arabinose operon and their detalied explanation about the operon conceptt and their regulation both positive and negative and the detailed explanation of the promoter ,operator,inducer,structural gene,arac protein
3. ARABINOSE
Operon is a unit of genetic material that functions in a coordinated manner by means of
an operator, a promoter and structural genes that are transcribed together
Operon model was first proposed by jacob and monod in 1961 to explain the regulation
OF genes encoding the enzymes required for lactose utilization in e.coli
First studied by Ellis Englesberg
Arabinose operon can be regulated both positively and negatively
Arabinose operon is the operon system found in bacterial cell which is used to
breakdown the arabinose
Provides energy to the cell by the breakdown of arabinose into xylulose phosphate
Provides energy in the form of carbon source
Main purpose of arabinose operon is to break the complex arabinose molecule into
xylulose phosphate which then enters the metabolic pathway namely the pentose
phosphate pathway
Carries the number of genes like regulatory ,promoter,operator,inducer and structural
genes for breakdown of L-Arabinose into xylulose phosphate
L-arabinose is an arabinose and is an aldopentose – a monosaccharide containing 5
carbon atoms, and including an aldehyde (CHO) functional group
4. It is a 5 carbon sugar used as alternative carbon and energy source by E.coli
Encoded by the araBAD operon and the ara operon is regulated by the Ara c protein
Operon encodes a single polycistronic mrna with 3 open reading frames
Catabolic pathway for arabinose
Expression of araDAB is highly regulated
Exhibits the phenomenon of catabolite repression
Conversion of arabinose into xylulose phosphate is carried by structural genes ara –
B,Aand D
Transcribes mRNA by the kinase,isomerase and epimerase
Structure of arabinose operon is linear consist of 4 specific genes with a catabolite active
site
The 3 structural genes are arranged in an operon that is regulated by the araC gene
product
The araC (regulatory protein) regulates its own synthesis by repressing transcription of
its gene. This phenomenon is called autoregulation.
5. Two transport system
Lower affinity transport system –
araE gene product is bound to the
inner membrane
Utilizes electrochemical potential
to transport arabinose
araFGH genes –arabinose –
specific components of high –
affinity transport system,an ABC
transporter
Cassette transporter family
AraF- periplamic arabinose –
binding protein,
AraH- membrane bound
component
araJ –induced by arabinose
7. ARABINOSE
Defines as the number of genes like
regulatory,promoter,operator,inducer and structural genes
For breakdown pg l-arabinose into xylulose phosphate
This conversion carried `out by structural genes (ara-B,Aand D)
Transcribes mRNA by kinase,isomerase,epimerase catalyze the
conversion and then enters the phosphate pathway
Three structural genes ara A,arB,araD
Its main function is to encode the metabolic enzymes
Metabolic enzymes are kinase,isomerase,epimerase
These metabolic enzymes breakdown the non glucose
Arabinose to produce a “multigenic or polycistronic mRNA.
Arabinose isomerase –encoded by araA-coverts arabinose to ribulose
8. STRUCTURAL GENES
Ribulokinase –encoded by araB – phosphorylates ribulose
Ribulose -5-phosphate epimerase –encoded by araD—converts ribulose -5-
phosphate to xylulose -5-phosphate which can then be metabolized via the
pentose phosphate pathway
the expression of l-arabinose opron is controllred as a single unit by the
product pf regulatory gene arac and the catabolite activator protein
(CAP-CAMP complex)
Regulator protein AraC is sensitive to level of arabinose
Plays a dual role as both activator in the presence of arabinose
Repressor in the absence of arabinose to regulate the expression of ara
BAD.
Ara c protein not only controls the expression of araBAD but also auto –
regulates its own expression at high AraC levels
Operator region (arao1 , arao2)
9. CONT…..
Initiator region (araI1,araI2)
Also a CAP binding site
CAP-cAMP complex binds to and facilitate catabolite repression
Resultin potArabinose isomerase –encoded by araA-coverts arabinose to ribulose
Ribulokinase –encoded by araB – phosphorylates ribulose
Ribulose -5-phosphate epimerase –encoded by araD—converts ribulose -5-phosphate to
xylulose -5-phosphate which can then be metabolized via the pentose phosphate pathway
E regulation of araBAD when the cell is starved of gulcose
Regulatory gene araC is located upstream of l-arabinose operon
Encodes arabinose-responsive regulatory protein regulatory protein Ara C.
Both araC and araBAD have a discrete promoter
RNA polymerase binds and initiates transcription
Transcribed in opposite directions from the ara BAD promoter (pBAD) and araC
promoter (PC)
10. Substrate enzymes Function Reversible Product
L-arabinose Ara A Isomerase Yes L-ribulose
L-ribulose AraB Ribulokinase No L-ribulose 5-
phosphate
L-ribulose 5-
phosphate
Ara D Epimerase Yes D-xylulose 5-
phosphate
11. INDUCER GENE
inducer gene in arabinose operon namely ara I
gene
Trancription
Inducer molecule is the arabinose
Binds with the repressor protein to induce the
transcription of the gene into mRNA
Presence of inducer the arabinose operon
works positively
Absence of inducer works negatively
12. CATABOLITE ACTIVE SITE
Activator site refers as “CAP”.
CAP stand for “catabolite activator protein”
Activates the efficiency pf transcription rate by promoting
the effective binding of RNA polymerase to promoter
region
When availability of glucose is high with low arabinose
ATP will not convert into cAMP.
Availability of glucose is low with high arabinose ATP will
convert to cAMP
Bind to CAP to activate transcription of mRNA
Cyclic cAMP plus CAP form a complex which binds to
CAPregion of the operon
13. OPERATOR GENE
Two operatpr gene o1 and o2
These operate araBAD Mrna synthesis
Positive regulation glucose is low and arabinose
is high the repressor protein (arac) will get
activated by the arabinose
Promote the synthesis of araBAD mRNA
Negative regulation glucose is high and
arabinose is low the repressor protein (araC)
Not promote the synthesis of araBAD Mrna.
14. PROMOTER GENE
o Two promoter genes in arabinose operon
(pBAD and Pc.
o pBAD is the promoter site of ara BAD structural
genes
o Promotes the synthesis of ara BAD Mrna
o Pc is the promoter site of araC regulatory gene
o Promotes the synthesis of AraC repressor
protein
o Ara C repressor exits in active P1state in the
absence of inducer
o Presence of inducer exist in inactive P2state
15. REGULATORY GENE
Ara c is the only regulatory gene
Encodes the Ara c protein acts as repressor
Ara c protein regulates the arabinose both positively and
negatively
When araC protein binds with operator it repress the synthesis of
araBAD mRNA
Not promote the binding of RNA polymerase to the promoter
region
Ara c repressor protein binds witg the inducer
Arabinose ,then complex bevcomes activated
This activation promote the attachment of RNA polymerase to the
promoter region
Leads to the synthesis of araBAD mRNA
16. REGULATION
L-arabinose is not only under the control of CAP-Camp activator but also
positively or negatively regulated
Through binding of Ara C protein
Ara c functions as a homodimer
Can control transcription of ara BAD through interaction with the operator and
the initiator region on l-arabinose operon
Each araC monomer is composed of 2 domains including DNA binding domain
and dimerisation domain
Dimerisation domain is responsible for arabinose –binding
Ara c undergoes conformational changes upon arabinose-binding in which has
two distinct conformations
Conformation is purely determined by binding of allosteric inducer arabinose
Ara c can also negatively autoregulate its own expression
When concentration of araC becomes too high
Ara C synthesis is represed through binding of dimeric Ara c to the operator
region (arao1)
17. POSITIVE REGULATION
Synthesis of mRNA
The araBAD mRNA will produce
In positive condition it is regulated by two conditions
Expression of araBAD operon is activated in the
absence of glucose and in the presence of arabinose
When arabinose is present ,both Ara c and CAP work
together and function as activators
CASE I: when both inducer and repressor protein is
adsent.
18. CASE I
When it is absent there will be no repression of the arabinose operon.
In this condition the RNA polymerase will bind with the specific promoter
region and transcribe ara-BAD genes to form mRNA.
Rate of transccribing mRNA is much slower
19. CASE II
When both inducer and repressor are present:
The inducer (arabinose) will bind with repressor protein to regulate the mRNA
transcription
Ara c protein by forming a complex with the inducer will not able to form a loop
Arabinose binds with ara c dimer and changes its structural configuration
This change will allow the RNA polymerase to transcribe the araBAD genes form
Mrna this will further translate into proteins
20. NEGATIVE REGULATION
When arabinose is absent cells do not need the ara BAD products for breaking
down arabinose
Negative term indicates that no transcription occurs
Case III: when only repressor protein is present :
Main role of arabinose operon is to breakdown the arabinose
21. CASE III
If there is no arabinose there will be no
transcription and translation of the DNA
molecules
Arabinose acts as an “Inducer” binds with
repressor pprotein ARA-C to inactivate it
But in absence of inducer the repressor protein
will produced by the ara-c gene
Ara-c repressor protein forms a dimer with the
operator and the inducer gene by forming a
loop
the loop formation will not allow the RNA
polymerase to transcribe the ara –BAD genes to
form mRNA.
22. CAP /CAMP (CATABOLITE REPRESSION)
Cap act as a transcriptional activator only in the
absence of e.coli preferred sugar ,glucose
Glucose is absent ,high level of cap protein /cAMP
complex bind to CAP binding site
Between ara I1 and ara o1
These binding is responsible for opening up the DNA
loop between ara I1 and ara o2
Increasing the binding affinity of araC protein for
araI2
Promoting RNA polymerase to bind to ara BAD
promoter to switch on the expression of the aea BAD
required for metabolising L-ARABINOSE
23. Operon system can be controlled by both
activation and repression
Arabinose will bind with repressor and
inactivates it into the p2 state which can bind
with the inducer and the operator gene
Arabinose operon is switched off in the
presence of repressor
Swtiched on in the presence of inducer
24. DUAL NATURED ARA C GENE PRODUCT
In absence of arabinose ara c protein acts as
a repressor (c rep) and binds to ara 0
locus,which blocks transcription (negative
control)
The addition of arabinose causes it to bind to
the protein to convert it to an activator protein
(c act)
This then binds to ara I and stimulate
transcription (positive control)
25. MUTATIONS IN ARABINOSE OPERON
A mutation in the ara A gene will cause the
bacterial cel to become arabinose negative
Bacteria no longer uses arabinose as
carbon source
araB gene mutation results the same
araD gene mutation results in cell death
Mutation of ara c gene causes the promoters
(pBAD) and pC to become inactive
permanently repressed
26. AUTOREGULATION OF ARA C
The expression of ara c is negatively
regulated by its own protein product ,araC
The excess araC binds to the operator of the
araC gene ,ara O1 at high Ara C levels which
physically blocks the RNA polymerase from
accessing the ara C promoter
araC protein inhibits its own expression at
high concentrations
27. USE IN PROTEIN EXPRESSION SYSTEM
Been focus for research in molecular biology
Extensively at its genetic
biochemical,physiology and biotechnical levels
L – arabinose operon has been commonly used
in protein expression system
araBAD promoter can be used for producing
targeted expression under tight regulation
For example the pGLO plasmid contains a
green fluorescent protein gene under the control
of the p BAD promoter allowing GFP production
to be induced by arabinose.
28. QUESTIONS
1. What is operon and who discovered
2. What is CAP and Camp
3. Role of repressor
4. Role of inducer
5. Positive regulation
6. Negative regulation
7. Role of ara c protein
8. Enzymes involved in arabinose operon
9. What is autoregulation
10. Role of operator gene and promoter gene