IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3. Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2000 Zhu Y., Tepperman JM., Fairchild CD., Quail PH. Note the deletions of PhyB indicate that deletion of 1-37 amino acids results in weaker interaction whereas deletion of 1-90 amino acids results in no interaction , therefore I took 1-90 as the binding region. Similarly 1181 deletions interaction is weaker , 1161 is abolished therfore c-terminal 1161-1121was used. The same approach was taken for PIF3 deletion analysis in Fig 4. Light treatments were carried out by inclusion of phycocyanobilin and light treatments as follows. For red light treatments the mixture received 10 minute red light treatment on ice (343 mmol meter squared per second at a wavelength of 664 nm). For far red light treatments the mixture received 1 minute far red light treatment (353 mmol meter squared per second at a wavelength of 748 nm) immediately after the red light treatment. All binding reactions were then incubated in darkness for 2 h at 4degree celsius , proir to assay. quail@nature.berkeley.edu 16-April-2008: Contacted by JYOTI. in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Zhu Y., Tepperman JM., Fairchild CD., Quail PH. Note the deletions of PhyB indicate that deletion of 1-37 amino acids results in weaker interaction whereas deletion of 1-90 amino acids results in no interaction , therefore I took 1-90 as the binding region. Similarly 1181 deletions interaction is weaker , 1161 is abolished therfore c-terminal 1161-1121was used. The same approach was taken for PIF3 deletion analysis in Fig 4. Light treatments were carried out by inclusion of phycocyanobilin and light treatments as follows. For red light treatments the mixture received 10 minute red light treatment on ice (343 mmol meter squared per second at a wavelength of 664 nm). For far red light treatments the mixture received 1 minute far red light treatment (353 mmol meter squared per second at a wavelength of 748 nm) immediately after the red light treatment. All binding reactions were then incubated in darkness for 2 h at 4degree celsius , proir to assay. quail@nature.berkeley.edu Accepted 2008-APR-3 by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2000 pif3_arath Transcription factor PIF3 Phytochrome-associated protein 3 Basic helix-loop-helix protein 8 PIF3 PAP3 BHLH8 At1g09530 F14J9.19 Transcription factor EN 100 bHLH transcription factor bHLH008 Phytochrome-interacting factor 3 EN100 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MPLFELFRLTKAKLESAQDRNPSPPVDEVVELVWENGQISTQSQSSRSRNIPPPQANSSRAREIGNGSKTTMVDEIPMSVPSLMTGLSQDDDFVPWLNHHPSLDGYCSDFLRDVSSPVTVNEQESDMAVNQTAFPLFQRRKDGNESAPAASSSQYNGFQSHSLYGSDRARDLPSQQTNPDRFTQTQEPLITSNKPSLVNFSHFLRPATFAKTTNNNLHDTKEKSPQSPPNVFQTRVLGAKDSEDKVLNESVASATPKDNQKACLISEDSCRKDQESEKAVVCSSVGSGNSLDGPSESPSLSLKRKHSNIQDIDCHSEDVEEESGDGRKEAGPSRTGLGSKRSRSAEVHNLSERRRRDRINEKMRALQELIPNCNKVDKASMLDEAIEYLKSLQLQVQIMSMASGYYLPPAVMFPPGMGHYPAAAAAMAMGMGMPYAMGLPDLSRGGSSVNHGPQFQVSGMQQQPVAMGIPRVSGGGIFAGSSTIGNGSTRDLSGSKDQTTTNNNSNLKPIKRKQGSSDQFCGSS DIP protein O80536 original sequence version: 116 DIP protein O80536 original sequence version: 85 1044AC01D598DE7C phyb_arath Phytochrome B PHYB HY3 OOP1 Protein LONG HYPOCOTYL 3 Protein OUT OF PHASE 1 MSF3.17 At2g18790 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MVSGVGGSGGGRGGGRGGEEEPSSSHTPNNRRGGEQAQSSGTKSLRPRSNTESMSKAIQQYTVDARLHAVFEQSGESGKSFDYSQSLKTTTYGSSVPEQQITAYLSRIQRGGYIQPFGCMIAVDESSFRIIGYSENAREMLGIMPQSVPTLEKPEILAMGTDVRSLFTSSSSILLERAFVAREITLLNPVWIHSKNTGKPFYAILHRIDVGVVIDLEPARTEDPALSIAGAVQSQKLAVRAISQLQALPGGDIKLLCDTVVESVRDLTGYDRVMVYKFHEDEHGEVVAESKRDDLEPYIGLHYPATDIPQASRFLFKQNRVRMIVDCNATPVLVVQDDRLTQSMCLVGSTLRAPHGCHSQYMANMGSIASLAMAVIINGNEDDGSNVASGRSSMRLWGLVVCHHTSSRCIPFPLRYACEFLMQAFGLQLNMELQLALQMSEKRVLRTQTLLCDMLLRDSPAGIVTQSPSIMDLVKCDGAAFLYHGKYYPLGVAPSEVQIKDVVEWLLANHADSTGLSTDSLGDAGYPGAAALGDAVCGMAVAYITKRDFLFWFRSHTAKEIKWGGAKHHPEDKDDGQRMHPRSSFQAFLEVVKSRSQPWETAEMDAIHSLQLILRDSFKESEAAMNSKVVDGVVQPCRDMAGEQGIDELGAVAREMVRLIETATVPIFAVDAGGCINGWNAKIAELTGLSVEEAMGKSLVSDLIYKENEATVNKLLSRALRGDEEKNVEVKLKTFSPELQGKAVFVVVNACSSKDYLNNIVGVCFVGQDVTSQKIVMDKFINIQGDYKAIVHSPNPLIPPIFAADENTCCLEWNMAMEKLTGWSRSEVIGKMIVGEVFGSCCMLKGPDALTKFMIVLHNAIGGQDTDKFPFPFFDRNGKFVQALLTANKRVSLEGKVIGAFCFLQIPSPELQQALAVQRRQDTECFTKAKELAYICQVIKNPLSGMRFANSLLEATDLNEDQKQLLETSVSCEKQISRIVGDMDLESIEDGSFVLKREEFFLGSVINAIVSQAMFLLRDRGLQLIRDIPEEIKSIEVFGDQIRIQQLLAEFLLSIIRYAPSQEWVEIHLSQLSKQMADGFAAIRTEFRMACPGEGLPPELVRDMFHSSRWTSPEGLGLSVCRKILKLMNGEVQYIRESERSYFLIILELPVPRKRPLSTASGSGDMMLMMPY DIP protein P14713 original sequence version: 107 DIP protein P14713 original sequence version: 131 7B5348CB1091B813 phya_arath Phytochrome A PHYA HY8 At1g09570 F14J9.23 FHY2 FRE1 Protein FAR RED ELONGATED HYPOCOTYL 2 Protein FAR RED ELONGATED 1 Protein ELONGATED HYPOCOTYL 8 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSGSRPTQSSEGSRRSRHSARIIAQTTVDAKLHADFEESGSSFDYSTSVRVTGPVVENQPPRSDKVTTTYLHHIQKGKLIQPFGCLLALDEKTFKVIAYSENASELLTMASHAVPSVGEHPVLGIGTDIRSLFTAPSASALQKALGFGDVSLLNPILVHCRTSAKPFYAIIHRVTGSIIIDFEPVKPYEVPMTAAGALQSYKLAAKAITRLQSLPSGSMERLCDTMVQEVFELTGYDRVMAYKFHEDDHGEVVSEVTKPGLEPYLGLHYPATDIPQAARFLFMKNKVRMIVDCNAKHARVLQDEKLSFDLTLCGSTLRAPHSCHLQYMANMDSIASLVMAVVVNEEDGEGDAPDATTQPQKRKRLWGLVVCHNTTPRFVPFPLRYACEFLAQVFAIHVNKEVELDNQMVEKNILRTQTLLCDMLMRDAPLGIVSQSPNIMDLVKCDGAALLYKDKIWKLGTTPSEFHLQEIASWLCEYHMDSTGLSTDSLHDAGFPRALSLGDSVCGMAAVRISSKDMIFWFRSHTAGEVRWGGAKHDPDDRDDARRMHPRSSFKAFLEVVKTRSLPWKDYEMDAIHSLQLILRNAFKDSETTDVNTKVIYSKLNDLKIDGIQELEAVTSEMVRLIETATVPILAVDSDGLVNGWNTKIAELTGLSVDEAIGKHFLTLVEDSSVEIVKRMLENALEGTEEQNVQFEIKTHLSRADAGPISLVVNACASRDLHENVVGVCFVAHDLTGQKTVMDKFTRIEGDYKAIIQNPNPLIPPIFGTDEFGWCTEWNPAMSKLTGLKREEVIDKMLLGEVFGTQKSCCRLKNQEAFVNLGIVLNNAVTSQDPEKVSFAFFTRGGKYVECLLCVSKKLDREGVVTGVFCFLQLASHELQQALHVQRLAERTAVKRLKALAYIKRQIRNPLSGIMFTRKMIEGTELGPEQRRILQTSALCQKQLSKILDDSDLESIIEGCLDLEMKEFTLNEVLTASTSQVMMKSNGKSVRITNETGEEVMSDTLYGDSIRLQQVLADFMLMAVNFTPSGGQLTVSASLRKDQLGRSVHLANLEIRLTHTGAGIPEFLLNQMFGTEEDVSEEGLSLMVSRKLVKLMNGDVQYLRQAGKSSFIITAELAAANK DIP protein P14712 original sequence version: 133 D8B359713430744B bhlh8-phyb-8 1 3 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain c-terminal required to bind necessary binding region required to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined [EBI-1639014]Certain and ragged-n-terminus positions must always be strictly superior to 0 and inferior or equal to the protein sequence length. Actual positions : 1211-1211, sequence length 1172 [EBI-1639014]1161-1211 in vitro In vitro 2 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined gal4 region GAL4 activation domain tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction PIF3 preferentially interacts with the Pfr (biologically active) form of PhyB Figures 1, 2, 3 In initial studies using this procedure, we found that full-length phyB bound strongly and specifically to PIF3 in the Pfr form as expected The authors concluded that " data indicate that phyB binds with up to 10-fold higher apparent affinity toPIF3 than does phyA". bhlh8-phya-1 1 2 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined gal4 region GAL4 activation domain tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 4 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction PIF3 preferentially interacts with the Pfr (biologically active) form of PhyA Figures 1, 2 PhyA also bound selectively to PIF3 in the Pfr form, The authors concluded that " data indicate that phyB binds with up to 10-fold higher apparent affinity toPIF3 than does phyA". phyb-bhlh8 1 3 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined gal4 tag GAL4 activation domain tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 2 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain O80536:p.Gly165Val mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain G V Wed May 02 20:32:40 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly165val' Tue Jun 30 16:00:43 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly165Val' in vitro In vitro direct interaction direct interaction Figures 4 The PAS domain of PIF3 is necessary but not sufficient for phyB binding