Regulation of gene expression in prokaryotes final

REGULATION OF GENE EXPRESSION
IN PROKAYOTES
BY
Dr. Ichha Purak
University Professor
Ranchi Women’s College,Ranchi
Email- purak.ichha@gmail.com
01/01/18 1Regulation of gene expression in prokaryotes

A gene is expressed through polypeptide synthesis. One gene directs
formation of one polypeptide chain. Concentration of particular protein
varies even in a simple bacterial cell ( E.coli) , with time and nature of
nutrients present in medium (environment) ,suggesting regulation of gene
expression.
NEED OF GENE REGULATION
The cells need to regulate gene expression to economize by not investing
energy and resources in synthesis of unnecessary RNAs (mRNA) and
proteins (enzymes), when not required.
Prokaryotes are relatively simple than complex multicellular eukaryotes,
where regulation is required for cell specialization, with specialized function

and behaviour of a cell is determined not only by what genes it possess, but
also by which of those genes are expressed at a given time.
Regulation of expression of gene is done by alternate switching on and
turning off of genes as per the need of the cell. This type of regulation
keeps order and prevent wastes.
Levels of Regulation
In prokaryotes the most significant level of regulation is at initiation of
transcription level whereas in eukaryotes gene expression is regulated at
several levels such as chromatin modeling, intiation of transcription, RNA
processing, Translation etc.

The basic concept for how transcription is controlled in bacteria was
given by Jacob and Monad (1961) by Operon model by studying
lactose metabolism.
According to this model, some prokaryotic genes involved in the
operation of a metabolic sequence are clustered.
This functionally related cluster of genes is regulated in a
co-ordinated fashion and constitute an operon.
The organization of different operons may vary, but generally all of
them include a regulator gene( closely or distantly placed ), operator
region, promoter site and a number of structural genes (cistron )

In prokaryotes there are two main types of regulation
1.Inducible Regulation- Generally for regulation of catabolic pathways
Inducer molecule is needed for switching on the operon. The product of
regulator gene ,the active repressor switch offs the operon .
Example –Lac (Lactose ) Operon: Negative control
2. Repressible Regulation Generally for regulation of anabolic
pathways, the product of regulator gene is inactive repressor ,So the
operon is switched.
Example Trp(Tryptophan) Operon: Positive control.

trp Operon Gene Gene Function
P/O
Promoter; operator sequence is found in the
promoter
trp L
Leader sequence; attenuator (A) sequence
is found in the leader
trp E Gene for anthranilate synthetase subunit
trp D Gene for anthranilate synthetase subunit
trp C Gene for glycerolphosphate synthetase
trp B Gene for tryptophan synthetase subunit
trp A Gene for tryptophan synthetase subunit
lac Operon Gene Gene Function
I Gene for repressor protein
P Promoter
O Operator
lac Z Gene for ß-galactosidase
lac Y Gene for ß-galactoside permease
lac A Gene for ß-galactoside transacetylase
Inducible system - the effector molecule interacts with the
repressor protein such that it can not bind to the operator
Repressible system - the effector molecule interacts with the repressor
protein such that it can bind to the operator
Components of
Lac Operon
Components
of
Trp operon

The addition of lactose leads to induction of operon because Lactose
(substrate) or allolactose binds to the Repessor , makes it inactive by
bringing conformational change and becomes unable to bind with
operator and so transcription of Lac Z,Y and A takes place to
synthesize polycistronic mRNA, which as a result complete translation
and synthesis of 3 enzymes -galactosidase, Permease andβ
Transacetylase required for lactose catabolism or degradation.
In this operon, when lactose is present , then only the enzymes are
required and so operon is induced to switch on.
In contrast when lactose is not present , the enzymes are not required
so operon is turned off. This regulation protects energy and resources.
THE LACTOSE OPERON

Repressor is not in a position to bind with the operator, RNA polymerase transcribes mRNA
Regulation of the lac operon in E. coli. The repressor of the operon is synthesized from the i gene.
The repressor protein binds to the operator region of the operon and prevents RNA polymerase
from transcribing the operon. In the presence of an inducer (such as the natural inducer,
allolactose) the necessary for utilization of -galactosides (such as lactose) as an energy source.β
repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the
operon and transcription proceeds. The resultant mRNA encodes the -galactosidase, permeaseβ
and transacetylase activities

The structural genes are :
Lac Z. Encoding ß-galactosidase, the enzyme which hydrolyses
disaccharide lactose to galactose and glucose
lac Y encoding a membrane bound enzyme ß-galactoside permease which
transports lactose across the cell membrane, thus enabling it to enter the
cell
lac A encoding a ß-galactoside transacetylase, an enzyme that transfers
acetyl group from Acetyl CoA to ß-galactoside. This enzyme is not directly
involved in lac catabolism.
Only lacz and lac y appear to be necessary for lactose catabolism
The repressor has a special affinity for 8 base pairs of operator , it
interacts with N7
and 2 amino sites of Guanine and 5 (CH3
) of thymine in
the central major groove of operator (O)

The Genetic control sequences are ;
Lac P . a promoter sequence, Approx 100 bp long , to which RNA
polymerase binds and initiates transcription
Lac O . an operator sequence, about 45 bp long overlapping the promoter ,
to which lac repressor can bind (Lac OP)
Lac t a terminator sequence signaling the termination of transcription.
lac i ( r ) the regulator gene lies at the left, has its own promoter and
terminator. Occupies first 26 bp of transcription unit. This regulatory gene
produces a protein termed as repressor. Lac i always expresses
constitutively
The long LacZ gene starts at base 39 and is followed by Lac Y and Lac A
and terminator. Promoter 100 bp, Operator 45 bp

Total length of Lac Operon is about 6100 nucleotides ( 0.2% of the genome ),
Regulator gene (1040 bp approx) and is closely linked to operon, but is
transcribed independently, proceeded by its own promoter and is followed
by its own terminator.
Structural gene cluster are co-ordinately controlled as a single unit that is
transcribed into a polycistronic mRNA
The Lac operon includes 3 regulator elements (control sequences ) and 3
protein coding structural genes.(Z,Y and A)
The Regulator gene ( i ) is not part of the Operon.

Repressor is diffusible protein and Regulator gene is not required to be
located adjacent to Operon it controls.

Repressor protein recognizes the operator site by covering it up,
prevents transcription.
The inducer can be a substrate of the operon (Allolactose ) or an
analogue of substrate.( Gratuitous inducer)
When the inducer is combined with repressor, the latter suffers a
conformational change which prevents it from blocking the operator
region, when the repressor is removed, transcription of structural
genes begin.
The lac operon is an example of negative control because the product
of regulator gene is active repressor which is able to block
transcription of structural genes by binding with until induction takes
place by an inducer.

COMPONENTS OF LAC OPERON
Total length – Regulator+ Promoter + Operator + Structural genes
+Terminator = 6100 bp of DNA , promoter-100 bp,Operator -45 bp
The Lac operon includes 3 regulator elements (control sequences ) and 3
protein coding structural genes.(Z,Y and A)

The lac operon is additionally regulated through binding of the cAMP-
receptor protein, CRP (also termed the catabolite activator protein, CAP) to
sequences near the promoter domain of the operon. The result is a 50 fold
enhancement of polymerase activity
Actually lac operon also contains some elements of positive control as well.
Positive control means some effector is required for transcription.
In lac system , the Catabolic Activator Protein (CAP ) is an effector. This
protein when combined with cyclic Adenosine Mono Phosphate (cAMP ),
facilitates the attachment of RNA Polymerase (Transcriptase ) to the operon.

The Promoter (P ) and Operator ( O ) region of Operon include CAP site ,
According to Schmitz (1981) CAP interacts not only with P region but also
at a second site with Operator ( O )
The lactose operon of E.coli. The lac Z,lac Y and lac A genes are
separated by short untranslated sequences

The cyclic AMP (cAMP ) has a hormone like action in Pro and Eukaryote
frequently, it is called the second messenger, a term denoting its
hormone type function.
When CAP is attached to CAP site , the transcriptase enzyme has a
chance to anchored to position P ( promoter ) site. The transcription is
initiated at the Operator sequence and continues through structural
genes until terminator is reached at the right end of the A gene.
The Lac repressor and CAP can not be simultaneously attached to the
operon.
Facilitating attachment of RNA Polymerase to Promoter site
Transcription begins at Operator, proceeds through Structural genes
Z,Y and A and then terminates.

Diagrammatic representation of role of CAP

Catabolite Repression of the lac Operon
Lactose is not the preferred carbohydrate source for E. coli. If lactose
and glucose are present, the cell will use all of the glucose before the
lac operon is turned on. This type of control is termed catabolite
repression. To prevent lactose metabolism, a second level of control of
gene expression exists. The promoter of the lac 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 the
catabolite activator protein (CAP) and cyclic AMP (cAMP). The binding
of the CAP-cAMP complex to the promoter site is required for
transcription of the lac operon.

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. This decrease in the complex inactivates the promoter, and
the lac operon is turned off. Because the CAP-cAMP complex is needed for
transcription, the complex exerts a positive control over the expression of
the lac operon.

THE TRYPTOPHAN OPERON
It is a good example of repressible Operon. In this type of Operon , the
structural genes are not transcribed in presence of end product
( Tryptophan) .
Trp R( i ) regulatory gene is not adjacent to the Trp Operon and its
immediate product , the trp R repressor is inactive and is unable to
recognize and bind to Operator sequence, thus the Operon is switched on.
However when tryptophan is produced ,it combines with Repressor to
produce active repressor complex which binds to the Operator and switch
off the structural genes. Thus in a repressible system, an inactive repressor
is activated by combining with the end product of the pathway. This end
product is some times refered as Co-repressor.
Neither lac or Trp repressor are subject to genetic regulation., Laci and Trpi
are transcribed all the time , irrespective of substrate or end product. These
are constitutive genes.

Regulation of the trp operon in E. coli. The trp operon is controlled by both a repressor protein
binding to the operator region as well as by translation-induced transcriptional attenuation. The
trp repressor binds the operator region of the trp operon only when bound to tryptophan. This
makes tryptophan a co-repressor of the operon. The trpL gene encodes a non-functional leader
peptide which contains several adjacent trp codons. The structural genes of the operon
responsible for tryptophan biosynthesis are trpE, D, C, B and A. When tryptophan level are high

The Biosynthesis of tryptophan in E.coli. Tryptophan is synthesized in five sequential reactions.
1.Anthranilic synthetase product of Gene Trp E converts Chorismic acid to Anthranilic acid
2.Phosphoribosyl transferase product of gene Trp D catalyzes conversion to Phosphoribosyl anthranilate
3.Indole glycerol phosphate synthetase product of gene Trp C catalyzes convesion to
carboxyphenylaminodeoxy ribulose phosphate
4.Tryptophan synthetase B product of gene TrpB protein catalyzes conversion to Indolglycerol phosphate
5.Tryptophan synthetase A protein product of Gene Trp A catalyzes conversion to Tryptophan

The trp operon is a repressible system. The primary difference between
repressible and inducible systems is the result that occurs when the
effector molecule binds to the repressor. With inducible systems, the
binding of the effector molecule to the repressor greatly reduces the
affinity of the repressor for the operator, the repressor is released and
transcription proceeds. The lac operon is an example of an inducible
system. With repressible systems, the binding of the effector molecule to
the repressor greatly increases the affinity of repressor for the operator
and the repressor binds and stops transcription. Thus, for the trp operon ,
the addition of tryptophan (the effector molecule) to the E. coli
environment shuts off the system because the repressors binds at the
operator

Attenuation
The trp operon is regulated in part by a repressor that, when bound to a
tryptophan (in its presence ) , blocks transcription.
However, transcriptional attenuation provide an additional level of control
that results in more stringent regulation, that could be achieved by
repression of initiation alone.
The site of attenuation is located 162 nucleotides downstream of the
transcription start site.
If tryptophan is abundant, most transcription terminate at this site, only if
tryptophan is scarce does transcription continue to yield functional Trp
mRNA.
The mechanism of attenuation depends on the fact that translation in
bacteria is coupled with transcription, so ribosome begin translating at
5’end of mRNA while still it is being transcribed

Thus the rate of translation can affect the structure of the growing RNA
chain, which in turn determines whether transcription can continue.
Transcription termination is signaled by a stem-loop structure that forms by
complementary base pairing between two specific sequence of the growing
Trp mRNA chain.
This structure forms if translation of the growing chain is proceeding at a
normal rate as it does when tryptophan is present in adequate supply.
If Tryptophan is scarce , however protein synthesis stalls at a critical region
of the message. If this occurs, ribosome bound to the mRNA block
formation of the transcription terminating stem loop, allowing Trp mrna
synthesis to continue
The critical region of Trp mRNA contains two adjacent tryptophan codons, so
the rate of translation is highly dependent on tryptophan levels, this is the link
between transcriptional attenuation and availability of tryptophan.

If Tryptophan levels in cell are low, ribosome stalls at this point and
transcription of mRNA(trp) continue. If tryptophan is abundant translation
continues and transcription is terminated
In addition to Repression, Attenuator sequence, prematurely terminates
transcription at high level of tryptophan
promoter leader sequence structural genes
Attenuator
Figure 1

Figure 2
Figure 3

Attenuation of the trp Operon
One element of the trp operon is the leader sequence (L) that in immediately 5'
of the trpE gene. This sequence about 160 bp is size also controls the
expression of the operon through a process called attenuation. This sequence
has four domains (1-4). Domain 3 (nucleotides 108-121) of the mRNA can base
pair with either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134).
If domain 3 pairs with domain 4, a stem and loop structure forms on the mRNA
and transcription stops. This structure forms when the level of tryptophan is high
in the cell. If domain 3 pairs with domain 2, then the stem and loop structure
does not form and transcription continues through the operon, and all of the
enzymes required for tryptophan biosynthesis are produce. These events occur
when tryptophan is low in the cell.

If domain 4 is deleted, the stem and loop structure can not form and
transcription of the remainder of the operon will occur even in the presence of
tryptophan. Domain 4 is called the attenuator because its presence is required
to reduce (attenuate) mRNA transcription in the presence of high levels of
tryptophan.
Domain 1 is also an important component of the attenuation process. The
section of the leader sequence encodes a 14 amino acid peptide that has two
tryptophan residues.
How does this entire attentuation process work? We will discuss the molecular
events that occur under conditions of high and low tryptophan.

trp Operon Transcription Under Low Levels of
Tryptophan
Under low cellular levels of tryptophan, the translation of the short peptide
on domain 1 is slow. Because of the slow translation, domain 2 does not
become associated with the ribosome. Rather domain 2 associates with
domain 3. This structure permits the continued transcription of the
operon. Then the trpE-A genes are translated, and the biosynthesis of
tryptophan occurs.

trp Operon Transcription Under High Levels
of Tryptophan
When the cellular levels of tryptophan are high, the levels of the
tryptophan tRNA are also high. Immediately after transcription, the
mRNA moves quickly through the ribosome complex and the small
peptide is translated. Translation is quick because of the high levels
of tryptophan tRNA. Because of the quick translation, domain 2
becomes associated with the ribosome complex. Then domain 3
binds with domain 4, and transcription is attenuated because of the
stem and loop formation.

THE END
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Regulation of gene expression in prokaryotes final

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