1. 1 GEN304 Lecture #4 The Arabinose operon, a new “twist” on negative and positive control of genes. No assigned reading

2. 2 operons can be regulated by both positive and negative nutritional signals, allowing cells to optimize their metabolic responses. both gal and lac operons are negatively regulated by repressor proteins (galR or lacI) that bind to operator sequences and prevent RNAP from transcribing structural genes (although RNAP can still bind and form an open complex). mutations in either lacI or galR lead to constitutive expression of the operon, as long as glucose levels are low (positive signal)

3. 3 mutations in operator sequences of these operons have the same phenotype - constitutive expression today we will discuss another variation of this theme : dual positive and negative controls that regulate the “BAD” operon the Ara operon, also called the “BAD” operon encodes genes whose products are used to metabolize the sugar arabinose as well as a transporter of this sugar. D A B P BAD araI CAP araO 1 araC araO 2 +1 -63 -92 -123 -148 -280 Pc

4. 4 mutational studies identified a closely linked gene, araC, encoding a regulatory protein footprinting studies identified 3 sites that araC binds: araI, araO 1 and araO 2 CAP-binding site located well away from promoter under conditions of low arabinose, and high glucose (non-inducing) araC binds to the operator sites So far, this looks just like the other operons we’ve studied - what’s different?

5. 5 first clue was that mutations in araC gene give a non-inducible phenotype, rather than a constitutive phenotype. compare mutant results of Lac vs Arabinose operons: repressor -inducer +inducer genotype lacI+ off on lacI- on on araC+ off on araC- off off ANY IDEAS HOW THESE RESULTS COULD BE OBTAINED?

6. 6 answer proposed was that araC is not only a repressor of the pBAD operon, but also an activator! presence or absence of arabinose determines which function of araC is seen. araC ara repressor function activator function when this explanation was first suggested, it was met with skepticism….

7. 7 Analysis of the  719 mutant: D A B P BAD araI CAP araO 1 araC araO 2 Pc [ ]  719 deletion: this mutant was particularly informative because it removed the entire araC gene (plus araO 2 ) remember: araC mutants can grow on glucose (only) but not on arabinose (only), because they are not inducible (or constitutive). “second- site suppressor” mutations of the  719 mutant were selected to see if they could shed some light on how araC might act both positively and negatively.

8. 8 How? Mutagenize  719 mutant cells and look for progeny cells that can grow (even weakly) on arabinose as a sole carbon source. Then characterize their levels of expression under inducing and non-inducing conditions Strainnon-inducedinduced WT0.133 (300X)  7190.10.2  719sup4 (*40X)4 (40X) suppressors had ~low levels of pBAD expression under both inducing conditions AND non-inducing (enough to live on) ANY IDEAS what these mutants are?

9. 9 What Next?? Map the locations of the suppressors (19 isolated). ara leu E. coli chromosome D A B araI araO1 araC araO2 leu to map the location of the suppressors, P1 transducing phage were used to first infect cells carrying the  719/suppressor mutants recombinant phage then used to infect leu-ara+ tester cells *

10. 10 the infected cells in the second cross were screened for leu+, to select for those that were infected by phage that had picked up DNA in the region of the BAD operon, and then they were analyzed for whether or not they were either : 1. inducible on arabinose (ie WT w/respect to BAD) 2. not inducible on arabinose (ie like  719 deletion mutant, don’t grow on arabinose) 3. weak constitutive pBAD (ie like  719/suppressor, grow slowly on arabinose)

11. 11 when you select for leu+ cells in the second phage cross, you are essentially selecting for homologous recombination events that have one cross-over on one side of the leu+ locus and the other cross-over on the other side : D A B araI araO1 araC araO2 leu D A B araI araO1 araC araO2 leu+ [ ]  719 deletion: “x” “y” 63% of leu+ were  719 (ie no araC made) 50% of leu+ didn’t make araC AND had the “slow growing arabinose”

12. 12 so of the cells where a recombination event included the  719 deletion, approximately 75% also included the suppressor mutation - suggesting that they are “farther away” from leu+ than the  719 (but in the same direction) D A B araI [ ] leu+ Possible leu+ recombinants: this mapping experiment established that the suppressor mutations were tightly linked to pBAD operon, to the “left” of araC

13. 13 further mapping experiments placed the suppressor mutants at the araI operator, hence these mutants were called araI c mutants so what do you think these mutations were? remember the model: araC plus arabinose acts as a positive regulator to help RNAP bind the promoter are the araI c mutants the same as “promoter up” mutations that act in cis or do they involve some kind of trans acting factor? to answer this question, a “cis-trans” test was performed

14. 14 remember, if you’re going to do a cis-trans test, you need to create a partial diploid : No Arabinose WT genotype: D+A+B+araI+araC+ (0.1 basal in absence of arabinose)  719/sup genotype: D+A+B+araI c  719 (4 - low level expression) diploid genotype: D+A+B+araI+araC+ D+A-*B+araI c  719 (0.1: ~basal, therefore does not work in trans, must be cis)

15. 15 so this data was consistent with an interpretation that the araI c mutants were indeed “promoter up” and somehow helped make the promoter more attractive to RNAP but this doesn’t really help us to understand a mechanism for how araC can act either as a negative OR a positive regulator of BAD transcription - let’s retest the “positive” function idea again: subsequent approaches used for studying BAD regulation: 1. in vitro transcription system 2. footprinting experiments

16. 16 D A B P BAD araI template + araC and RNAP proteins RNAP + template = no transcription RNAP+ arabinose = no transcription RNAP+template+araC= no transcription all of above+ arabinose = YES so this experiment says that you do need both araC AND arabinose for induction 1. in vitro transcription assay: 2. Biochemical footprinting assay: using purified template + araC protein DAB P araI CAP araO 1 araC araO 2

17. 17 Footprinting, cont.: a surprising result: the araI site is bound differently, depending on whether arabinose is present or not! in the absence of arabinose araC only binds part of the araI site, plus the araO 2 site ~200 bp away. in the presence of arabinose araC binds to the whole araI site and not to araO 2 araC ara araC ara araC araIaraO 2 araIaraO 2

18. 18 So how might these results explain what is going on? one suggestion was that in the absence of arabinose, araC forms a dimer that links the araI site to the araO2 site and in this configuration araC acts as a repressor araC araI2 araI1 araC binding studies established that the interaction of araC with the two sites is cooperative

19. 19 binding studies also established that the stoichiometry of araC was as a dimer binding to the two sites affinity studies established that the loop formed in the absence of arabinose is extremely stable so how does the activation function of araC work? mutations in araI 2 (not bound in absence of arabinose) do not affect repression - but these do interfere with induction. does the arabinose-bound form of araC “help” RNAP bind to the promoter?

20. 20 the idea here is that in the absence of arabinose, araC has high affinity for the araI 1 and araO 2 sites, and under these conditions RNAP cannot bind to the promoter (due to a poor consensus sequence) the repression function of araC then is in “not helping” RNAP to bind, rather than in preventing already bound RNAP from proceeding along the template. araC ara araC ara RNAP go to next slide for a mihnute here:

21. 21 in the presence of arabinose, the affinity of araC changes to lose affinity for araO 2 and gain affinity for araI 2 this “unloops” the DNA and makes the promoter “attractive” to RNAP simultaneously this may not actually involve dissociation of the araC dimer, it could simply flip to transition from the looped to unlooped forms much but not all of regulation of the BAD operon is explained by the story above what is the rest?

22. 22 remember that the operon also has a CAP binding site, suggesting that it is responsive to glucose levels via cAMP metabolism the CAP site is far upstream of the pBAD promoter though, so it is difficult to see how CAP-cAMP can help RNAP bind (like lac) instead, CAP binding seems to de-stabilize the “loop” structure under inducing conditions (low glucose, + arabinose). Recall that CAP binding to DNA has the effect of “bending” the DNA - this is the force that helps with this function. in addition, CAP binding probably helps to prevent reformation of the loop.

23. 23 this function of CAP provides another level of regulatory input so that the BAD operon won’t be turned on by arabinose if glucose is present - pretty smart! one final regulatory twist to this story has to do with the transcription of the araC gene itself: recall there is an operator site that we haven’t yet discussed: araO 1, another site that araC can bind D A B P BAD araI CAP araO 1 araC araO 2 +1 -63 -92 -123 -148 -280 Pc

24. 24 when araC is bound to this site, RNAP cannot bind to the Pc promoter, and araC transcription is prevented. this is an example of autogenous gene regulation, where a gene product regulates its own synthesis araC no new araC transcription araC Pc araO 1 RNAP transcription

25. 25 what is the consequence of araC regulating its own synthesis? basically it provides a way of modulating the levels of araC so that they are “just enough” and not more than that (recall that lacI levels are kept low by a different means - by having a very poor translation initiation codon on the mRNA) protein transcription

26. 26 araC transcription is also enhanced by CAP binding, presumably by the steric changes to DNA that result in Pc being more “attractive” to RNAP since araO 2 is within araC, the gene willl not be transcribed under repressing conditions araC transcription is also stimulated by arabinose - so if the cell is switched to arabinose (low glucose) it can quickly increase araC levels to stimulate pBAD transcription

27. 27 Summary of Regulatory Inputs 1. Repression: araC dimer binds DNA in loop via araI 1 /araO 2 sites. RNAP can’t bind promoter. Absence of CAP binding stabilizes loop, 2. Activation: araC-arabinose dimer “switches” to araI 2 / 1 -bound configuration, releasing loop and stimulating RNAP binding. Binding of CAP-cAMP helps de-stabilize loop. 3. Autogenous regulation of araC: araC protein levels determine the occupancy of the araC promoter through interactions with araO 1. CAP and arabinose also modulate RNAP affinity for promoter.