Arabinose operon - Structure, function, postive, negative and auto regulation

1. ara operon
Dr. s. sivasankara narayani
Assistant professor
Department of Microbiology
Ayya nadar Janaki ammal college
sivakasi
19-01-2021Dr.SS ., MRSB (UK)

2. UNIT - IV
Regulation of gene expression in bacterial system : Operon concept
– lac, trp, ara operon in E. coli - *DNA methylation and
heterochromatization. Transposons (brief account only).
19-01-2021Dr.SS ., MRSB (UK)

3. L-arabinose operon
• The L-arabinose operon, also called the ara or araBAD operon, is an operon required for the breakdown of the five-carbon sugar L-
arabinose in Escherichia coli.[1]
• The L-arabinose operon contains three structural genes: araB, araA, araD (collectively known as araBAD), which encode for
three metabolic enzymes that are required for the metabolism of L-arabinose.[2]
• AraB (ribulokinase), AraA (an isomerase), AraD (an epimerase) produced by these genes catalyse conversion of L-arabinose to
an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.[2]
• The structural genes of the L-arabinose operon are transcribed from a common promoter into a single transcript, a mRNA.[3]
• The expression of the L-arabinose operon is controlled as a single unit by the product of regulatory gene araC and the catabolite
activator protein (CAP)-cAMP complex.[4]
• The regulator protein AraC is sensitive to the level of arabinose and plays a dual role as both an activator in the presence of arabinose
and a repressor in the absence of arabinose to regulate the expression of araBAD.[5]
• AraC protein not only controls the expression of araBAD but also auto-regulates its own expression at high AraC levels
19-01-2021Dr.SS ., MRSB (UK)

4. Structure
•
L-arabinose operon is composed of structural genes and regulatory regions including
the operator region (araO1, araO2) and the initiator region (araI1, araI2).[7] The
structural genes, araB, araA and araD, encode enzymes for L-arabinose catabolism.
There is also a CAP binding site where CAP-cAMP complex binds to and
facilitates catabolite repression, and results in positive regulation of araBAD when
the cell is starved of glucose.
• The regulatory gene, araC, is located upstream of the L-arabinose operon and
encodes the arabinose-responsive regulatory protein AraC.
Both araC and araBAD have a discrete promoter where RNA polymerase binds and
initiates transcription.[4] araBAD and araC are transcribed in opposite directions
from the araBAD promoter (PBAD) and araC promoter (PC) respectively.
19-01-2021Dr.SS ., MRSB (UK)

5. 19-01-2021Dr.SS ., MRSB (UK)

6. Function
• araA encodes L-arabinose isomerase, which
catalyses isomerization between L-arabinose and L-ribulose.
• araB encodes ribulokinase, which catalyses phosphorylation of L-
ribulose to form L-ribulose-5-phosphate.
• araD encodes L-ribulose-5-phosphate 4-epimerase, which
catalyses epimerization between L-ribulose 5-phosphate and D-
xylulose-5-phosphate
19-01-2021Dr.SS ., MRSB (UK)

7. 19-01-2021Dr.SS ., MRSB (UK)
Substrate Enzyme(s) Function Reversible Product
L-arabinose AraA Isomerase Yes L-ribulose
L-ribulose AraB Ribulokinase No
L-ribulose-5-
phosphate
L-ribulose-5-
phosphate
AraD Epimerase Yes
D-xylulose-5-
phosphate
Metabolic pathway of L-arabinose via the action of three enzymes, which are encoded by
the araBAD operon
Catabolism of arabinose in E. coli
Both L-ribulose 5-phosphate and D-xylulose-5-phosphate are metabolites of the pentose
phosphate pathway, which links the metabolism of 5-carbon sugars to that of 6-carbon sugars

8. Regulation
• The L-arabinose system is not only under the control of
CAP-cAMP activator, but also positively or negatively
regulated through binding of AraC protein.
• AraC functions as a homodimer, which can control
transcription of araBAD through interaction with the
operator and the initiator region on L-arabinose operon.
• Each AraC monomer is composed of two domains
including a DNA binding domain and
a dimerisation domain.[9] The dimerisation domain is
responsible for arabinose-binding.
• AraC undergoes conformational change upon arabinose-
binding, in which, it has two distinct conformations.[6]
• The conformation is purely determined by the binding
of allosteric inducer arabinose.[11]
• AraC can also negatively autoregulate its own expression
when the concentration of AraC becomes too high.
• AraC synthesis is repressed through binding of dimeric
AraC to the operator region (araO1 )
19-01-2021Dr.SS ., MRSB (UK)

9. Negative regulation of araBAD
• When arabinose is absent, cells do not need the araBAD products
for breaking down arabinose. Therefore, dimeric AraC acts as a
repressor: one monomer binds to the operator of the araBAD gene
(araO2), another monomer binds to a distant DNA half site known
as araI1.[12] This leads to the formation of a DNA loop.[13] This
orientation blocks RNA polymerase from binding to
the araBAD promoter.[14] Therefore, transcription of structural
gene araBAD is inhibited
19-01-2021Dr.SS ., MRSB (UK)

10. 19-01-2021Dr.SS ., MRSB (UK)

11. Positive regulation of araBAD
• Expression of the araBAD operon is activated in the absence of
glucose and in the presence of arabinose. When arabinose is
present, both AraC and CAP work together and function as
activators
19-01-2021Dr.SS ., MRSB (UK)

12. Via AraC
• AraC acts as an activator in the presence of arabinose. AraC undergoes a
conformational change when arabinose binds to the dimerization domain
of AraC. As a result, the AraC-arabinose complex falls off from araO2 and
breaks the DNA loop. Hence, it is more energetically favourable for
AraC-arabinose to bind to two adjacent DNA half sites: araI1 and araI2 in
the presence of arabinose. One of the monomers binds araI1, the
remaining monomer binds araI2 - in other words, binding of AraC
to araI2 is allosterically induced by arabinose. One of the AraC monomers
places near to the araBAD promoter in this configuration, which helps to
recruit RNA polymerase to the promoter to initiate transcription
19-01-2021Dr.SS ., MRSB (UK)

13. Via CAP/cAMP (catabolite repression)
• CAP act as a transcriptional activator only in the absence of E.
coli's preferred sugar, glucose.[18] When glucose is absent, high
level of CAP protein/cAMP complex bind to CAP binding site, a site
between araI1 and araO1.[19] Binding of CAP/cAMP is responsible
for opening up the DNA loop between araI1 and araO2, increasing
the binding affinity of AraC protein for araI2 and thereby
promoting RNA polymerase to bind to araBAD promoter to switch
on the expression of the araBAD required for metabolising L-
arabinose
19-01-2021Dr.SS ., MRSB (UK)

14. 19-01-2021Dr.SS ., MRSB (UK)

15. Autoregulation of AraC
• The expression of araC is negatively regulated by its own protein
product, AraC. The excess AraC binds to the operator of
the araC gene, araO1, at high AraC levels, which physically blocks
the RNA polymerase from accessing the araC promoter.
• Therefore, the AraC protein inhibits its own expression at high
concentrations
19-01-2021Dr.SS ., MRSB (UK)

16. 19-01-2021Dr.SS ., MRSB (UK)

17. Use in protein expression system
• The L-arabinose operon has been a focus for research in molecular biology since 1970,
and has been investigated extensively at its genetic, biochemical, physiological and
biotechnical levels.[3] The L-arabinose operon has been commonly used in
protein expression system, as the araBAD promoter can be used for producing targeted
expression under tight regulation. By fusing the araBAD promoter to a gene of interest,
the expression of the target gene can be solely regulated by arabinose: for example,
the pGLO plasmid contains a green fluorescent protein gene under the control of
the PBAD promoter, allowing GFP production to be induced by arabinose
19-01-2021Dr.SS ., MRSB (UK)

18. sivasan91@gmail.com
19-01-2021Dr.SS ., MRSB (UK)

19. QUESTIONS TO THINK
• Operon
• Structural genes of arabinose operon
• Functional genes of arabinose operon
• Mechanism behind positive regulation of ara operon
• Mechanism behind negative regulation of ara operon
19-01-2021Dr.SS ., MRSB (UK)

20. 19-01-2021Dr.SS ., MRSB (UK)