The L-arabinose operon , also called fig or araBAD operon , is a operon required for the breakdown of five carbon sugars, L-arabinose, at Escherichia coli . Operon L-arabinose contains three structural genes: araB , araA , araD (collectively known as araBAD ), which encodes three metabolic enzymes necessary for L-arabinose metabolism. Ara (ribulokinase), AraA (isomerase), Ara (epimerase) produced by this gene catalyze the conversion of L-arabinose into the intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.
The structural genes of the L-arabinose operon are transcribed from the general promoter to a single transcript of mRNA. The expression of the L-arabinose operon is controlled as a single unit by the product of the regulatory gene araC and the protein catabolite activator complex (CAP) -cAMP. AraC regulating proteins are sensitive to arabinose levels and play multiple roles both as activators in the presence of arabinose and repressors in the absence of arabinose to regulate expression araBAD . The araC protein not only controls the expression araBAD , but also self-regulates the self-expression at a high AraC level.
Video L-arabinose operon
Structure
Operon L-arabinose consists of structural genes and regulator regions including the operator region ( araO 1 , araO 2 ) and region of initiator ( araI 1 , araI 2 ). Structural genes, araB , araA and araD , encode enzymes for L-arabinose catabolism. There is also a CAP binding site in which the CAP-cAMP complex binds and facilitates catabolite repression, and generates positive regulation when the cells are deprived of glucose.
The regulatory gene, araC , lies in the upper L-arabinose operon and encodes the Arab-responsive AraC protein regulatory protein regulator. Both araC and araBAD have discrete promoters in which RNA polymerase binds and initiates transcription. araBAD and araC are transcribed in the opposite direction of the promoter araBAD ( P BAD ) and araC promoters ( P C ) respectively.
Maps L-arabinose operon
Function
- encodes L-arabinose isomerase, which catalyzes the isomerization between L-arabinose and L-ribulose.
- araB encodes ribulokinase, which catalyzes the phosphorylation of L-ribulose to form L-ribulose-5-phosphate.
- araD encodes L-ribulose-5-phosphate 4-epimerase, which catalyzes the epimerization between L-ribulose 5-phosphate and D-xylulose-5-phosphate.
Both L-ribulose 5-phosphate and D-xylulose-5-phosphate are metabolites of the pentose phosphate pathway, which links the 5-carbon sugar metabolism with 6-carbon sugar.
Rule
The L-arabinose system is not only under the control of CAP-cAMP activators, but is also positively or negatively regulated by binding of the AraC protein. AraC serves as a homodimer, which can control transcription of araBAD through interaction with the operator and the initiator region of the L-arabinose operon. Each AraC monomer consists of two domains including DNA binding domains and dimerization domains. Domain dimerisation is responsible for arabinose-binding. AraC undergoes a conformational change in arabinose-binding, in which, it has two different conformations. This conformation is purely determined by the binding of the arabinose allosteric inducer.
AraC can also negatively mengegegulasi its own expression when the concentration of AraC becomes too high. AraC synthesis is suppressed by binding of AraC dimer to operator region ( araO 1 ).
Negative settings araBAD
When arabinose is absent, cells do not require fig BAD products to break up arabinose. Therefore, dimeric AraC acts as a repressor: one monomer binds to the gene carrier araBAD ( araO 2 ), another monomer binds to a DNA half site known as araI 1 . This leads to the formation of DNA loops. This orientation prevents RNA polymerase from binding to the promoter araBAD . Therefore, structural gene transcription araBAD is inhibited.
Positive settings araBAD
The expression of the operon araBAD is activated in the absence of glucose and in the presence of arabinose. When arabinose is present, AraC and CAP work together and function as activators.
Via AraC
AraC acts as an activator in the presence of arabinose. AraC undergoes a conformational change when arabinose binds the dimerized domain of AraC. As a result, the AraC-arabinose complex falls from araO 2 and breaks the circle of DNA. Therefore, it is more advantageous for AraC-arabinose to bind to two adjacent half-DNA sites: araI 1 and araI 2 in the presence of arabinose. One monomer binds araI 1 , the rest of the binding monomer araI 2 - in other words, the binding of AraC to araI 2 allosterically caused by arabinose. One AraC monomer locates near the araBAD promoter in this configuration, which helps recruit RNA polymerase to the promoter to initiate transcription.
Via CAP/cAMP (catabolite repression)
CAP acts as a transcriptional activator only in the absence of E. coli ' preferably sugar, glucose. When glucose is absent, high levels of complex CAP protein/cAMP bind to the CAP binding site, a site between araI 1 and araO 1 . Binding CAP/cAMP is responsible for opening the DNA loop between araI 1 and araO 2 , increasing binding affinity of protein AraC for araI 2 and thus promoting RNA polymerase to bind the promoter araBAD to enable the expression araBAD required to metabolize L- arabinose.
AraC Autoregulation
The expression araC is negatively regulated by its own protein product, AraC. Excess AraC binds to the gene operator araC , araO 1 , at the high AraC level, which physically blocks the RNA polymerase from accessing araC promoter. Therefore, the AraC protein inhibits its own expression at high concentrations.
Use in protein expression system
Operon L-arabinose has been the focus of molecular biology research since 1970, and has been extensively studied at the genetic, biochemical, physiological and biotechnical levels. L-arabinose operon has been commonly used in protein expression systems, since promoters can be used to produce targeted expression under strict regulation. By combining the araBAD promoter to the desired gene, the target gene expression can be self-regulated by arabinose: for example, the plasmid pGLO contains the green fluorescent protein gene under control. > Promoter P BAD , enabling GFP production to be induced by arabinose.
See also
- Operon
- Catabolism
- Cursing of wordbolit
Other operon systems in E. coli :
- gal operon
- gab operon
- lac operon
- trp operon
References
External links
- Modern Genetic Analysis by Griffiths, A. J et al. (online textbook)
- Biochemistry by Berg, J.M et al. (online textbook)
- An Introduction to Genetic Analysis by Griffiths, A. J et al. (online textbook)
Source of the article : Wikipedia
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