UniProt UniProt http://www.uniprot.org Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Frontiers in Molecular Biosciences 2014 imex curation Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro enzymatic footprint enzymatic footprinting enzymatic footprint weight by comassie molecular weight estimation by coomasie staining weight by comassie Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. Proteins were degraded by addition of thermolysin, complex formation protected proteins from the activity of this protease d.dougan@latrobe.edu.au Accepted 2020-APR-17 AT 07:32 BST AT 07:32 BST by LIVIAP Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Frontiers in Molecular Biosciences 2014 imex curation Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column weight by comassie molecular weight estimation by coomasie staining weight by comassie Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Accepted 2020-APR-17 AT 07:32 BST AT 07:32 BST by LIVIAP Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Frontiers in Molecular Biosciences 2014 imex curation Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Accepted 2020-APR-17 AT 07:32 BST AT 07:32 BST by LIVIAP Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP. Frontiers in Molecular Biosciences 2014 imex curation Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au 04-05-20: Contacted by IntAct-Help. Only protein-protein interactions imex curation ecoli Escherichia coli (strain K12) pull down pull down western blot western blot Immuno blot Immuno blot Micevski D., Zammit JE., Truscott KN., Dougan DA., Micevski D., Zammit JE., Truscott KN., Dougan DA. d.dougan@latrobe.edu.au Accepted 2020-APR-17 AT 07:32 BST AT 07:32 BST by LIVIAP Frontiers in Molecular Biosciences 2014 Only protein-protein interactions imex curation imex curation rpos_ecoli RNA polymerase sigma factor RpoS Sigma-38 rpoS appR katF nur otsX sigS b2741 JW5437 Sigma S protein protein ecoli Escherichia coli (strain K12) MSQNTLKVHDLNEDAEFDENGVEVFDEKALVEQEPSDNDLAEEELLSQGATQRVLDATQLYLGEIGYSPLLTAEEEVYFARRALRGDVASRRRMIESNLRLVVKIARRYGNRGLALLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHIVKELNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSVDTPLGGDSEKALLDILADEKENGPEDTTQDDDMKQSIVKWLFELNAKQREVLARRFGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLREILQTQGLNIEALFRE DIP protein P13445 original sequence version: 126 DIP protein P13445 original sequence version: null 4E6E675E4CA6BC6F iram_ecoli Anti-adapter protein IraM iraM elb1 elbA ycgW Enhancing lycopene biosynthesis protein 1 b1160 JW1147 protein protein ecoli Escherichia coli (strain K12) MKWIVIDTVIQPTCGISFSAIWGNMKMIIWYQSTIFLPPGSIFTPVKSGIILKDKEYPITIYHIAPFNKDLWSLLKSSQECPPGESKITNKCLHNSCIIKICPYGLK 27F8A8C64FA18984 irap_ecoli Anti-adapter protein IraP iraP b0382 JW0373 yaiB protein protein ecoli Escherichia coli (strain K12) MKNLIAELLFKLAQKEEESKELCAQVEALEIIVTAMLRNMAQNDQQRLIDQVEGALYEVKPDASIPDDDTELLRDYVKKLLKHPRQ 1C2EE018BFB16BEE rssb_ecoli Regulator of RpoS rssB hnr sprE ychL JW1223 b1235 protein protein ecoli Escherichia coli (strain K12) MTQPLVGKQILIVEDEQVFRSLLDSWFSSLGATTVLAADGVDALELLGGFTPDLMICDIAMPRMNGLKLLEHIRNRGDQTPVLVISATENMADIAKALRLGVEDVLLKPVKDLNRLREMVFACLYPSMFNSRVEEEERLFRDWDAMVDNPAAAAKLLQELQPPVQQVISHCRVNYRQLVAADKPGLVLDIAALSENDLAFYCLDVTRAGHNGVLAALLLRALFNGLLQEQLAHQNQRLPELGALLKQVNHLLRQANLPGQFPLLVGYYHRELKNLILVSAGLNATLNTGEHQVQISNGVPLGTLGNAYLNQLSQRCDAWQCQIWGTGGRLRLMLSAE AB962EF94BC7B470 irad_ecoli Anti-adapter protein IraD iraD yjiD b4326 JW5782 protein protein ecoli Escherichia coli (strain K12) MMRQSLQAVLPEISGNKTSSLRKSVCSDLLTLFNSPHSALPSLLVSGMPEWQVHNPSDKHLQSWYCRQLRSALLFHEPRIAALQVNLKEAYCHTLAISLEIMLYHDDEPLTFDLVWDNGGWRSATLENVS AA07645C39525D90 irad-rssb-1 3 9 unspecified role unspecified role bait bait purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 2C To confirm if this relief of IraD inhibition on sigmaS degradation was due to a specific interaction between IraD and the N-domain of RssB, a series of pull-down experiments were performed. irad-rssb-2 2 9 unspecified role unspecified role neutral component neutral component purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 2D rpos-rssb-1 2 8 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 2F Addition of iraD caused dissociation of this complex irap-rssb-1 3 7 unspecified role unspecified role bait bait purified purified c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P0AEV1:p.Asp58Pro mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain D P Tue Jun 30 16:16:15 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp58Pro' P0AEV1:p.Asp58Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain D A Tue Jun 30 18:06:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp58Ala' ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3C, 3F Addition of iraD caused dissociation of this complex To examine more directly the mode of interaction between IraP and RssB, we monitored the binding of IraP with each RssB domain, using a series of pull-down experiments, in which purified recombinant IraP was immobilized to Ni-NTA agarose beads, via a C-terminal His10-tag (Figure ​(Figure3C).3C). iram-rssb-1 4 6 unspecified role unspecified role bait bait purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 8 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig. 4 As expected, full-length RssB was specifically recovered, from the column containing immobilized His6-IraM (Figure ​(Figure4A,4A, lane 3) irad-rssb-3 1 9 unspecified role unspecified role neutral component neutral component purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 5A, 5B irap-rssb-2 1 7 unspecified role unspecified role neutral component neutral component purified purified c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 5C Addition of iraD caused dissociation of this complex irad-1 2 9 unspecified role unspecified role neutral component neutral component purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 2D