transcription
and requires RNAP and several transcription factors
to proceed.
 Unlike the prokaryotic RNA polymerase that can bind
to a DNA template on its own, eukaryotes require
several other proteins, called transcription
factors, to first bind to the promoter region and then
help recruit the appropriate polymerase.
 The completed assembly of transcription factors and
RNA polymerase bind to the promoter, forming a
transcription pre-initiation complex (PIC).
 The most-extensively studied core promoter element
in eukaryotes is a short DNA sequence known as a
TATA box, found 25-30 base pairs upstream from the
start site of transcription.
 Only about 10-15% of mammalian genes contain
TATA boxes, while the rest contain other core
promoter elements, but the mechanisms by which
transcription is initiated at promoters with TATA boxes
is well characterized.
 TBP: Tata binding protein Transcription preinitiation complex

68
EUKARYOTIC Transcription: Initiation
 The TATA box, as a core promoter element, is
the binding site for a transcription factor known
as TATA-binding protein (TBP), which is
itself a subunit of another transcription factor:
Transcription Factor II D (TFIID).
 After TFIID binds to the TATA box via the
TBP, five more transcription factors and RNA
polymerase combine around the TATA box in a
series of stages to form a pre-initiation
complex.
 One transcription factor, Transcription Factor
II H (TFIIH), is involved in separating
opposing strands of double-stranded DNA
to provide the RNA Polymerase access to a
single-stranded DNA template.
 Proteins known as activators and
repressors, along with any associated
coactivators or corepressors, are
responsible for modulating transcription rate.

69
EUKARYOTIC Transcription: Elongation
 RNA Polymerase II is a complex of 12 protein subunits.
 Specific subunits within the protein allow RNA Polymerase II to act as its own helicase, sliding clamp,
single-stranded DNA binding protein, as well as carry out other functions.
 As the RNA Polymerases II travel along the template DNA strand in the 3′ to 5′ direction, and catalyse
the synthesis of new RNA strands towards the 5′ to 3′ direction by adding new nucleotides to the 3′
end of the growing m-RNA strand.
 RNA Polymerases unwind the double stranded DNA ahead of them and allow the unwound DNA
behind them to rewind.
 As a result, RNA strand synthesis occurs in a transcription bubble of about 25 unwound DNA base
pairs.
 Only about 8 nucleotides of newly-synthesized RNA remain base paired to the template DNA. The
rest of the RNA molecules falls off the template to allow the DNA behind it to rewind.
 RNA Polymerases use the DNA strand below them as a template to direct which nucleotide to add to
the 3′ end of the growing RNA strand at each point in the sequence.
 The RNA Polymerase travels along the template DNA one nucleotide at a time. Whichever RNA
nucleotide is capable of base pairing to the template nucleotide below the RNA Polymerase is the
next nucleotide to be added.
 Once the addition of a new nucleotide to the 3′ end of the growing strand has been catalysed, the
RNA Polymerase moves to the next DNA nucleotide on the template below it. This process continues
until transcription termination occurs.

Initiation process (Eukaryotic): Formation of transcription
initiation complex
70
ATG
TAC
Promoter
5’
3’
+1
Template
strand
RNA Polymerase
3’
5’

Elongation process (Eukaryotic): Transcription
bubble and elongation
71
TATA Box ATG
TAC
Promoter
5’
3’
+1
AUG
preRNA molecule
RNA Polymerase
3’
5’
8 nucleotide of
RNA Molecule
25 unwound DNA base pairs
TAG
ATC

Elongation process (Eukaryotic): Termination
72
ATG
TAC
Promoter
5’
3’
+1
AUG
preRNA molecule
RNA Polymerase
3’
5’
8 nucleotide of
RNA Molecule
25 unwound DNA base pairs
TAG
ATC

AUG UAG
5’
3’
3’
5’
5’ 3’
Start
codon
Stop codon
UAA UGA
ATG TAG TAA TGA
TAC ATC ATT ACT
DNA
Codon mRNA
Non template strand
Template strand
Transcription termination
Transcription
Initiation
Promoter Other sequence
Other sequence
Other sequence
Transcription: Start codon and stop codon
Methionine
Start and stop codons are important because they tell the cell machinery where to
begin and end translation, the process of making a protein.
UAC
5’
Anticodon tRNA
Amino acid
73

74
EUKARYOTIC Transcription: Termination (mRNA)
 The protein-encoding, structural RNA, and regulatory RNA genes transcribed by
RNA Polymerse II lack any specific signals or sequences that direct RNA
Polymerase II to terminate at specific locations.
 RNA Polymerase II can continue to transcribe RNA anywhere from a few bp to
thousands of bp past the actual end of the gene.
 However, the transcript is cleaved at an internal site before RNA
Polymerase II finishes transcribing. This releases the upstream portion of the
transcript, which will serve as the initial RNA prior to further processing (the pre-
mRNA in the case of protein-encoding genes). This cleavage site is
considered the “end” of the gene.
 The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in
humans) while it is still being transcribed by the RNA Polymerase II.
 When the 5′-exonulease “catches up” to RNA Polymerase II by digesting
away all the overhanging RNA, it helps disengage the polymerase from its
DNA template strand, finally terminating that round of transcription.

75
EUKARYOTIC Transcription: Termination (rRNA): Extra notes
The termination of transcription is different for the three different eukaryotic RNA
polymerases.
• The ribosomal rRNA genes transcribed by RNA Polymerase I contain a specific
sequence of base pairs (11 bp long in humans; 18 bp in mice) that is recognized
by a termination protein called TTF-1 (Transcription Termination Factor for
RNA Polymerase I.)
• This protein binds the DNA at its recognition sequence and blocks further
transcription, causing the RNA Polymerase I to disengage from the template
DNA strand and to release its newly-synthesized RNA.

76
EUKARYOTIC Transcription: Termination (tRNA) Extra notes
 The tRNA, 5S rRNA, and structural RNAs genes transcribed by RNA
Polymerase III have a not-entirely-understood termination signal.
 The RNAs transcribed by RNA Polymerase III have a short stretch of four to
seven U’s at their 3′ end.
 This somehow triggers RNA Polymerase III to both release the nascent RNA
and disengage from the template DNA strand.

77
EUKARYOTIC Transcription: mRNA Processing
Key points:
 A 7-methyl-guanosine cap is added to the 5′ end of the pre-mRNA while
elongation is still in progress. The 5′ cap protects the nascent mRNA from
degradation and assists in ribosome binding during translation.
 A poly (A) tail is added to the 3′ end of the pre-mRNA once elongation is
complete. The poly (A) tail protects the mRNA from degradation, aids in the
export of the mature mRNA to the cytoplasm, and is involved in binding
proteins involved in initiating translation.
 Introns are removed from the pre-mRNA before the mRNA is exported to the
cytoplasm.

78
EUKARYOTIC Transcription: mRNA Processing
Pre-mRNA Processing:-
 The eukaryotic pre-mRNA undergoes extensive processing before it is ready to
be translated.
 The additional steps involved in eukaryotic mRNA maturation.
 Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the
pre-mRNA from degradation while it is processed and exported out of the
nucleus.
 The three most important steps of pre-mRNA processing are the addition of
stabilizing and signalling factors at the 5′ and 3′ ends of the molecule, and
the removal of intervening sequences that do not specify the appropriate
amino acids.

79
EUKARYOTIC Transcription: mRNA Processing (5′ Capping)
 While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is
added to the 5′ end of the growing transcript by a 5′-to-5′ phosphate
linkage.
 This moiety protects the nascent mRNA from degradation.
 In addition, initiation factors involved in protein synthesis recognize the cap
to help initiate translation by ribosomes (Signalling).

80
EUKARYOTIC Transcription: mRNA Processing (3′ Poly-A Tail)
 While RNA Polymerase II is still transcribing downstream of the proper end of a gene, the
pre-mRNA is cleaved by an endonuclease-containing protein complex between an
AAUAAA consensus sequence and a GU-rich sequence.
 This releases the functional pre-mRNA from the rest of the transcript, which is still attached
to the RNA Polymerase.
 An enzyme called poly (A) polymerase (PAP) is part of the same protein complex that
cleaves the pre-mRNA and it immediately adds a string of approximately 200 A
nucleotides, called the poly (A) tail, to the 3′ end of the just-cleaved pre-mRNA.
 The poly (A) tail protects the mRNA from degradation, aids in the export of the mature
mRNA to the cytoplasm, and is involved in binding proteins involved in initiating
translation (Signalling).

81
EUKARYOTIC Transcription: mRNA Processing (Pre-mRNA Splicing)
 Eukaryotic genes are composed of exons, which correspond to protein-coding
sequences (exon signifies that they are expressed), and intervening sequences
called introns (intron denotes their intervening role), which may be involved in
gene regulation, but are removed from the pre-mRNA during processing.
 Intron sequences in mRNA do not encode functional proteins.
 Hence, all introns in a pre-mRNA must be completely and precisely removed before
protein synthesis.
 If the process makes mistakes by even a single nucleotide, the reading frame of the
re-joined exons would shift, and the resulting protein would be dysfunctional.
 The process of removing introns and reconnecting exons is called splicing.
 Introns are removed and degraded while the pre-mRNA is still in the nucleus.
 Splicing occurs by a sequence-specific mechanism that ensures introns will be removed
and exons re-joined with the accuracy and precision of a single nucleotide.
 The splicing of pre-mRNAs is conducted by complexes of proteins and RNA molecules
called spliceosome.

82
EUKARYOTIC Transcription: mRNA Processing (Spliceosome)
 Each spliceosome is composed of five subunits called snRNPs (for small nuclear
ribonucleoparticles, and pronounced “snurps”.).
 Each snRNP is itself a complex of proteins and a special type of RNA found only in the
nucleus called snRNAs (small nuclear RNAs).
 Spliceosomes recognize sequences at the 5′ end of the intron because introns
always start with the nucleotides GU and they recognize sequences at the 3′ end of
the intron because they always end with the nucleotides AG.
 The spliceosome cleaves the pre-mRNA’s sugar phosphate backbone at the G that
starts the intron and then covalently attaches that G to an internal A nucleotide within
the intron.
 Then the spliceosme connects the 3′ end of the first exon to the 5′ end of the
following exon, cleaving the 3′ end of the intron in the process.
 This results in the splicing together of the two exons and the release of the intron.

83
EUKARYOTIC Transcription: rRNA Processing Extra notes
 The four rRNAs in eukaryotes are first transcribed as two long precursor molecules.
 One contains just the pre-rRNA that will be processed into the 5S rRNA; the other spans the
28S, 5.8S, and 18S rRNAs.
 Enzymes then cleave the precursors into subunits corresponding to each rRNA.
 Some of the bases of pre-rRNAs are methylated for added stability.
 Mature rRNAs make up 50-60% of each ribosome.
 Some of a ribosome’s RNA molecules are purely structural, whereas others have catalytic
or binding activities.
 The eukaryotic ribosome is composed of two subunits: a large subunit (60S) and a small
subunit (40S). The 60S subunit is composed of the 28S rRNA, 5.8S rRNA, 5S rRNA, and
50 proteins. The 40S subunit is composed of the 18S rRNA and 33 proteins.
 The two subunits join to constitute a functioning ribosome that is capable of creating
proteins

84
EUKARYOTIC Transcription: tRNA Processing Extra notes
 Each different tRNA binds to a specific amino acid and transfers it to the ribosome. Mature
tRNAs take on a three-dimensional structure through intramolecular basepairing to position
the amino acid binding site at one end and the anticodon in an unbasepaired loop of
nucleotides at the other end. The anticodon is a three-nucleotide sequence, unique to each
different tRNA, that interacts with a messenger RNA (mRNA) codon through
complementary base pairing.
 There are different tRNAs for the 21 different amino acids. Most amino acids can be carried
by more than one tRNA.
 In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple
processing steps before the mature tRNA is ready for use in translation. In bacteria, multiple
tRNAs are often transcribed as a single RNA. The first step in their processing is the
digestion of the RNA to release individual pre-tRNAs. In archaea and eukaryotes, each pre-
tRNA is transcribed as a separate transcript.

85
EUKARYOTIC Transcription: tRNA Processing Extra notes
The processing to convert the pre-tRNA to a mature tRNA involves five steps.
1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.
2. The 3′ end of the pre-tRNA is cleaved off.
3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides
is added to the 3′ end of the pre-tRNA after the original 3′ end is trimmed off. Some bacteria and archaea pre-
tRNAs already have the CCA encoded in their transcript immediately upstream of the 3′ cleavage site, so they
don’t need to add one. The CCA at the 3′ end of the mature tRNA will be the site at which the tRNA’s amino
acid will be added.
4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitorgen bases. On average about
12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to
pseudouridine (ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D).
But over 100 other modifications can occur.
5. A significant number of eukaryotic and archaeal pre-tRNAs have introns that have to be spliced out. Introns are
rarer in bacterial pre-tRNAs, but do occur occasionally and are spliced out
After processing, the mature pre-tRNA is ready to have its allied amino acid attached. The allied amino acid for a
tRNA is the one specified by its anticodon.

Prokaryotic
and Eukaryotic
Transcription
COMPARISION
86

SIMILARITIES
PROKARYOTIC
 Both group of DNA act as a template for RNA synthesis
 Both group transcription produces RNA molecule
 Chemical composition of transcription is similar in both group.
 Transcription is facilitated by the enzyme RNA polymerase in both
group.
 In both groups, one strand of DNA duplex act as the template.
EUKARYOTIC
88

Transcription and translation are
continuous process and occur
simultaneously in cytoplasm.
Transcription initiation machinery is simple
since DNA is not associated with histone
protein.
Only 1 type of RNA polymerase enzyme ,
which synthesize all type RNA in the
cell.(mRNA , Rrna , trna)
There are 2 separate processes,
transcription occur in nucleus where
translation occur in the cytoplasm.
Transcription initiation machinery is very
complex since the genetic material is
associated with proteins.
Three type RNA polymerase in the cell.
RNA polymerase 1 for RNA synthesis,
RNA polymerase 2 for mRNA synthesis,
RNA polymerase 3 for tRNA & 5s rRNA
synthesis.
DIFFERENCES
Prokaryotic transcription Eukaryotic transcription
89

RNA polymerase with 5 subunits, Tow α
subunits, one β subunit, one β΄ subunit,
one ω subunit. Functional RNA
polymerase is 2α, 1β, 1β΄ω.
σ factor present , which is essential for
transcription initiation. RNA polymerase
can recognize and bind to the promoter
region with the help of σ factor.
Promoter region contain pribnow box at -
10 position.
TATA box and CAT box are absent in
promoter region of prokaryotes.
RNA polymerase 1 with 14 subunit,RNA
polymerase 2 with 10 -12 subunits
σ absent and it is not required for
transcription initiation. Initiation of
transcription is facilitated by initiation
factor.
RNA polymerase cannot recognize the
promoter region directly unless to
promoter is pre-occupied by transcription
initiation factor.
Promoter region contain ; TATA box
located 35 to 25 upstream ; CAT box
located ~70 nucleotide upstream ; GC box
located~110 nucleotide upstream.
Pribnow box absent in eukaryotes.
DIFFERENCES
90

Termination of transcription is done either
by rho dependent mechanism or rho
independent mechanism.
Usually there is no post transcriptional
modification of the primary transcript.
RNA capping absent, mRNA is devoid of 5’
guanosine cap Poly A tailing of mRNA is
absent
Introns absent in the mRNA
A termination mechanism of transcription
is not completely known. It may be direct
by the poly A signal or by the presence of
termination sequence in the DNA.
Primary transcript undergo post
transcriptional modification (RNA editing).
RNA capping present, capping occur at 5’
position of mRNA
Mature mRNA with a poly A tail at the 3’
position. Poly A tail added enzymatically
without the complementary strand.
Introns present in the primary transcript
DIFFERENCES
91

Splicing of mRNA absent since introns are
absent.
Genes usually polycistronic & hence single
transcript may contains sequence for
many polypeptides.
SD sequence (shine dalgarno sequence)
present about 8 nucleotide upstream of a
start codon in the mRNA SD sequence act
as the ribosome binding site.
Splicing present, introns in the primary
transcript are removed and exons are re-
joined by a variety of splicing mechanisms.
Genes are monocistronic thus single
transcript code for only one polypeptide.
SD sequence is absent.
DIFFERENCES
92

 The purpose of replication is to conserve the
entire genome for next generation.
 The purpose of transcription is to make RNA
copies of individual genes that the cell can use
in the biochemistry.
Replication & Transcription: Differences
Purpose
Replication Transcription
 DNA replication is the replication of a strand of
DNA into two daughter strands, each daughter
strand contains half of the original DNA double
helix.
 Uses the genes as templates to produce several
functional forms of RNA
Definition
 One strand of DNA becomes 2 daughter strands.  mRNA, tRNA, rRNA and non-coding RNA( like
microRNA)
Product
 In eukaryotes complementary base pair nucleotides
bond with the sense or antisense strand. Thesre are
then connected with phosphodiester bonds by DNA
helix to create a complete strand.
 A 5’ cap is added, a 3’ poly A tail is added and
introns are spliced out.
Product
processin
g
93

 Since there are 4 bases in 3-letter
combinations, there are 64 possible codons
(43 combinations).
 RNA transcription follows base pairing rules.
The enzyme makes the complementary strand
by finding the correct base through
complementary base pairing, and bonding it
onto the original strand.
Base
pairing
 These encode the twenty standard amino acids,
giving most amino acids more than one possible
codon. There are also three 'stop' or 'nonsense'
codons signifying the end of the coding region;
these are the UAA, UAG and UGA codons.
 DNA polymerases can only extend a DNA strand in
a 5′ to 3′ direction, different mechanisms are used
to copy the antiparallel strands of the double helix.
In this way, the base on the old strand dictates
which base appears on the new strand.
Codons
 In replication, the end result is DNA molecule.  While in transcription, the end result is a RNA
molecule.
Result
 Replication is the duplication of two-strands of
DNA.
 Transcription is the formation of single, identical
RNA from the two-stranded DNA.
Product
94

 The two strands are separated and then each
strand's complementary DNA sequence is
recreated by an enzyme called DNA
polymerase.
 In transcription, the codons of a gene are
copied into messenger RNA by RNA
polymerase. This RNA copy is then decoded
by a ribosome that reads the RNA sequence
by base-pairing the messenger RNA to transfer
RNA, which carries amino acids.
Enzymes
 DNA Helicase, DNA Polymerase.  Transcriptase (type of DNA Helicase), RNA
polymerase.
Enzyme
types
95

REPLICATION
 Replication and transcription involves a parental DNA strand that
is the foundation on which the products are built on.
 Replication and transcription both have initiation step which
involve the breakage of the parental DNA strand.
 Replication and transcription both have specific proteins that keep
the polymerase molecule attached to the parental DNA strand.
 There are elongation factors for transcription and sliding clamp for
replication.
 Both processes use DNA topoisomerases to relieve supercoiling.
 Both processes only proceed in the 5' to 3' direction.
TRANSCRIPTION
96
Replication & Transcription: Similarities

REPLICATION
 Replication and transcription both involve the addition of specific 3'
endings. In replication, it is the addition of the GGGTTA sequence
by telomerase. In transcription, it is the addition of the poly-A tail.
 Both processes used nucleotides as the language on which the
daughter strands come from.
 Replication and transcription involve the hydrolysis of a
phosphodiester bonds to begin their process.
 Both processes take place in the nucleus.
TRANSCRIPTION
97
Replication & Transcription: Similarities

Regulation of Gene
Expression
Prokaryotes and Eukaryotes
98

REGULATION OF GENE EXPRESSION: Key points
 Gene expression is the mechanism at molecular level by which a gene is able to
express itself in the phenotype of an organism.
 Gene regulation is the mechanism of switching off and switching on of the genes
depending upon the requirement of the cells and the state of development.
 Experiments have shown that many of the genes within the cells of organisms are
inactive much or even all of the time. Thus, at any time, in both eukaryotes and
prokaryotes, it seems that a gene can be switched on or off. The regulation of genes
between eukaryotes and prokaryotes differs in important ways. The process by which
genes are activated and deactivated in bacteria is well characterized.
 There are two types of genes, house keeping genes and regulated genes.
 Housekeeping genes or Constitutive genes: are the one which are continuously
expressing themselves in all the cells of body. It is because their product is always
required. E.g. genes for glycolysis.
 Luxury genes/ non-constitutive genes: Their activity is regulated and, therefore,
they are called regulated genes.
99

Gene regulations: negative or positive
 Negative the gene continuously expressing their effect till their activity is
suppressed. The negative gene expression is also called repressible
regulation. Repression is due to product of regulatory gene.
 Positive gene regulation is the one in which the gene remain non-expressed
unless and until they are induced to it. It has therefore, inducible regulation.
Here a product removes a biochemical that keeps the gene in non-expressed
state.
 As genes express their effect through enzymes, their enzymes are also called
inducible enzyme and repressible enzymes.
100

REGULATION OF GENE EXPRESSION: Bacteria
 Bacteria have three types of genes: structural, operator, and regulator.
 Structural genes code for the synthesis of specific polypeptides.
 Operator genes contain the code necessary to begin the process of
transcribing the DNA message of one or more structural genes into mRNA.
 Thus, structural genes are linked to an operator gene in a functional unit called
an operon.
 Ultimately, the activity of the operon is controlled by a regulator gene, which
produces a small protein molecule called a repressor.
 The repressor binds to the operator gene and prevents it from initiating the
synthesis of the protein called for by the operon.
 The presence or absence of certain repressor molecules determines whether
the operon is off or on. As mentioned, this model applies to bacteria.
101

REGULATION OF GENE EXPRESSION: Eukaryotes
In Eukaryotes, the regulation of gene expression is completed at four levels:-
1. Transcriptional level: Formation of primary transcript
2. Processing level: Regulation of splicing
3. Transport of mRNA from nucleus to cytoplasm
4. Translational level
102

REGULATION OF GENE EXPRESSION: Eukaryotes
 Mostly, they do not have operons, which are regulated independently.
 The series of events associated with gene expression in higher organisms involves
multiple levels of regulation and is often influenced by the presence or absence of
molecules called transcription factors. These factors influence the fundamental level of
gene control, which is the rate of transcription, and may function as activators or
enhancers. Specific transcription factors regulate the production of RNA from genes at
certain times and in certain types of cells.
 Transcription factors often bind to the promoter, or regulatory region, found in the genes
of higher organisms. Following transcription, introns (noncoding nucleotide sequences)
are excised from the primary transcript through processes known as editing and splicing.
 The result of these processes is a functional strand of mRNA. For most genes this is a
routine step in the production of mRNA, but in some genes there are multiple ways to
splice the primary transcript, resulting in different mRNAs, which in turn result in different
proteins. Some genes also are controlled at the translational and posttranslational levels.
103

Operon Concept
 Francois Jacob and Jacques Monad proposed a model of
gene regulation known as operon model.
 Examples: Lac, Trp, ara, his,val etc.
104

Operon
 Francois Jacob and Jacques Monod proposed a model of gene regulation,
known as Operon model.
 Operon is a cluster of structural genes that is expressed or controlled by a
single promoter and is considered as the functional unit of genomic DNA.
 This feature allows protein synthesis to be controlled coordinately in response
to the needs of the cell.
 By providing the means to produce proteins only when and where they are
required, the operon allows the cell to conserve energy (which is an important
part of an organism’s life strategy).
 A typical operon consists of a group of structural genes that code for enzymes
involved in a metabolic pathway, such as the biosynthesis of an amino acid.
105

Operon
 These genes are located adjacent to promoter (a short segment of DNA to
which the RNA polymerase binds to initiate transcription).
 A single unit of messenger RNA (mRNA) is transcribed from the operon and is
subsequently translated into separate proteins.
 The promoter is controlled by various regulatory elements that respond to
environmental cues.
 One common method of regulation is carried out by a regulator protein that
binds to the operator region, which is another short segment of DNA found
between the promoter and the structural genes.
 The regulator protein can either block transcription, in which case it is
referred to as a repressor protein; or as an activator protein it can stimulate
transcription.
106

Operon
 These genes are located adjacent to promoter (a short segment of DNA to
which the RNA polymerase binds to initiate transcription).
 A single unit of messenger RNA (mRNA) is transcribed from the operon and is
subsequently translated into separate proteins.
 The promoter is controlled by various regulatory elements that respond to
environmental cues.
 One common method of regulation is carried out by a regulator protein that
binds to the operator region, which is another short segment of DNA found
between the promoter and the structural genes.
 The regulator protein can either block transcription, in which case it is
referred to as a repressor protein; or as an activator protein it can stimulate
transcription.
107

Operon
 Further regulation occurs in some operons: a molecule called an inducer can
bind to the repressor, inactivating it; or a repressor may not be able to bind to
the operator unless it is bound to another molecule, the corepressor.
 Some operons are under attenuator control, in which transcription is initiated
but is halted before the mRNA is transcribed. This introductory region of the
mRNA is called the leader sequence; it includes the attenuator region, which
can fold back on itself, forming a stem-and-loop structure that blocks the RNA
polymerase from advancing along the DNA.
108

Operator?
 An operator is found in prokaryotic gene structure.
 It is the main region of DNA in which the regulatory molecules of an operon
system binds to. The lac operator is the operator sequence present in the lac
operon of many prokaryotic bacteria.
 In the case of the lac operon, the repressor molecule binds to the operator
region. This binding will prevent RNA polymerase from transcribing the genes
present downstream of the operator.
 Eukaryotes do not possess operator regions. Instead, their transcription factors
involved in regulation of transcription are bind to the promoter regions. Thus,
the main function of the operator in prokaryotes is to regulate gene expression.
109

Similarities Between Promoter and Operator?
 Both Promoter and Operator are composed of
deoxyribose nucleic acids (DNA).
 Both Promoter and Operator sequences are important
in the transcription process.
110

Operator
1. Operators are the sites in
which the regulatory molecule
binds into an operon model.
Type of Organism
2. Operators are found only in
prokaryotes.
3. Operators regulate the gene
expression by facilitating the
binding of the regulatory
molecule to the operon.
1. Promoters are the sites in which
RNA polymerase binds and they
are present upstream of the
transcription start site of a gene.
2. Promoters are found in both
prokaryotes and eukaryotes.
3. Promoter facilitates the binding
of the RNA polymerase and
transcription factors (only in
eukaryotes) to the gene for gene
transcription. In prokaryotes,
promoter region facilitates the
binding of sigma factor of RNA
Polymerase (in prokaryotes).
Promoter
Difference Between Promoter and Operator?
111

Inducible operon (lac operon):
 In Escherichia coli, breakdown of lactose requires three enzymes.
 These enzymes are synthesized together in a co-ordinated manner and the unit is known as lac
operon.
 Since the addition of lactose itself stimulates the production of required enzymes, it is also known as
inducible system. 112

Inducible operon (lac operon): Consists of………..
113
Structural genes. These genes code for the proteins needed by the cell which include
enzymes or other proteins having structural functions.
In lac-operon, there are following three structural genes:
 lac a -gene coding for enzyme transacetylase
 lac y -gene coding for enzyme permease
 lac z -gene coding for enzyme β-galactosidase

Operon structure:
An operon is made up of 3 basic DNA components:
1. Promoter – a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by
RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should
be used for messenger RNA creation – and, by extension, control which proteins the cell produces.
2. Operator – a segment of DNA to which a repressor binds. It is classically defined in the lac operon as a
segment between the promoter and the genes of the operon. The main operator (O1) in the lac operon is
located slightly downstream of the promoter; two additional operators, O1 and O3 are located at -82 and
+412, respectively. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase
from transcribing the genes.
3. Structural genes – the genes that are co-regulated by the operon.
Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed
gene which codes for repressor proteins. The regulatory gene does not need to be in, adjacent to, or even near
the operon to control it. An inducer (small molecule) can displace a repressor (protein) from the operator site
(DNA), resulting in an uninhibited operon. Alternatively, a corepressor can bind to the repressor to allow its binding
to the operator site. A good example of this type of regulation is seen for the trp operon.
114

Operon regulation:
Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various
genes depending on environmental conditions. Operon regulation can be either negative or positive by induction
or repression.
Negative control involves the binding of a repressor to the operator to prevent transcription.
 In negative inducible operons, a regulatory repressor protein is normally bound to the operator, which
prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the
repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression
of the operon. The lac operon is a negatively controlled inducible operon, where the inducer molecule is
allolactose.
 In negative repressible operons, transcription of the operon normally takes place. Repressor proteins are
produced by a regulator gene, but they are unable to bind to the operator in their normal conformation.
However, certain molecules called corepressors are bound by the repressor protein, causing a conformational
change to the active site. The activated repressor protein binds to the operator and prevents transcription.
The trp operon, involved in the synthesis of tryptophan (which itself acts as the corepressor), is a negatively
controlled repressible operon.
115

Operon regulation:
Operons can also be positively controlled. With positive control, an activator protein stimulates transcription by
binding to DNA (usually at a site other than the operator).
 In positive inducible operons, activator proteins are normally unable to bind to the pertinent DNA. When an
inducer is bound by the activator protein, it undergoes a change in conformation so that it can bind to the DNA
and activate transcription.
 In positive repressible operons, the activator proteins are normally bound to the pertinent DNA segment.
However, when an inhibitor is bound by the activator, it is prevented from binding the DNA. This stops
activation and transcription of the system.
116

Lac Operon: An Inducible Operon
 Inducible operons have proteins that can bind to either activate or repress transcription depending
on the local environment and the needs of the cell.
 E. coli is able to use other sugars as energy sources when glucose concentrations are low.
 To do so, the cAMP–CAP protein complex serves as a positive regulator to induce transcription.
One such sugar source is lactose.
 The lac operon encodes the genes necessary to acquire and process the lactose from the local
environment, which includes the structural genes lacZ, lacY, and lacA.
 lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide
lactose into glucose and galactose.
 lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps
lactose into the cell.
 lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group
from acetyl-CoA to β-galactosides.
 Only lacZ and lacY appear to be necessary for lactose catabolism.
117

Lac Operon: An Inducer Operon
 CAP binds to the operator sequence upstream of the promoter that initiates transcription of the lac
operon.
 The lac operon uses a two-part control mechanism to ensure that the cell expends energy
producing β-galactosidase, β-galactoside permease, and thiogalactoside transacetylase (also
known as galactoside O-acetyltransferase) only when necessary.
 However, for the lac operon to be activated, two conditions must be met.
 First, the level of glucose must be very low or non-existent.
 Second, lactose must be present.
• If glucose is absent, then CAP can bind to the operator sequence to activate transcription.
• If lactose is absent, then the repressor binds to the operator to prevent transcription.
• If either of these requirements is met, then transcription remains off.
• The cell can use lactose as an energy source by producing the enzyme b-galactosidase to digest
that lactose into glucose and galactose.
• Only when both conditions are satisfied is the lac operon transcribed, such as when glucose is
absent and lactose is present.
• This process is beneficial and makes most sense for the cell as it would be energetically wasteful
to create the proteins to process lactose if glucose were plentiful or if lactose were not available.
118

LAC Operon: structure
119
Operator LacZ LacY LacA
CAP site
RNA
Polymerase
Promoter
β-galactosidase
Glucose Galactose
Lactose permease
Transport lactose inside the cell
Thiogalactoside transacetylase
Removes toxic thiogalactosides
Transported by LacY
Lactose

LAC Operon: Turn OFF
120
CAP site
RNA
Polymerase
Promoter
β-galactosidase
Glucose Galactose
Lactose permease
Transported by LacY
Turn OFF
If Lactose is absent
Repressor
Lactose

LAC Operon: Turn OFF
121
LacZ LacY LacA
CAP site
RNA
Polymerase
Promoter
β-galactosidase
Lactose permease
Turn OFF
Repressor
Operator
• It would be energetically wasteful for E. coli if the lac genes were expressed
when lactose was not present.
• The effect of the Lac repressor on the lac genes is referred to as negative
regulation.

Repressor
LAC Operon: Turn ON
122
CAP site
RNA
Polymerase
Promoter
Turn ON
If Lactose is present
 When lactose is present, the lac genes are expressed because
allolactose binds to the Lac repressor protein and keeps it from
binding to the lac operator.
Allolactose

Repressor
LAC Operon: Turn ON
123
CAP site
RNA
Polymerase
Promoter
Turn ON
 Allolactose is an isomer of lactose. Small amounts of allolactose are
formed when lactose enters E. coli.
 Allolactose binds to an allosteric site on the repressor protein causing a
conformational change.
 As a result of this change, the repressor can no longer bind to the operator
region and falls off. RNA polymerase can then bind to the promoter and
transcribe the lac genes.
Allolactose
Allolactose
Lactose

LAC Operon: Turn ON
124
CAP site
RNA
Polymerase
Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn ON
Repressor
Glucose Galactose
Lactose
Allolactose

RNA
Polymerase
LAC Operon: Turn ON
125
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn ON
Repressor
Glucose Galactose
Lactose
Allolactose

RNA
Polymerase
LAC Operon: Turn ON
126
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn ON
Repressor
Glucose Galactose
Lactose
Allolactose

RNA
Polymerase
LAC Operon: Turn ON
127
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn ON
Repressor
Glucose Galactose
Lactose
Allolactose

RNA
Polymerase
LAC Operon: Turn ON
128
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn ON
Repressor
Glucose Galactose
Lactose
Allolactose
When the enzymes
encoded by the lac
operon are produced,
they break down
lactose and allolactose,
eventually releasing the
repressor to stop
additional synthesis of
lac mRNA.

RNA
Polymerase
LAC Operon: Turn ON/Off
129
LacZ LacY LacA
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Turn OFF
As Lactose is utilized
Repressor
Glucose Galactose
Lactose
When the enzymes
encoded by the lac
operon are produced,
they break down
lactose and allolactose,
eventually releasing the
repressor to stop
additional synthesis of
lac mRNA.
Operator

RNA
Polymerase
130
LacZ LacY LacA
CAP site Promoter
β-galactosidase
Lactose permease
Turn ON
Repressor
Operator
LAC Operon: (BASAL LEVEL TRANSCRIPTION)
✓
 Occasionally repressor may fall of from the operator allowing
transcription of lac gene is referred as Basal level transcription.
 Because of level transcription, there is some amounts of lactose
permiase, β-galactosidase and Thiogalactoside transacetylase are
available inside the cell.
Allolactose

RNA
Polymerase
131
LacZ LacY LacA
CAP site Promoter
Turn OFF
Operator
LAC Operon: Role of CAP Site
 CAP- Catabolite/cyclic
activator protein
 c-AMP receptor protein
 As the level of Glucose inside
the cell decreases, it will
increases the level of cAMP
c-AMP receptor protein
CAP-Catabolite activator protein

RNA
Polymerase
132
LacZ LacY LacA
CAP site Promoter
Turn ON
Operator
 cAMP binds CAP protein and
activates it.
 Which will create cAMP-CAP
complex.
CAMp-CAP Complex

RNA
Polymerase
133
LacZ LacY LacA
CAP site Promoter
Turn ON
Operator
 cAMP and CAP protein
complex then binds to CAP
site and interacts with C-
terminal α subunit of RNA
polymerase, increasing the
transcription of lac genes.
CAMp-CAP Complex

LAC Operon: Energy Source Preferences of E. coli
 Whenever glucose is present, E. coli
metabolizes it before using alternative energy
sources such as lactose, arabinose, galactose,
and maltose.
 Glucose is the preferred and most frequently
available energy source for E. coli. The enzymes
to metabolize glucose are made constantly by E.
coli.
 When both glucose and lactose are available,
the genes for lactose metabolism are transcribed
at low levels.
 Only when the supply of glucose has been
exhausted does RNA polymerase start to
transcribe the lac genes efficiently, which allows
E. coli to metabolize lactose.
134

LAC Operon: Glucose/lactose
 When both glucose and lactose are present, the
genes for lactose metabolism are transcribed to
a small extent.
 Maximal transcription of the lac operon occurs
only when glucose is absent and lactose is
present. The action of cyclic AMP and a
catabolite activator protein produce this effect.
135

LAC Operon: The Effect of Glucose and Cyclic AMP
 The presence or absence of glucose affects
the lac operon by affecting the concentration
of cyclic AMP.
 The concentration of cyclic AMP in E. coli is
inversely proportional to the concentration of
glucose: as the concentration of glucose
decreases, the concentration of cyclic AMP
increases.
136

LAC Operon: The Effect of Lactose in the Absence of
Glucose
 In the presence of lactose and absence of
glucose, cyclic AMP (cAMP) joins with a
catabolite activator protein that binds to
the lac promoter and facilitates the
transcription of the lac operon.
 In some texts, the catabolite activator protein
(CAP) is called the cAMP-receptor protein.
 When the concentration of glucose is low, cAMP
accumulates in the cell. The binding of cAMP
and the catabolite activator protein to
the lac promoter increases transcription by
enhancing the binding of RNA polymerase to
the lac promoter.
137

RNA
Polymerase
Quiz: You may refer to this illustration while answering any of
the questions in the Self-Quiz.
138
CAP site Promoter
β-galactosidase
Lactose permease
Transported by LacY
Repressor
Glucose Galactose
Lactose
Allolactose
When the enzymes encoded by the lac operon
are produced, they break down lactose and
allolactose, eventually releasing the repressor to
stop additional synthesis of lac mRNA.
Mutations typically disable a
gene. Predict the phenotype of
the mutants as asked in next
slides.

LAC Operon
1. Predict the phenotype of a lacI mutant.
a. The lac genes would be expressed efficiently only in the absence of lactose.
b. The lac genes would be expressed efficiently only in the presence of lactose.
c. The lac genes would be expressed continuously.
d. The lac genes would never be expressed efficiently.
139
The correct response is c. In a lacI mutant, the mutant lac repressor protein cannot
bind to the operator. In the absence of the repressor, RNA polymerase can bind to the
lac promoter, and the lac genes will be transcribed continually regardless of whether
the inducer, allolactose, is present or not. This is referred to as a constitutive
phenotype. Remember, though, that the level of lac gene expression will depend on
whether glucose is present in the medium.

LAC Operon
2. If a second wild type or normal copy of the lacI gene (just lacI and
not lacZ, lacY, or lacA) is introduced into the lacI mutant cell, what would be the
phenotype of this partial diploid (also referred to as a merodiploid)?
140
The correct response is b. Constitutive mutants in the lacI gene are recessive. The
lac repressor protein is diffusible. Therefore the wild type copy of the repressor
protein will be able to diffuse and bind to the operator in the chromosomal copy of the
lac operon and will block expression of the chromosomal lac genes.

LAC Operon
3. Predict the phenotype of a lacI S or "super-repressor" mutant. A lacI S mutant
synthesizes a repressor that cannot bind to the inducer.
.
141
The correct response is d. The "super repressor" has lost its binding site for the
inducer (allolactose). Therefore, the inducer cannot bind to the "super repressor,"
which binds permanently to the operator.

LAC Operon
4. Predict how a lacI S mutant would be affected by the construction of a
merodiploid that has a second normal copy of the lacI gene.
.
142
The correct response is d. The wild-type repressor can be bound and inactivated by
the inducer (allolactose). However, the mutant "super-repressor" will bind to the lac
operator and will not be inactivated because allolactose cannot bind to the mutant
repressor.

LAC Operon
5. Predict the phenotype of an operator mutant (O c) which would prevent the
binding of the repressor.
.
143
The correct response is c. Active repressor cannot bind to the mutant O c operator.
Therefore, the repressor cannot prevent RNA polymerase from binding to the lac
promoter, causing the lac operon to be transcribed continuously (or constitutively).
Once again, the level of expression is inversely proportional to the level of glucose
present.

LAC Operon
6. Predict the phenotype of a promoter mutant (lacP) which has a mutation in the
promoter for the lac operon.
.
144
The correct response is d. If the promoter region is mutated, RNA polymerase will not
be able to recognize it and bind to it. Therefore, the lac genes will not be transcribed
even when the repressor protein is inactivated (in the presence of allolactose) and
glucose is absent from the medium (the catabolite activator protein is bound to cyclic
AMP and to the promoter).

LAC Operon
7. Predict the phenotype of a lacZ mutant, which has a mutation in the gene for β-
galactosidase.
a. The production of all protein products would be affected.
b. The production of β-galactosidase would be affected, but other protein products
would be unaffected.
c. The production of β-galactosidase would be affected, and the production of some
other protein products might also be affected.
d. The production of β-galactosidase would be unaffected, but other protein products
would be affected.
145
The correct response is c. If the lacZ gene carries a mutation in an essential part of the gene, the β-galactosidase
protein produced will be not be functional. Other protein products may not be functional as well. Such a mutation
might cause:
1. substitution of an essential amino acid resulting in a loss of activity due to, for example, improper folding of the
β-galactosidase protein.
2. substitution of many amino acids (caused, for example, by a frameshift mutation) leading to complete loss of
protein function.
3. premature termination of protein synthesis leading to a truncated, nonfunctional β-galactosidase protein and no
lactose permease or transacetylase.

LAC Operon
8. Predict the phenotype of a lacY mutant, which has a mutation in the gene for
lactose permease.
b. The lac genes would be expressed efficiently until the lactose supply in the cell is
exhausted.
d. Expression of the lac genes would cease immediately.
146
• The correct response is b. lactose permease is involved with the transport of lactose into the cell
through the cell membrane. Therefore,
• No lactose permease. → lactose cannot enter cell.
• This means that when the cell's supply of lactose has been used up, no more lactose will be able to
enter the cell. As a result, the lac repressor protein will remain permanently bound to the operator
region in the lac promoter because there will be no allolactose available in the cell to inactivate it,
even though there may be abundant lactose present in the surrounding medium.
• RNA polymerase will be permanently blocked. → NO expression of lac genes.

TRP Operon: Key points
 The trp operon, found in E. coli bacteria, is a group of genes that encode
biosynthetic enzymes for the amino acid tryptophan.
 The trp operon is expressed (turned "on") when tryptophan levels are low
and repressed (turned "off") when tryptophan level is high.
 The trp operon is regulated by the trp repressor. When bound to
tryptophan, the trp repressor blocks expression of the operon.
 Tryptophan biosynthesis is also regulated by attenuation (a mechanism
based on coupling of transcription and translation).
147

TRP Operon: Introduction
 Bacteria such as Escherichia coli (a friendly inhabitant of our gut) need amino
acids to survive—because, like us, they need to build proteins.
 One of the amino acids they need is tryptophan.
 If tryptophan is available in the environment, E. coli will take it up and use it to
build proteins.
 However, E. coli can also make their own tryptophan using enzymes that are
encoded by five genes.
 These five genes are located next to each other in what is called the trp
operon.
 If tryptophan is present in the environment, then E. coli bacteria don't need to
synthesize it, so transcription of the genes in the trp operon is switched "off."
When tryptophan availability is low, on the other hand, the operon is switched
"on," the genes are transcribed, biosynthetic enzymes are made, and more
tryptophan is produced.
148

TRP Operon: structure
 The trp operon includes five genes that encode enzymes needed for tryptophan
biosynthesis, along with a promoter (RNA polymerase binding site) and an
operator (binding site for a repressor protein). The genes of the trp operon are
transcribed as a single mRNA.
149
RNA
Polymerase
Operator
Promoter trpE trpD trpC trpB trpA
Repressor
Tryptophane
The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription
only when tryptophan is present. When tryptophan is around, it attaches to the repressor
molecules and changes their shape so they become active. A small molecule like trytophan,
which switches a repressor into its active state, is called a corepressor.
What does the operator do? This stretch of DNA is recognized by a regulatory protein known as the trp
repressor. When the repressor binds to the DNA of the operator, it keeps the operon from being transcribed by
physically getting in the way of RNA polymerase, the transcription enzyme.

RNA
Polymerase
TRP Operon:
150
Operator
Repressor
In the absence of tryptophan
Tryptophan
Turn ON

RNA
Polymerase
TRP Operon:
151
Operator
Repressor
In the absence of tryptophane
Tryptophan
Turn ON

RNA
Polymerase
TRP Operon:
152
Operator
Repressor
Tryptophan
Turn ON

RNA
Polymerase
TRP Operon:
153
Operator
Repressor
Tryptophan
Turn ON

RNA
Polymerase
TRP Operon:
154
Operator
Repressor
Tryptophan
Turn ON

RNA
Polymerase
TRP Operon:
155
Operator
Tryptophan
m-RNA
Ribosome
Repressor
Turn OFF

Repressor
RNA
Polymerase
TRP Operon:
156
Operator
When Tryptophane is present
Tryptophan
Where does the trp repressor come from?
The trp repressor protein is encoded by a gene called trpR. This gene is not part of the trp operon, and it's located
elsewhere on the bacterial chromosome, where it has its own promoter and other regulatory sequences.
Turn OFF

TRP Operon: A Repressor Operon
 The trp operon, found in E. coli, is a group of genes that encode biosynthetic enzymes for
the amino acid tryptophan.
 The trp operon is expressed (turned "on") when tryptophan levels are low and repressed
(turned "off") when they are high.
 The trp operon is regulated by the trp repressor. When bound to tryptophan, the trp
repressor blocks expression of the operon.
 Tryptophan biosynthesis is also regulated by attenuation (a mechanism based on
coupling of transcription and translation).
 If tryptophan is available in the environment, E. coli will take it up and use it to build
proteins. However, E. coli can also make their own tryptophan using enzymes that are
encoded by five genes. These five genes are located next to each other in what is called
the trp operon.
 If tryptophan is present in the environment, then E. coli bacteria don't need to synthesize
it, so transcription of the genes in the trp operon is switched "off." When tryptophan
availability is low, on the other hand, the operon is switched "on," the genes are
transcribed, biosynthetic enzymes are made, and more tryptophan is produced.
157

TRP Operon: structure
 The trp operon includes five genes that encode enzymes needed for tryptophan biosynthesis,
along with a promoter (RNA polymerase binding site) and an operator (binding site for a repressor
protein). The genes of the trp operon are transcribed as a single mRNA.
Turning the operon "on" and "off“
 What does the operator do? This stretch of DNA is recognized by a regulatory protein known as
the trp repressor. When the repressor binds to the DNA of the operator, it keeps the operon from
being transcribed by physically getting in the way of RNA polymerase, the transcription enzyme.
 The trp repressor does not always bind to DNA. Instead, it binds and blocks transcription only
when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and
changes their shape so they become active. A small molecule like trytophan, which switches a
repressor into its active state, is called a corepressor.
 When there is little tryptophan in the cell, on the other hand, the trp repressor is inactive (because
no tryptophan is available to bind to and activate it). It does not attach to the DNA or block
transcription, and this allows the trp operon to be transcribed by RNA polymerase.
 In this system, the trp repressor acts as both a sensor and a switch. It senses whether tryptophan
is already present at high levels, and if so, it switches the operon to the "off" position, preventing
unnecessary biosynthetic enzymes from being made.
158

TRP Operon: Attenuation
trp operon regulation: Attenuation
 Like regulation by the trp repressor, attenuation is a mechanism for reducing expression
of the trp operon when levels of tryptophan are high.
 However, rather than blocking initiation of transcription, attenuation prevents completion
of transcription.
 When levels of tryptophan are high, attenuation causes RNA polymerase to stop
prematurely when it's transcribing the trp operon.
 Only a short, mRNA is made, one that does not encode any tryptophan biosynthesis
enzymes.
159

Inducible operon
 Turn of only in the presence
of substrate.
 Example: Lac operon.
 The inducible operons
function in catabolic
pathways.
160
 End product serve as a
feedback inhibitor of the
operon.
 Example: Trp operon.
 The repressible operons
function in anabolic
pathways.
Repressible operon

Anti-termination
 Anti-termination factors are proteins that prevent termination of
transcription at specific sites.
 The RNA polymerase therefore continues on its way and
transcribes the region of DNA beyond the terminator.
 This mechanism for controlling gene expression is common in
bacterial viruses and is also found for a few bacterial genes.
 Anti-termination factors attach themselves to the RNA polymerase
before it reaches the terminator.
 The recognition sequences for anti-termination are found in the
DNA well upstream of the terminator.
 As the RNA polymerase passes by, the anti-termination factors are
loaded on. They remain attached and allow the RNA polymerase to
travel past the stem and loop region of the terminator without
pausing. Consequently, termination is suppressed.
161
Figure. Operation of Anti-Termination Factor A) Transcription of the DNA
begins with the RNA polymerase bound to the promoter. B) In the
absence of an anti-terminator factor, the RNA polymerase reaches the
terminator region and drops off, having transcribed a short RNA. C) In the
presence of an anti-termination factor, the RNA polymerase binds the
factor when it reaches the recognition site. The factor allows the RNA
polymerase to transcribe through the termination site.
Source: Molecular Cell Biology by David P. Clark and Nanette J. Pazdernik

Anti-termination
 Anti-termination in E. coli involves several Nus proteins (The N-
utilizing substance A).
 NusA protein is probably attached to the core RNA polymerase
shortly after the sigma factor is lost just after initiation.
 NusA itself actually promotes termination, apparently by increasing
the duration of pauses at hairpin structures.
 NusA and sigma cannot both bind to the core enzyme
simultaneously.
 As long as RNA polymerase is attached to DNA, the Nus proteins
cannot be dislodged.
 However, addition of sigma displaces NusA from free RNA
polymerase.
 Thus, RNA polymerase cycles between initiation mode (with sigma
bound) and termination mode (with NusA bound).
162
Figure. Sigma NusA Cycle of RNA Polymerase RNA polymerase needs the sigma
subunit to recognize and bind to the promoter. Once RNA polymerase moves
forward and starts transcribing, it releases sigma and picks up NusA protein. After
termination, NusA protein is displaced by sigma..
Source: Molecular Cell Biology by David P. Clark and Nanette J. Pazdernik

Group Discussion
Questions and answers
163

The process of synthesis of RNA from the DNA
molecules is referred as:-
A. Central Dogma of Biology
B. Translation
C. Transcription
D. Anti-termination
164

Promoter region of a prokaryotic gene:-
A. TATA Box
B. CAT Box
C. Dog Box
D. Pribnow Box
165

Identify correct match from the following:-
A. Prokaryotes : Single RNA Polymerase
B. Eukaryotes: Single RNA Polymerase
C. Dog Box: Prokayotes
D. Pribnow Box: Eukaryotes
E. Eukaryotes: TATA Box
F. Prokaryotes: Splisosome
G. Eukaryotes: Polycistronic RNA
H. Prokaryotes: Monocistronic RNA
166

Identify incorrect match from the following:-
A. Prokaryotes : Single RNA Polymerase
B. Eukaryotes: Single RNA Polymerase
C. Dog Box: Prokayotes
D. Pribnow Box: Eukaryotes
E. Eukaryotes: TATA Box
F. Prokaryotes: Splisosome
G. Eukaryotes: Polycistronic RNA
H. Prokaryotes: Monocistronic RNA
167

Unit 1 transcription

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