L-arabinose operon

L-arabinose operon is composed of structural genes and regulatory regions including the operator region (araO₁, araO₂) and the initiator region (araI₁, araI₂).{{cite journal|last1=Schleif|first1=Robert|last2=Lis|first2=John T.|title=The regulatory region of the l-arabinose operon: A physical, genetic and physiological study|journal=Journal of Molecular Biology|date=1975|volume=95|issue=3|pages=417–431|doi=10.1016/0022-2836(75)90200-4|pmid=168391}} 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.{{cite journal|last1=Ogden|first1=S|last2=Haggerty|first2=D|last3=Stoner|first3=CM|last4=Kolodrubetz|first4=D|last5=Schleif|first5=R|title=The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=1980|volume=77|issue=6|pages=3346–3350|pmid=6251457|doi=10.1073/pnas.77.6.3346|pmc=349612|bibcode=1980PNAS...77.3346O|doi-access=free}}

File:Structure of L-arabinose operon of E. coli.tif

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. araBAD and araC are transcribed in opposite directions from the araBAD promoter (P_BAD) and araC promoter (P_C) respectively.

• 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.

File:Process of L-arabinose catabolism.png

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.

File:Structure of AraC protein.png

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.{{cite journal|last1=Bustos|first1=S. A|last2=Schleif|first2=R. F|title=Functional domains of the AraC protein.|journal=Proceedings of the National Academy of Sciences of the United States of America|date=1993|volume=90|issue=12|pages=5638–5642|pmid=8516313|doi=10.1073/pnas.90.12.5638|pmc=46776|bibcode=1993PNAS...90.5638B|doi-access=free}} The dimerisation domain is responsible for arabinose-binding.{{cite journal|last1=Saviola|first1=B|last2=Seabold|first2=R|last3=Schleif|first3=R. F|title=Arm-domain interactions in AraC.|journal=Journal of Molecular Biology|date=1998|volume=278|issue=3|pages=539–548|doi=10.1006/jmbi.1998.1712|pmid=9600837|doi-access=free}}

AraC undergoes conformational change upon arabinose-binding, in which, it has two distinct conformations. The conformation is purely determined by the binding of allosteric inducer arabinose.{{cite book|last1=Griffiths|first1= Anthony J.|last2=Wessler|first2= Susan R.|title=Introduction to genetic analysis|date=2015|publisher=Freeman|location=New York, NY|isbn=9781429276344|pages=413–414|edition= 11th}}

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).

File:Negative regulation of L-arabinose operon via AraC protein.png

When arabinose is absent, cells do not need the ara_BAD 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.{{cite journal|last1=Casadaban|first1=Malcolm J.|title=Regulation of the regulatory gene for the arabinose pathway, araC|journal=Journal of Molecular Biology|date=1976|volume=104|issue=3|pages=557–566|doi=10.1016/0022-2836(76)90120-0|pmid=781294}} This leads to the formation of a DNA loop.{{cite journal|last1=Seabold|first1=Robert R|last2=Schleif|first2=Robert F|title=Apo-AraC actively seeks to loop|journal=Journal of Molecular Biology|year=1998|volume=278|issue=3|pages=529–538|doi=10.1006/jmbi.1998.1713|pmid=9600836|doi-access=free}} This orientation blocks RNA polymerase from binding to the araBAD promoter.{{cite journal|last1=Hendrickson|first1=William|last2=Schleif|first2=Robert|title=Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay|journal=Journal of Molecular Biology|date=1984|volume=178|issue=3|pages=611–628|doi=10.1016/0022-2836(84)90241-9|pmid=6387154}} Therefore, transcription of structural gene araBAD is inhibited.{{cite book|last1=Weaver|first1=Robert Franklin|title=Molecular biology|url=https://archive.org/details/molecularbiology00weav_632|url-access=limited|date=2012|publisher=McGraw-Hill|location=New York|isbn=9780071316866|pages=[https://archive.org/details/molecularbiology00weav_632/page/n205 183]–186|edition= 5th int. student}}

File:Positive regulation of L-arabinose operon via AraC and CAP.png

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.{{cite book|last1=Snyder|first1=Larry|title=Molecular genetics of bacteria|date=2013|publisher=ASM Press|location=Washington, DC|isbn=9781555816278|pages=487–494|edition= 4th.}}

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.{{cite book|last1=Hartwell|first1=Leland|last2=Hood|first2=Leroy|title=Genetics : from genes to genomes|date=2010|publisher=McGraw-Hill Education|location=Boston|isbn=9780071102155|page=[https://archive.org/details/genetics00lela_0/page/528 528]|edition=4th|url=https://archive.org/details/genetics00lela_0/page/528}}

CAP act as a transcriptional activator only in the absence of E. coli's preferred sugar, glucose.{{cite book|last1=Cox|first1=Michael M. |last2=Doudna|first2=Jennifer A.|last3=O'Donnell|first3=Michael E.|title=Molecular biology : principles and practice|date=2012|publisher=W.H. Freeman|location=New York|isbn=9781464102257|pages=707–708|edition= International}} When glucose is absent, high level of CAP protein/cAMP complex bind to CAP binding site, a site between araI₁ and araO₁.{{cite book|last1=Griffiths|first1=Anthony J.F.|title=Modern genetic analysis: integrating genes and genomes|date=2002|publisher=W.H. Freeman|location=New York|isbn=0716743825|pages=[https://archive.org/details/lccn_2001059225/page/432 432–433]|edition=2nd.|url-access=registration|url=https://archive.org/details/lccn_2001059225/page/432}} 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.

File:Autoregulation of araC expression.png

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.{{cite journal|last1=Lee|first1=N. L|last2=Gielow|first2=W.O|last3=Wallace|first3=R. G|title=Mechanism of araC autoregulation and the domains of two overlapping promoters, P_c and P_BAD, in the L-arabinose regulatory region of Escherichia coli.|journal=Proceedings of the National Academy of Sciences of the United States of America|year= 1981|volume=78|issue=2|pages=752–756|pmid=6262769|doi=10.1073/pnas.78.2.752|pmc=319880|bibcode=1981PNAS...78..752L|doi-access=free}} Therefore, the AraC protein inhibits its own expression at high concentrations.

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. 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 P_BAD promoter, allowing GFP production to be induced by arabinose.

• Operon
• Catabolism
• Catabolite repression

Other operon systems in E. coli:

• gal operon
• gab operon
• lac operon
• trp operon

{{Reflist}}

• [https://www.ncbi.nlm.nih.gov/books/NBK21277/ Modern Genetic Analysis] by Griffiths, A.J et al. (online textbook)
• [https://www.ncbi.nlm.nih.gov/books/NBK22512/ Biochemistry] by Berg, J.M et al. (online textbook)
• [https://www.ncbi.nlm.nih.gov/books/NBK21849/ An Introduction to Genetic Analysis] by Griffiths, A.J et al. (online textbook)

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Category:Gene expression

Category:Operons

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