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/ Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol. Cell. Biol. (0270-7306) 2001 imex curation Moretti P., Shore D. David.Shore@molbio.unige.ch 23-Feb-2010: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Moretti P., Shore D. David.Shore@molbio.unige.ch Accepted 2010-FEB-6 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2001 orchard Only protein-protein interactions imex curation Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Multiple interactions in Sir protein recruitment by Rap1p at silencers and telomeres in yeast. Mol. Cell. Biol. (0270-7306) 2001 imex curation Moretti P., Shore D. David.Shore@molbio.unige.ch 23-Feb-2010: Contacted by JYOTI. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Moretti P., Shore D. David.Shore@molbio.unige.ch Accepted 2010-FEB-6 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Mol. Cell. Biol. (0270-7306) 2001 orchard Only protein-protein interactions imex curation rap1_yeast DNA-binding protein RAP1 RAP1 GRF1 TUF1 YNL216W N1310 SBF-E Repressor/activator site-binding protein TUF protein protein yeast Saccharomyces cerevisiae Baker's yeast MSSPDDFETAPAEYVDALDPSMVVVDSGSAAVTAPSDSAAEVKANQNEENTGATAAETSEKVDQTEVEKKDDDDTTEVGVTTTTPSIADTAATANIASTSGASVTEPTTDDTAADEKKEQVSGPPLSNMKFYLNRDADAHDSLNDIDQLARLIRANGGEVLDSKPRESKENVFIVSPYNHTNLPTVTPTYIKACCQSNSLLNMENYLVPYDNFREVVDSRLQEESHSNGVDNSNSNSDNKDSIRPKTEIISTNTNGATEDSTSEKVMVDAEQQARLQEQAQLLRQHVSSTASITSGGHNDLVQIEQPQKDTSNNNNSNVNDEDNDLLTQDNNPQTADEGNASFQAQRSMISRGALPSHNKASFTDEEDEFILDVVRKNPTRRTTHTLYDEISHYVPNHTGNSIRHRFRVYLSKRLEYVYEVDKFGKLVRDDDGNLIKTKVLPPSIKRKFSADEDYTLAIAVKKQFYRDLFQIDPDTGRSLITDEDTPTAIARRNMTMDPNHVPGSEPNFAAYRTQSRRGPIAREFFKHFAEEHAAHTENAWRDRFRKFLLAYGIDDYISYYEAEKAQNREPEPMKNLTNRPKRPGVPTPGNYNSAAKRARNYSSQRNVQPTANAASANAAAAAAAAASNSYAIPENELLDEDTMNFISSLKNDLSNISNSLPFEYPHEIAEAIRSDFSNEDIYDNIDPDTISFPPKIATTDLFLPLFFHFGSTRQFMDKLHEVISGDYEPSQAEKLVQDLCDETGIRKNFSTSILTCLSGDLMVFPRYFLNMFKDNVNPPPNVPGIWTHDDDESLKSNDQEQIRKLVKKHGTGRMEMRKRFFEKDLL 67C4AFB03B3C0713 67C4AFB03B3C0713 sir4_yeast Regulatory protein SIR4 STE9 SIR4 ASD1 UTH2 YDR227W YD9934.12 Silent information regulator 4 protein protein yeast Saccharomyces cerevisiae Baker's yeast MPNDNKTPNRSSTPKFTKKPVTPNDKIPEREEKSNEVKTPKIPLFTFAKSKNYSRPSTAIHTSPHQPSDVKPTSHKQLQQPKSSPLKKNNYNSFPHSNLEKISNSKLLSLLRSKTSAGRIESNNPSHDASRSLASFEQTAFSRHAQQQTSTFNSKPVRTIVPISTSQTNNSFLSGVKSLLSEEKIRDYSKEILGINLANEQPVLEKPLKKGSADIGASVISLTKDKSIRKDTVEEKKEEKLNIGKNFAHSDSLSVPKVSAGDSGISPEESKARSPGIAKPNAIQTEVYGINEESTNERLEINQEKPVKLDENSANSTVASALDTNGTSATTETLTSKKIVPSPKKVAIDQDKITLHDEKTLAPSKHQPITSEQKMKEDADLKRMEILKSPHLSKSPADRPQGRRNSRNFSTRDEETTKLAFLVEYEGQENNYNSTSRSTEKKNDMNTSAKNKNGENKKIGKRPPEIMSTEAHVNKVTEETTKQIQSVRIDGRKVLQKVQGESHIDSRNNTLNVTPSKRPQLGEIPNPMKKHKPNEGRTPNISNGTINIQKKLEPKEIVRDILHTKESSNEAKKTIQNPLNKSQNTALPSTHKVTQKKDIKIGTNDLFQVESAPKISSEIDRENVKSKDEPVSKAVESKSLLNLFSNVLKAPFIKSESKPFSSDALSKEKANFLETIASTEKPENKTDKVSLSQPVSASKHEYSDNFPVSLSQPSKKSFANHTEDEQIEKKKICRGRMNTIITHPGKMELVYVSDSDDSSSDNDSLTDLESLSSGESNEIKVTNDLDTSAEKDQIQAGKWFDPVLDWRKSDRELTKNILWRIADKTTYDKETITDLIEQGIPKHSYLSGNPLTSVTNDICSVENYETSSAFFYQQVHKKDRLQYLPLYAVSTFENTNNTEKNDVTNKNINIGKHSQEQNSSSAKPSQIPTVSSPLGFEETKLSTTPTKSNRRVSHSDTNSSKPKNTKENLSKSSWRQEWLANLKLISVSLVDEFPSELSDSDRQIINEKMQLLKDIFANNLKSAISNNFRESDIIILKGEIEDYPMSSEIKIYYNELQNKPDAKKARFWSFMKTQRFVSNMGFDIQKSCEPVSISTSVKPHVVEPEHMADAKIMPKDILQITKKPLMVKNVKPSSPPDVKSLVQLSTMETKTLPEKKQFDSIFNSNKAKIIPGNGKHASENISLSFSRPASYGYFSVGKRVPIVEDRRVKQLDDITDSNTTEILTSVDVLGTHSQTGTQQSNMYTSTQKTELEIDNKDSVTECSKDMKEDGLSFVDIVLSKAASALDEKEKQLAVANEIIRSLSDEVMRNEIRITSLQGDLTFTKKCLENARSQISEKDAKINKLMEKDFQVNKEIKPY 9C698765964F094E 9C698765964F094E sir3_yeast Regulatory protein SIR3 SIR3 CMT1 MAR2 STE8 L9753.10 YLR442C Silent information regulator 3 protein protein yeast Saccharomyces cerevisiae Baker's yeast MAKTLKDLDGWQVIITDDQGRVIDDNNRRRSRKRGGENVFLKRISDGLSFGKGESVIFNDNVTETYSVYLIHEIRLNTLNNVVEIWVFSYLRWFELKPKLYYEQFRPDLIKEDHPLEFYKDKFFNEVNKSELYLTAELSEIWLKDFIAVGQILPESQWNDSSIDKIEDRDFLVRYACEPTAEKFVPIDIFQIIRRVKEMEPKQSDEYLKRVSVPVSGQKTNRQVMHKMGVERSSKRLAKKPSMKKIKIEPSADDDVNNGNIPSQRGTSTTHGSISPQEESVSPNISSASPSALTSPTDSSKILQKRSISKELIVSEEIPINSSEQESDYEPNNETSVLSSKPGSKPEKTSTELVDGRENFVYANNPEVSDDGGLEEETDEVSSESSDEAIIPVNKRRGAHGSELSSKIRKIHIQETQEFSKNYTTETDNEMNGNGKPGIPRGNTKIHSMNENPTPEKGNAKMIDFATLSKLKKKYQIILDRFAPDNQVTDSSQLNKLTDEQSSLDVAGLEDKFRKACSSSGRETILSNFNADINLEESIRESLQKRELLKSQVEDFTRIFLPIYDSLMSSQNKLFYITNADDSTKFQLVNDVMDELITSSARKELPIFDYIHIDALELAGMDALYEKIWFAISKENLCGDISLEALNFYITNVPKAKKRKTLILIQNPENLLSEKILQYFEKWISSKNSKLSIICVGGHNVTIREQINIMPSLKAHFTEIKLNKVDKNELQQMIITRLKSLLKPFHVKVNDKKEMTIYNNIREGQNQKIPDNVIVINHKINNKITQLIAKNVANVSGSTEKAFKICEAAVEISKKDFVRKGGLQKGKLVVSQEMVPRYFSEAINGFKDETISKKIIGMSLLMRTFLYTLAQETEGTNRHTLALETVLIKMVKMLRDNPGYKASKEIKKVICGAWEPAITIEKLKQFSWISVVNDLVGEKLVVVVLEEPSASIMVELKLPLEINYAFSMDEEFKNMDCI CA2503D7645397AC CA2503D7645397AC rap1-sir3 2 5 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 3 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 8 physical association physical association 1,6 table 1, 2 From these results, we place the amino-terminal endpoint of the minimal region of Sir3p required for the two-hybrid interaction with Rap1p between amino acids 455 and 481. rap1-sir3-1 1 5 unspecified role unspecified role prey prey region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 3 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 13 16 direct interaction direct interaction Fig. 2 wild-type Sir3p and Sir3p(440-454) are both able to bind to the GST/Rap1p(562-827) fusion. In contrast, Sir3p(456-479) and Sir3p(440-480) show a strong impairment in binding to GST/Rap1p. rap1-sir4 1 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 region required to bind necessary binding region required 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 human-ws Homo sapiens Werner Syndrom (WS) cell line transformed with SV40 (AG11395 cell line) ws Human Werner Syndrome (WS) cell line transformed with SV40 (AG11395 cell line) 3 unspecified role unspecified role bait bait 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 5 the Sir4p(737-1358) fragment can interact with GST/Rap1p(562-827), whereas the shorter Sir4p(737-840) fragment cannot bind specifically to Rap1p even when larger amounts of the 35S-labeled protein are used. rap1-sir4-1 2 4 unspecified role unspecified role prey prey region required to bind necessary binding region required 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 3 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient 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 31 27 physical association physical association 6 As shown in the middle column of Fig. 6, deletion of the last 83 amino acids of Sir4p completely abolishes the two-hybrid interaction with all LexA/Rap1p fusions.