Arabinose operon and their regulation and arac

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

1. Presented by G.Vijayalakshmi 20PY10 I MSC., Microbiology Department of microbiology Ayya nadar janaki ammal college Sivakasi , Tamilnadu -India

2. Presented to DR.S.Munisamy Assistant professor Department of microbiology Ayya nadar janaki ammal college Sivakasi , Tamilnadu .

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

6. Structure of 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 po t Arabinose 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 Two promoter genes in arabinose operon ( pBAD and Pc. pBAD is the promoter site of ara BAD structural genes Promotes the synthesis of ara BAD Mrna Pc is the promoter site of araC regulatory gene Promotes the synthesis of AraC repressor protein Ara C repressor exits in active P1state in the absence of inducer 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 protei n 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 . I n 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 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 What is operon and who discovered What is CAP and Camp Role of repressor Role of inducer Positive regulation Negative regulation Role of ara c protein Enzymes involved in arabinose operon What is autoregulation Role of operator gene and promoter gene