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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] 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 (Isomerase), AraD (Epimerase) produced by these genes catalyze conversion of L-arabinose to an intermediate of pentose phosphate pathway, D-xylulose-5-phosphate.Cite error: The opening <ref> tag is malformed or has a bad name (see the help page).

The structural genes of the L-arabinose operon are transcribed from a common promoter into a single transcript i.e. mRNA.[3] The expression of 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 regulates 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.[6]

Structure

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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 binds to and facilitates catabolite repression. i.e. positive regulation of araBAD.[8]

Structure of L-arabinose operon of E.coli

The regulatory gene,araC, is located upstream of L-arabinose operon and is encoded for arabinose-responsive regulatory protein AraC. Both araC and araBAD have a discrete promoter where RNA polymerase bound and initiate transcription.Cite error: The opening <ref> tag is malformed or has a bad name (see the help page). In which, araBAD and araC are transcribed in opposite direction from the araBAD promoter (PBAD) and araC promoter (PC) respectively.Cite error: The opening <ref> tag is malformed or has a bad name (see the help page).

Function

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Metabolic pathway of L-arabinose via the action of three enzymes, which are encoded by Ara operon gene.
Catabolism of arabinose in E. coli
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

In which, both L-ribulose 5-phosphate and D-xylulose-5-phosphate are involved in the pentose phosphate pathway and produce reducing power. Cite error: The opening <ref> tag is malformed or has a bad name (see the help page).

Regulation

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Structure of AraC monomer

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 composed of two domains including a DNA binding domain and a dimerization domain.[9] The dimerization domain is responsible for arabinose-binding.[10] AraC undergoes conformational change upon arabinose-binding, in which, it has two distinct conformations.Cite error: The opening <ref> tag is malformed or has a bad name (see the help page). The conformation is purely determined by the binding of allosteric inducer arabinose. [11]

AraC can also negatively autoregulates 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).

Negative regulation of araBAD

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Negative regulation of L-arabinose operon via AraC protein

When arabinose is absent, cells do not need PBAD product for breaking down arabinose. Therefore, dimeric AraC acts as a suppressor by which 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.[15]

Positive regulation of araBAD

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Positive regulation of L-arabinose operon via dimeric AraC and CAP

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.[16]

Via AraC

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AraC acts as an activator in the presence of arabinose. AraC undergoes a conformational change when arabinose bind to the dimerization domain of AraC. As a result, AraC-arabinose complex falls off from araO2 and breaks the DNA loop. Hence, it is more energetically favorable for AraC-arabinose to bind to two adjacent DNA half sites: araI1 and araI2 in the presence of arabinose. In which, 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.[17]

Via CAP/cAMP (Catabolite Repression)

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CAP act as a transcriptional activator only in the absence of 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, also increase the binding affinity of AraC protein for araI2. Thereby, promoting RNA polymerase to bind to araBAD promoter and switches on expression of araBAD required for metabolising L-arabinose.

Autoregulation of araC expression

Autoregulation of AraC

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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. [20] Therefore, prevents the transcription of araC from araC promoter. i.e. AraC protein inhibits its own expression at high concentrations. Cite error: The opening <ref> tag is malformed or has a bad name (see the help page).

Use in protein expression system

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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. The L-arabinose operon has been commonly used in protein expression system, as the araBAD promoter can be used for producing an excessive level of targeted expression under tight regulation. By fusing araBAD promoter to a gene of interest, the expression of the target gene can be solely regulated by arabinose. i.e. constitutive expression of the gene of interest is permitted as long as arabinose is present. Cite error: The opening <ref> tag is malformed or has a bad name (see the help page).

See also

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Other Operon system

Reference

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  1. ^ Voet, Donald & Voet, Judith G. (2011). Biochemistry (4th ed. ed.). Hoboken, NJ: John Wiley & Sons. pp. 1291–1294. ISBN 978-0470-57095-1. {{cite book}}: |edition= has extra text (help)CS1 maint: multiple names: authors list (link)
  2. ^ Schleif, Robert (2000). "Regulation of the L-arabinose operon of Escherichia coli". Trends in Genetics. 16 (12): 559–565. doi:10.1016/S0168-9525(00)02153-3.
  3. ^ Watson, James D. (2008). Molecular biology of the gene (6th ed. ed.). Harlow: Addison-Wesley. pp. 634–635. ISBN 9780321507815. {{cite book}}: |edition= has extra text (help)
  4. ^ Schleif, Robert (2010). "AraC protein, regulation of the l-arabinose operon in, and the light switch mechanism of AraC action". FEMS Microbiology Reviews. 34 (5): 779–796. doi:10.1111/j.1574-6976.2010.00226.x.
  5. ^ Lobell, R. B.; Schleif, R. F. (1990). "DNA looping and unlooping by AraC protein". Science (New York, N.Y.). 250 (4980): 528–532. PMID 2237403.
  6. ^ Schleif, Robert (2003). "AraC protein: A love-hate relationship". BioEssays. 25 (3): 274–282. doi:10.1002/bies.10237.
  7. ^ Schleif, Robert; Lis, John T. (1975). "The regulatory region of the l-arabinose operon: A physical, genetic and physiological study". Journal of Molecular Biology. 95 (3): 417–431. doi:10.1016/0022-2836(75)90200-4.
  8. ^ Ogden, S; Haggerty, D; Stoner, CM; Kolodrubetz, D; Schleif, R (1980). "The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation". Proceedings of the National Academy of Sciences of the United States of America. 77 (6): 3346–3350. PMID 6251457.
  9. ^ Bustos, S. A; Schleif, R. F (1993). "Functional domains of the AraC protein". Proceedings of the National Academy of Sciences of the United States of America. 90 (12): 5638–5642. PMID 8516313.
  10. ^ Saviola, B; Seabold, R; Schleif, R. F (1998). "Arm-domain interactions in AraC". Journal of molecular biology. 278 (3): 539–548. doi:10.1006/jmbi.1998.1712. PMID 9600837.
  11. ^ Griffiths, Anthony J.; Wessler, Susan R. (2015). Introduction to genetic analysis (11. ed. ed.). New York, NY: Freeman. pp. 413–414. ISBN 9781429276344. {{cite book}}: |edition= has extra text (help)
  12. ^ Casadaban, Malcolm J. (1976). "Regulation of the regulatory gene for the arabinose pathway, araC". Journal of Molecular Biology. 104 (3): 557–566. doi:10.1016/0022-2836(76)90120-0.
  13. ^ Seabold, Robert R; Schleif, Robert F (1998). "Apo-AraC actively seeks to loop". Journal of Molecular Biology. 278 (3): 529–538. doi:10.1006/jmbi.1998.1713.
  14. ^ Hendrickson, William; Schleif, Robert (1984). "Regulation of the Escherichia coli l-arabinose operon studied by gel electrophoresis DNA binding assay". Journal of Molecular Biology. 178 (3): 611–628. doi:10.1016/0022-2836(84)90241-9.
  15. ^ Weaver, Robert Franklin (2012). Molecular biology (5th int. student ed. ed.). New York: McGraw-Hill. pp. 183–186. ISBN 9780071316866. {{cite book}}: |edition= has extra text (help)
  16. ^ Snyder, Larry (2013). Molecular genetics of bacteria (4th ed. ed.). Washington, DC: ASM Press. p. 487-494. ISBN 9781555816278. {{cite book}}: |edition= has extra text (help)
  17. ^ Hartwell, Leland; Hood, Leroy (2010). Genetics : from genes to genomes (4. ed., ed.). Boston: McGraw-Hill Education. p. 528. ISBN 9780071102155.{{cite book}}: CS1 maint: extra punctuation (link)
  18. ^ Cox, Michael M.; Doudna, Jennifer A.; O'Donnell, Michael E. (2012). Molecular biology : principles and practice (International ed. ed.). New York: W.H. Freeman. p. 707-708. ISBN 9781464102257. {{cite book}}: |edition= has extra text (help)
  19. ^ Griffiths, Anthony J.F. (2002). Modern genetic analysis :b integrating genes and genomes (2nd ed. ed.). New York: W.H. Freeman. pp. 432–433. ISBN 0716743825. {{cite book}}: |edition= has extra text (help)
  20. ^ Lee, N. L; Gielow, W.O; Wallace, R. G (1981). "Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America. 78 (2): 752–756. PMID 6262769.
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