CHAPTER 8

Regulations of gene activity

At the end of the lecture, students

should be able to:
Describe the mechanism of transcription in

prokaryotes
Describe control of transcription

The lac operon

The trp operon
The ara operon

Prokaryotic transcription
In E.coli, the RNA polymerase consisted of

the core enzyme and a - factor.

The core polymerase enzyme consisted of
the , , 2 and (basic transcription
machinery).
The holoenzyme included the - factor.
This causes the ability to transcribe specific
genes.

RNA polymerase subunits

Prokaryotic transcription
The -factor allows initiation of the

transcription process by binding to specific

promoters.
Therefore, the -factor can select the genes
to be transcribed.
Without the -factor , the core enzyme will
lack specificity.

Prokaryotic transcription
The core enzyme binds loosely to a closed

promoter complex (promoter searching)

The -factor will allow the holoenzyme to
bind tightly to the promoter complex (10-17
bp), causing it to be opened from a closed
double stranded form.
The opening process involved the melting
of a short region of the DNA and followed
by the tight binding of the open promoter
complex.

Prokaryotic transcription
There are two core promoter

regions, -35 and -10, a common

sequence as TTGACA and TATAAT.
Some very strong promoters had an
extra sequence further upstream
called the UP element (enhancing
RNA polymerase binding).

Prokaryotic transcription

Prokaryotic transcription
The core subunit lies near the active

site of the RNA polymerase where

phosphodiester bonds are formed.
The subunit had independently folded Nand C-terminal domains.
The C-terminal domains can recognized
and bind to the promoters UP elements.
This caused tight binding of the promoterpolymerase complex.
DNA is bound to the -subunit

Transcription
initiation

Binding
The binding of polymerase core
enzyme to the -factor will cause
the complete holoenzyme to open
the DNA at the specific initiation
site.
Initiation
The binding will cause the double
helix DNA to separate and initiate
transcription at the proper start
site.
Elongation
As the RNA chain is elongated,
the -factor will be released.

Transcription initiation
-factor only initiates the
transcription process, but not the
elongation.
It is released from the open promoter
complex randomly (stochastic model)
leaving the core enzyme to carry the
transcription process.
The -factor can be used by a
different core enzyme.
The

Transcription elongation
The RNA polymerase causes the

melting of a 10-17 bp of the

transcription start site.
This created a transcription bubble
that will move during transcription,
each time exposing the template
strand to be transcribed.
Topoisomerases relaxes the
transcribing DNA strand.

Transcription elongation
As it moves, the polymerase maintains a

short melted region aproximately 11-16

bases long and contains a DNA-RNA hybrid
about 9 bp long
The movements allows the DNA
unwinding ahead of the advancing
polymerase.
The DNA closes up again behind it.
Binding happens one at a time

Transcription termination
-independant

terminators consist of
an inverted repeats
that caused a hairpin
to form.
This destabilizes the
RNA-DNA complex
A string of TTTTTs in
the non template DNA
strand results in a
weak rU-dA base pairs

Transcription termination

Transcription termination
Whenever the transcript has grown long

enough, the Rho protein binds at the

upstream rut site.
The Rho protein translocates down the
mRNA
Interaction with the RNA polymerase
complex will stimulate release of the
transcript.

Transcription termination

Operons : Fine control of bacterial

transcription

lac operon
Thelacoperon(lactose operon) is

anoperonrequired for the transport

andmetabolismoflactoseinEscherichia
coliand some otherenteric bacteria.
It has three adjacent structural
genes,lacZ,lacY, andlacA.
The genes encode-galactosidase,lactose
permease, andgalactoside acetyltransferase,
respectively.

lac operon

The lacI gene encodes for the repressor

protein.
Further downstream there are the promoter
region and operator region.

lac operon
In its natural environment, thelacoperon

allows for the effective digestion of lactose.

Lactose permease, which is embedded in the
cytoplasmic membrane, transports lactose
into the cell.
-galactosidase, a cytoplasmic enzyme,
subsequently cleaves lactose
intoglucoseandgalactose.
Together with galactoside transacetylase, their
transcription can produced a mRNA called
polycistronic message.

lac operon Negative control

In cell, it would be wasteful to produce the

enzymes when there is no lactose available

or if there is a more preferable energy source
available, such as glucose.
Without lactose in the cell, the repressor
protein binds to the operator and prevents
the read through of RNA polymerase into the
three structural genes (lac Z, lacY and lacA).

lac operon Positive control

With lactose in the cell, lactose in a form of

allolactose binds to the repressor. This

causes a structural change in the repressor
and it loses its affinity for the operator.
RNA polymerase can then bind to the
promoter and transcribe the structural
genes.
In this system lactose acts as an effector
molecule.

lac operon
Lactose is not the preferred carbohydrate

source forE. coli. If lactose and glucose are

present, the cell will use all of the glucose
before thelacoperon is turned on.
This type of control is termed catabolite
repression.

lac operon
To prevent lactose metabolism, a second level

of control of gene expression exists.

The promoter of thelac operon has two
binding sites. One site is the location where
RNA polymerase binds. The second location
is the binding site for a complex between
thecatabolite activator protein (CAP)and
cyclic AMP (cAMP).

lac operon Negative control

The binding of the CAP-cAMP complex to the

promoter site is required for transcription of

thelacoperon.
The presence of this complex is closely
associated with the presence of glucose in
the cell.
As the concentration of glucose increases the
amount of cAMP decreases.
As the cAMP decreases, the amount of
complex decreases.

lac operon Positive control

This decrease in the complex inactivates the

promoter, and thelacoperon is turned off.

Because the CAP-cAMP complex is needed for
transcription, the complex exerts apositive
controlover the expression of the lacoperon.
Due to usage, concentration of glucose
decreases causing the amount of cAMP start to
increase.
As the cAMP increases, the amount of complex
increases and the lacoperon turns on.

trp operon

trp operon
The trp operon produces products that are used

to manufacture an amino acid, tryptophan.

The pathway controlled by the trp operon is an
example of an anabolic pathway.
The lac operon controls a catabolic pathway,
because it breaks down complex molecules to
release energy for biological work.
The lac operon is controlled both by the
abundance of its substrate, lactose, and by the
abundance of glucose, an alternative to glucose.
The trp operon is controlled by the amino acid
itself.

trp operon
This operon contains five structural genes:

trp E, trp D, trp C, trp B, and trp A, which

encodetryptophan synthetase.
It also contains a promoter where RNA
polymerase binds to a repressor gene (trp R)
which synthesizes a specific protein.
The protein that is synthesized by trp R then
binds to the operator which then causes the
transcription to be blocked.

trp operon
In thelac operon, allolactose binds to the

repressor protein, allowing gene transcription,

while in thetrpoperon, tryptophan binds to the
repressor protein effectively blocking gene
transcription.
In both situations, repression is that of RNA
polymerase transcribing the genes in the operon.
Also unlike thelacoperon, thetrpoperon
contains a leader peptide and
anattenuatorsequence which allows for graded
regulation.

trp operon

trp operon
Within the operon's regulatory sequence,

theoperatoris blocked by
therepressorprotein in the presence of
tryptophan (thereby
preventingtranscription) and is released in
tryptophan's absence (thereby allowing
transcription).
It is an example ofrepressible negative
regulationof gene expression.

trp operon
Attenuation is a second mechanism of

negative feedback in thetrpoperon.

The repression system targets the
intracellular trp concentration whereas the
attenuation responds to the concentration of
charged tRNAtrp.
The trpR repressor decreases gene
expression by altering the initiation of
transcription, while attenuation does so by
altering the process of transcription that's
already in progress.

trp operon
At the beginning of the transcribed genes of

thetrpoperon is a sequence of at least 130

nucleotides termed the leader transcript
(trpL).
The attenuation efficiency is correlated with
the stability of a secondary structure
embedded in trpL, this transcript includes
four short sequences designated 1-4, each of
which is partially complementary to the next
one.

trp operon
Thus, three distinct secondary structures (hairpins)

can form: 1-2, 2-3 or 3-4.

The hybridization of sequences 1 and 2 to form the 12 structure is rare because the RNA Polymerase waits
for a ribosome to attach before continuing
transcription past sequence 1.
The formation of a hairpin loop between sequences 23 prevents the formation of hairpin loops between
both 1-2 and 3-4.
The 3-4 structure is atranscription
terminationsequence (abundant in G/C and
immediately followed by several uracil residues), once
it forms RNA polymerase will disassociate from the
DNA and transcription of the structural genes of the
operon can not occur

trp operon
Attenuation is made possible by the fact that in

prokaryotes(which have nonucleus),

theribosomesbegintranslatingthemRNAwhile
RNA polymeraseis still transcribingthe DNA
sequence.
This allows the process of translation to affect
transcription of the operon directly.

ara operon

Thearaoperon codes for three enzymes that are required to

catalyze the metabolism of arabinose.

The three structural genes are arranged in an operon that is
regulated by thearaCgene product.
The operators arearaO1andaraO2. The operators lie
between thearaC and inducer site.
araI lies between the structural genes and the operator.
ThearaI1andaraI2are DNA-binding sites that, when
occupied by AraC, induce expression.

ara operon
Regulation of the arabinose operon is much more

complex than the lactose operon.

It is anoperonthat encodesenzymesneeded for
thecatabolismofarabinoseinEscherichia coli.
When arabinose is absent, there is no need to
express the structural genes.
AraCdoes this by having its repressor protein
binding simultaneously toaraIandaraO2.
As a result the intervening DNA islooped.
These two events block access to
thePBADpromoter which is a very weak promoter.

ara operon

ara operon
When arabinose is present, it binds

toAraCrepressor protein and allosterically

induces it to bind toaraIinsteadaraO2.
This complex is needed for RNA polymerase to bind
to the promoter and transcribe thearaoperon.
An additional feature needed is the CRP (formerly
known as CAP) + cyclic AMP that binds to the
initiator protein.
Therefore, in the presence of arabinose, both
theCRPcAMP complex and the AraCarabinose
complex must bind to the initiator region in order
forRNA polymeraseto bind to thepromoterand
transcribe thearaoperon.

ara operon
The structural genes (araB, araA,andaraD)

encode the metabolic enzymes that break

down arabinose are transcribed as a
multigenic mRNA.

ara operon