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1 DOI:.8/ncb65 Chromosom XII cdc RDN IGS RDN5- RDN7 Figur S Cdc4 is rquird for rdna silncing. Yast tiling array intnsitis along 5kb of chromosom XII containing IGS rgions ( and IGS) (x axis). Plottd is th log ratio of transcript abundanc in cdc5- (blu) and (grn) at 7ºC rlativ to 5ºC for th Watson and Crick strands (y axis). Macmillan Publishrs Limitd. All rights rsrvd.
2 a Input (%) immunoprcipitatd Input (%) immunoprcipitatd 5 wild typ Sirp (5ºC) Sirp (7ºC) b RNA Lvls G/M arrst (7 ºC) **** *** wild typ sirδ sirδ ** * S Promotr Enhancr 5S Promotr Enhancr RDN7 IGS RDN7 RDN7 IGS RDN5- RDN7 c RNA Lvls 5 G arrst (7 ºC) **** *** ** * wild typ trf4δ trf4δ S Promotr Enhancr RDN7 IGS RDN7 d kda 5 λ-ppas control GST-Cdc4 GST-Cdc4(CR) GST-Ssu7 GST-Pph GST-Msg Quantity ( )(μg) Rpb 5 S5 P 5 S P GST kda CTD SP (G arrst 7 ºC) f CTD S5P (G arrst 7 ºC).5 wild typ.5 wild typ Fold nrichmnt.5.5 Fold nrichmnt ACT ACT 5S Promotr Enhancr 5S Promotr Enhancr RDN7 IGS RDN7 RDN7 IGS RDN7 Figur S Transcription of sub-tlomric rgions lacking Y lmnts ar unaffctd by Cdc4 inactivation. Yast tiling array intnsitis along 4 kb of th lft tlomr rgion of chromosom XV (x axis), which dos not contain Y lmnts. Plottd is th log ratio of transcript abundanc in rlativ to cdc5- at 5ºC (blu) and 7ºC (rd) for th Watson and Crick strands (y axis). Macmillan Publishrs Limitd. All rights rsrvd.
3 a Chromosom XII b Chromosom IV 7ºC /cdc5- ratio (log) /cdc5- ratio (log) Trminal rpats ((TG)-6TG)n 5 5 * * YLL65w Y lmnt Y lmnt X-rpats YLL64c AYT Chr XII rdna 7ºC /cdc5- ratio (log) /cdc5- ratio (log) - YDR54C Watson strand YDR54W ( ) YDR54C YDR544C YRF- YDR545C-A YDR54c 4R 4R-XC 4R-XC 4R-XC 4R-XR 4R-TR Y lmnt 4R-YP kb YDR54w X-rpats Right tlomr (IV) * Y lmnt Trminal rpats ((TG)-6TG)n 7ºC c /cdc5- ratio (log) /cdc5- ratio (log) Watson YOL66C strand ( ) AAD5 YOL64W-A Trminal rpats ((TG)-6TG)n 5L Crick 5L-TR strand (-) 5L-XR 5L-XC 5L-XC 5L-XC YOLCdlta YOLWtau - - X-rpats Chromosom XV kb YOL66c AAD5 LTR LTR YOL64w-a d RNA lvls 5 4 wild typ 4 YDR54w G arrst (7 ºC) X-rpats Y lmnt Trminal rpats ((TG)-6TG)n Chr XV RNA lvls 5 4 cdc5- Tlophas arrst (7 ºC) f Fold nrichmnt Cdc4-9myc 4 YDR54w X-rpats Y lmnt Trminal rpats ((TG)-6TG)n 4 YDR54w X-rpats Y lmnt Trminal rpats ((TG)-6TG)n g.5 (7ºC/5ºC) CTD SP h.5 (7ºC/5ºC) CTD S5P Fold nrichmnt.5.5 cdc5- (7ºC/5ºC) Fold nrichmnt.5.5 cdc5- (7ºC/5ºC) 4 YDR54w X-rpats Y lmnt Trminal rpats ((TG)-6TG)n 4 YDR54w X-rpats Y lmnt Trminal rpats ((TG)-6TG)n Figur S Transcription up-rgulation of sub-tlomric Y lmnts is causd by Cdc4 inactivation. Yast tiling array intnsitis of th distal 5kb of th lft tlomr of chromosom XII containing two Y lmnts (x axis). Plottd is th log ratio of transcript abundanc in cdc5- (blu) and (rd) at 7ºC rlativ to 5ºC for th Watson and Crick strands (y axis). Y lmnts ar indicatd (*). Macmillan Publishrs Limitd. All rights rsrvd.
4 Supplmntary Figur 4 a b c d f g Figur S4 Top-bottom gls of croppd immunoblots. (a), (b) and (c) Phosphatas assays on yast Rpb using a varity of phosphatass as shown and antibodis against myc (a), CTD srin phosphorylation (b), CTD srin 5 phosphorylation (c). Corrsponds to Fig. d. (d) Validation of GST-quantitis addd to th in vitro ractions. Not that th gl was cut into two, on half was visualisd with antibodis against GST (bottom half as indicatd), th othr half with myc to confirm prsnc of th Rpb substrat (top half as indicatd) in th raction. Corrsponds to GST blot of Fig. d. () (f) and (g) Phosphatas assays on human Rpb using hcdc4a and hcdc4acs and antibodis against human Rpb (), CTD srin phosphorylation (f), CTD srin 5 phosphorylation (g). Corrsponds to Fig. 5c. 4 Macmillan Publishrs Limitd. All rights rsrvd.
5 Supplmntary Matrials Tabl S. Yast strains usd Strain Rlvant gnotyp Rfrnc CCG87 CCG4 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 SMCHA LEU CDC4-9myc MATa lu ura his trp ad bar::natmx4 CCG85 MATa bar:hisg ura- trp- lu, his- ad- can- GAL+ cdc5- CCG8 CCG4 CCG4748 CCG55 CCG5499 CCG6 CCG776 CCG8 CCG859 CCG86 CCG86 CCG874 CCG949 CCG98 CCG95 CCG944 CCG989 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 TtR-YFP ADE TtO(5.6Kb):487Kb ChrXII HIS sir::kanmx4; :9myc:TRP MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 MATa lu ura his trp ad lys bar pp4:his sir::kanmx4 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 sir KanMX4 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 CDC4- GPF Kan; TUB4-CFP TRP; NET-CFP Hyg:MX4; GalCDC::NatMX4 MATa; his ; lu ; mt5 ; ura ; trf4::kanmx4 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 RPB:9myc KanMX4 MATa bar lu, ura-5 his- trp- 6 ad- lys-8 pp4 GAL- CDC4 URA MATa bar lu, ura-5 his- trp- 6 ad::ade ttr-gfp lys-8 pp4 PRP:(Tl-IV lft):tto:ura (ARM-dot) MATa bar lu, ura-5 his- trp- 6 ad::ade ttr-gfp lys-8 pp4 RAD55:(4 lft):tto:ura (4-dot) MATa bar lu, ura-5 his- trp- 6 ad::ade ttr-gfp lys-8 pp4 4:(MiddlChrm IV):ttO:URA (ARM-dot) MATα lu ura his trp ad bar::natmx4 MATa bar lu, ura-5 his- trp- 6 ad::ade ttr-gfp lys-8 pp4 chrmiv:5kb:(tlomr IV):ttO:URA (4R-dot) MATa, trf4::kanmx,, ADE, his, LEU, TRP, ura MATa, lu, ura, his, trp, ad, lys, bar, pp4:his, SMC-6HA::KanMX MATa,, lu, ura, his, trp, ad, SMC-6HA::KanMX MATa bar lu, ura-5 his- trp- 6 ad::ade ttr-gfp lys-8 pp4 chrmiv:5kb:(tlomr IV):ttO:URA (4L-dot) Macmillan Publishrs Limitd. All rights rsrvd.
6 Tabl S. Primrs usd in this study. rdna--f rdna TTTGAATGAACCATCGCCAGC rdna--r GATCCTGCCAGTAGTCATATG Pair 4 rdna--f TTTCCCACCTATTCCCTCTTG rdna--r CTTCAAGTGTAACCTCCTCTC Pair4 rdna-f GGCTATTCAACAAGGCATTCC rdna-r TGTAAATGGCCTCGTCAAACG Pair rdna-4-f AGCCTACTCGAATTCGTTTCC rdna-4-r ATAGTGAGGAACTGGGTTACC Pair rdna-5-f GGAAGCGGAAAATACGGAAAC rdna-5-r TCTGAAGCGTATTTCCGTCAC Pair rdna-6-f ATTTCCGCACCTTTTCCTCTG rdna-6-f AAGACAAATGGATGGTGGCAG Pair rdna-7-f ACCTGTCACCTTGAAACTACC rdna-7-r AACGGAAACGCAGGTGATATG Pair 9 rdna-8-f GCCATATCTACCAGAAAGCAC rdna-8-r TCTTCAGAAGAAGAGTGCAGC Pair 7 rdna-9-f CCATTATGCTCATTGGGTTGC rdna-9-r AGTAAATTTTTGGCGACGCGG Pair 6 rdna--f TCGCCAACCATTCCATATCTG rdna--r TTTTTTCCGCACCATCAGAGC Pair 5 rdna--f CTCTGATGGTGCGGAAAAAAC rdna--r TCTTTCTAAGTGGGTACTGGC Pair 4 rdna--f AGGCTTAATCTCAGCAGATCG rdna--r ATTGGTTTTTGCGGCTGTCTG Pair Tlomrs Tl4R--F CGAGTTACAAGTCATGGTACC Tl4R--R ACAAGACGATACGGTGATAGG Pair Tl4R--F TGCTGGATGGTGTTAGACAAG Tl4R--R TATTAGGTATACGACCTCGCG Pair Tl4R-4-F TGGGATTTGAGGATCCAGATC Tl4R-4-R TGAACGGAATGATTGGCCATG Pair4 Tl4R-6-F AATGTGGCCCCTGTAAGAAAC Tl4R-6-R CTGTGCCGCTCAAAAAGATTG Pair6 Tl4R-8-F AAAAAACAGTTGGGCGGCAAG Tl4R-8-R TATTGCTCTCCTGGAAGCTAG Pair8 Tl4R--F AGAGTCGTATAAGCGGAAAGG Tl4R--R CTCCGATACGATACTTTCTGG Pair Tl4R--F ATCTAAAGTGCCGCATTGGAC Tl4R--R TTCACTCTCCAACTTCTCTGC Pair Controls 4-R TTACTGTAAAACTGTGACGATAAAACCG 4-F TTTCAGAATGTATGTCCATGATTCGCCGG ACT-R TTGCATTTCTTGTTCGAAGTCCA ACT-F GGTATTGTTTTGGATTCCGGTGA Macmillan Publishrs Limitd. All rights rsrvd.
Supplementary Figures
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