Pa_TrmU ......................................................................Pf_TrmU ......................................................................Tk_TrmU ......................................................................

Pa_TrmU ......................................................................Pf_TrmU ......................................................................Tk_TrmU ......................................................................

1 10 20 30 40 50

Pa_TrmU RG I L D G G ILVP APGD V K A V RK N VL FS EII R ER KR V S KLVR........M D F KG........... E V V R QWPf_TrmU RG I L D G G ILVP APGD V K A I KR N IL FS EVE K EK RA I T RLLR........M D F YN..........K L T Q K EWTk_TrmU RG I L D G G ILVP APGD V K A T ER S TI YT VVE R RR KK I T KVIE.......MI E L RA.......SRRE A W K L DF

60 70 80 90

Pa_TrmU SP R C FG CGGC LQH Y Q EFK KL GS L V R T L L RK KR L . GP.. KA K N DY I F........................Pf_TrmU SP R C FG CGGC LQH Y Q EFK KL GS I V R I I L KE RK L . GA.. KV K N NS I R........................Tk_TrmU SP R C FG CGGC LQH Y Q EFK KL GE I T K L L I SE SR I . EP.. PY V P EK Y T........................

β1 β2 β3 β4 β5

α1

Ec_TrmA M Y L K L EQ A VVR T................PEHLPT E Q AE ................................Hi_TrmA M Y L K L SQ E LEK ...................QLPI N L QK ................................Pp_TrmA M Y L K L SS T VAR .................SAAFDP A Q DA ................................

Ec_TrmA ......................................................................Hi_TrmA ......................................................................Pp_TrmA ......................................................................

Ec_TrmA PF V QSMM E................................... A SDLVP .....................Hi_TrmA PF V TALL Q................................... H NAPDI .....................Pp_TrmA PF V RELL A................................... A GAPEP .....................

Ec_TrmA ......................................................................Hi_TrmA ......................................................................Pp_TrmA ......................................................................

Sc_trm2 M P S S V T KRL S L S NR ...........TG TEM P.......................... TMKH VDN P TD G RTSp_Trm2 M P S S I A RAI S I A NK YPYCLKYLTNGLR KQM NFLCIQNILSARTLKICRFRFLPTPSH THKM QET . SE A RAHs_Trm2 M P S N E S ESS A S T SV ...........SE LDN G.......................... KPME CGQ L CP V PP

Sc_trm2 LKK RK VE V EV LK L P....KL YKAKK TTSPM.G LEF ND ....... SQN SREQVLNDVTSILNDKSSTDGPISp_Trm2 LRK RR VE V EI IE L L....SK AK.KP EGSSG.H LLL EN ....... KPV AN....................VHs_Trm2 LAA EK AT L IR LE V PAALEEV EGAGA GPGPQPG YSY DD FTSEIFK LQN PRHASFSDVRRFL.......GRF

Sc_trm2 K V G Q V S N K V I V IKV S V EVL YHREVKNV LEIT NGNGLALID PVETEK Q IPFGLP DV N FKTHPYYVE DLLD V Sp_Trm2 K V G E I S N Q A V V AKL A V SPF RF.QEIEV SYLS SGDGIGLVC .....D Y VPFTLP DI K HFLADTYAL DFLE I Hs_Trm2 K V G Q T Q S A R L L VRL R E EGL PH...... KLFG PPCAFVTFR AAERDK L HGALWK RP S ARPKADPMA RRRQ G

Sc_trm2 Y QL K S R IR L R T A K AK P.M RDDL DKYFGKSSGSQLEF T DD EL K IMN Y FF P....RLVAE............Sp_Trm2 Y QL K S R IK L K V A Q SP E.D DDTL CPYFAKCGGCQYQM S DK LQ R VEK F YY K....LD..S............Hs_Trm2 Y QL K E T VV V Q E V Q AS PPV RVAD TP.........LWT P AE ER L CEQ L KL KEIGSTNRALLPWLLEQRHKHN

Bs_RumA M K K VEK ..................... M PP N.......................................Lp_RumA M K K LTS ..................RKV P LN ........................................Ec_RumA M K S TTT .................AQFY A RR R.......................................

1 10

Bs_RumA ......................................................................Lp_RumA ......................................................................Ec_RumA ......................................................................

Bs_RumA L G G A G F LP E T KK VT A V K Q I V A AQ R F RLIEL E......EYYD FED THE V....... FP PN E K IKV V G AFG K Lp_RumA L G G A G F LP E T KK AR R I R N T I A VE R F KLLSI E........QT IVN SHD V....... KA QG G V FQY V D DEG V Ec_RumA L G G A G F LP E T KK VS Q V R N L I L AE E Y KVVRR S......QIIT VND DSF H....... KT PG Q N VTV D Q ARA L

20 30 40 50 60 70

Bs_RumA S R CP CGGC QH Q K T Y Q T QK V VLE P.H DA..P I KQ L M YEG LLF Q KD ERIGK....LD..L............Lp_RumA S R CP CGGC QH Q K V Y S S QS L LLP T.L EP..K H QM L M AEE IRF H LD SRYGH....TE..P............Ec_RumA S R CP CGGC QH Q K E F Q S SA L LMD P.E TP..R H GV Q A VDL QRS A AR KH........D..V............

80 90 100 110

β1 β2 β3 β4 β5

α1

* * *

... 100 110 120 130

Pa_TrmU V PS P I GHRNRID IGFR G WW VV K F LAIT E I.....E E ... ..........KDG... R K ............K Pf_TrmU V PS P I GHRNRID IGFR G WW II R F LAVT E V.....E E ... ..........VNA... K K ............S Tk_TrmU V PS P I GHRNRID IGFR G WW VI V Y VVIS R A.....D D ... ..........TGG... Y T ............D

. 140 150 160 170 180 190

Pa_TrmU VD ECPVFGKTS L E IE EK I GFL RY V REGKFT E MVN VT EG LP I REAIER K F SV KK E L V K D E I WN D . M E F . N .Pf_TrmU VD ECPVFGKTS L E IE EK I GFL RY V REGKFT E MVN VT EG LP V KKAIER R Y ST RE S L I K E E V WK D . M G F . E .Tk_TrmU VD ECPVFGKTS L E IE EK I GFL RY V REGKFT E MVN VT EG LP I RKVLKS R F TL RK A M L S E D P YE N . I G L . K .

200 210 220 230 240

Pa_TrmU Y F SIYWS NR SDVSYG D E WG F I E LD V YLI N F V SK I RF K R........DP.T ....... D .D .. E .. R D.. DPf_TrmU Y F SIYWS NR SDVSYG D E WG F I E LD V YLI S F I SK I KY K E........DP.T ....... D AD .. E .. M G.. KTk_TrmU Y F SIYWS NR SDVSYG D E WG F I E LD V YLI E Y V TE V RY E K........ESFP ....... P AT .. P .. R D.. T

250 260 270 280 290 300

Pa_TrmU HPNSFFQTNSYQAVNLV V V E GEK D YSGVGTFG YLAK G V G N FA R SE IL M I R S I K L ....... ... FN K FD EPf_TrmU HPNSFFQTNSYQAVNLV V V E GEK D YSGVGTFG YLAK G V G N FA K EK VV M V K S I T F ....... ... MK V FD ATk_TrmU HPNSFFQTNSYQAVNLV V V E GEK D YSGVGTFG YLAK G V G N FA R AE VL L I R I V K L ....... ... FK E IE P

β6 β7 β8

β9 α2 β10 β11

β12 β13 β14 β15

α3 β16 α4 β17

Ec_TrmA F SP HYR RAEFR W F VS M I I T R......... R .... . HDG....DDLYH... I DQQ KS ..............Hi_TrmA F SP HYR RAEFR W F TS M I I T R......... D .... . HEQ....DDFYH... M DQA LQ ..............Pp_TrmA F SP HYR RAEFR W F RE L L A E K......... D .... . RED....GQRHY... M APG KH .............A

Ec_TrmA D FP AS IN M L LFQ L TL YH L W V A QL AM RNN V K I Y T S A VSL K E IR S EL T IAGV P .... RH .....D NQ V L K DDHi_TrmA D FP AS IN M L LFQ L TL YH L W V I RM TL KQQ V K I Y S S I VSL K E YR E ML Q LPLL E .... HK .....D NK I L T TEPp_TrmA D FP AS IN M L LFQ L TL YH L W I I AL RL QAS E R V F T A A ITM R A IL D ER P KAAW E .... GN .....E GD M C P DE

Ec_TrmA A L L V IGR DY E L V G YRQRQE EA R A L AT KI Q I R A K I D . RA.......QNLN H KT ...........ELD D P EMHi_TrmA A L L V IGR DY E L V G YRQESA KN K L I AS KI Q V V N R V D . EK.......QDFD Q KQ ...........CFE D P NYPp_TrmA A L L V IGR DY E L V G YRQEVE RQ A A V SK RL R A K A R S E . ............G S GK ...........VIG V D VF

Ec_TrmA E FTQPN N ML WA DLLELYCGNGNF LA VLATEI K SV S A Q E T SK SL R R AV N A M I LDV .......K.G G A NFD PHi_TrmA E FTQPN N ML WA DLLELYCGNGNF LA VLATEI K SV S T K E T SE SI Q K AV N A V C IDC .......Q.N G A NFR PPp_TrmA E FTQPN N ML WA DLLELYCGNGNF LA VLATEI K SV A A K S I RE TL T Q SP G G V Q FEA .......G.E D P RVR T

Sc_trm2 S P Q YR K GKL TV T V L SI LPPFDT A ... L FG T I PHFD.MPKR.KQKELS RPP FGQKGRPQWRKDTLDIGGHGSp_Trm2 S P Q YR K GSL TV T I I RV LPAIKD G ... L YN T I PHFD.VPKG.GTKGPL ... FQEKGRR.............Hs_Trm2 S P Q YR K GKA VR E R L AV CCPLEG P ... Q TE N C FLVGVGVDGEDNTVGC ... K.......YKGGTC......

Sc_trm2 A EVL K RR EQE KK A R TTLDIDECVL T N GLTNE KF ...FKNY G TILL EN ..................ILDPSSp_Trm2 A KTI E IE QSR KR A R ATMDIEECPI T N EYPKI DV ...ANTF G TILM DS .......................Hs_Trm2 I EAT Q QE RST ET T K TVAAPFDTVH P K VVKAF FI PYSAYDP Y GHW. QL RTSRRHQAMAIAYFHP.QKLSPE

Sc_trm2 E E F K T V TKPTLEQLTEEASRD.......ENG.DISYVEV DK NNVRLA..KTCV NPRQI.. T YVDG..Y NFSp_Trm2 E E F K T V S................................ DG HCV........I DHKMI.. R QFGD..L TFHs_Trm2 E E F R V I TE..LAELKTSLAQHFTAGPGRASGVTCLYFVE GQ KTPSQEGLPLEH AGDRC.. H DLLG..L RI

Sc_trm2 FFQ N D CG G VIGVE V E IL K VR L K K V A S I SK K I S SAG N NS PIVT Y DN QAP.AKGDDN T FL Y LFS CS GVD SADSp_Trm2 FFQ N D CG G VIGVE V A IL S VR L R K V A S V SK S I S PAG N NS EKFT Y EQ LNPFGRKEHQ P YF Y LFS AC GFL SADHs_Trm2 FFQ N D CG G VIGVE V A AA T IQ A A S L V T L AR R L A SPH V TP EVLY V DW .......QLD G MV C TIG AL KVK CPE

Bs_RumA W YR A GF IS V T K QV V R QQ S ....KVT HP LGMEDP N N P G..E ....EGGLV...A Y R H.............D Lp_RumA W YR A GF IQ S S K RL T K ER N ....... VL PLTSHH N N S RFVE ....KQSTM...V R N PR............F Ec_RumA W YR A GF IS E A R RL L K KA S ....... VI DV...P G R S NYLP ....TQQLQ...M R G SD.............

120 130 140 150 ...

Bs_RumA C I II A Q K E V VK N N I VR T E M R S DMS L Q S ND A QA DICA YGVKAY EERHKGWL.RH M YGVV G M VFIT .T DFPHLp_RumA C I IT Q N K T I LR T E I VA N V L S L EIN P L S ID D VH KLID M..... DKHCIA....Q E AG.D E A FRNL .P TEQDEc_RumA C I IV Q A Q A L VR S Q V LV S T M A L DVK P L P LE L PK ACLG L..... AMRHLG....H E QA.T G L LRHT .P SSAD

160 170 180 190 200 210 ....

Bs_RumA KK I E T Q I N T E I VK A A ...I DI A .......FPHVKS VQ INPNK NVIFG.NETNVIWG EY..IYDL GD.. F ILp_RumA KE I R A Q V Q S A L IT Q L .... EF Q .......FQY..K FL PGGLD VFC......FYPSD HA.YLSYE PDYQ F FEc_RumA KR L E S S L A S E S LR T E .... RF H .......EGL..D YL P...D EIL......ETVSG MP.W..YD NG.. L F

220 230 240 250 .... ....... .. ... ...... ..

Bs_RumA F QVN A V D CG G L A V GVE K L K LE LQ E T I AY I S L KK ISARS Y PEQT V YD YA.......E G E TI F KQA Y IVPEA Lp_RumA F QVN A V D CG G L A V GVE R M Q IQ LK S I L LF L S M SR VHPND T AELN K VT LM.......E N D NF P KHC I GNKNM Ec_RumA F QVN A V D CG G L A V GVE Q M R LE VQ E R L LF M T L AS VSPRD I AGVN K VA WL.......D P D NF P TQA V GVPAL

260 270 280 290 300 310 320

β6 β7 β8

β9 α2 β10 β11

α3 β12 β13 β14 β15

α4 β16 α5 β17

*

Motif X Motif I Motif II

310 320 330 340 350

Pa_TrmU EMA N IN V AEF V D V DTV VDPPRAGLHP LV R VE S R VK F I R KRL R N D. E A E S.................. G NRPf_TrmU EMA N IN V AEF V D V DTV VDPPRAGLHP LV K AE S R IK F I K KRL N N E. F A E D.................. G NDTk_TrmU EMA N IN V AEF V D V DTV VDPPRAGLHP LV K AQ E K LS Y V K RKI K G D. R G D N.................S E LK

360 370 380 390 400

Pa_TrmU P VYVSCNP T L Y I E LDMFPHTPH E V KL VI E AR VK IV V L VEK .G F D M .D.......... R D A A .........Pf_TrmU P VYVSCNP T L Y I E LDMFPHTPH E V KL TI K KV IE LI I L RHG .E F N Q .N.......N.. I E A G RLV......Tk_TrmU P VYVSCNP T L Y I E LDMFPHTPH E V KL TL K SQ LK AV V A RDR .E L N E .S.......EG. K E G L KV.......

Pa_TrmU .............................Pf_TrmU .............................Tk_TrmU .............................

α5 β18 β19 α6

β20 α7 β21 β22

Ec_TrmA AA N N DN R AEE TQA N VR F RL GIDLKSY T FVDPPR G D TA I A I VQII MA M Q Q I S L KMVQ QY A H F G E N CE SE E AHi_TrmA AA N N DN R AEE TQA N VR F RL GIDLKSY T FVDPPR G D TA I E V LQII MS M K E I A L KLVQ QF A K F G A N CN PD V NPp_TrmA AA N N DN R AEE TQA N VR F RL GIDLKSY T FVDPPR G D TN L E V VRLV LS L E Q V A M ELTR LS D A L E P R FG PD C R

Ec_TrmA RILYISCNP TL N L TH E ALFDQFPYT HME G LY LET S KV R V TAK ...P E CK . ........Q L H C L .......Hi_TrmA RILYISCNP TL N L TH E ALFDQFPYT HME G LY LVE S RI K L IRK ...D H CD . ........K A D S W .......Pp_TrmA RILYISCNP TL N L TH E ALFDQFPYT HME G LF IAQ Q RI R V VRR ...E E AA . ........D C H S L .......

Ec_TrmA .............................Hi_TrmA .............................Pp_TrmA .............................

Sc_trm2 A NA N N F G AE DPPR GS E K VE R K L SI T E S IL K E L QL A F K A G C IV K FE ............D P.S NT V CD LF K A Sp_Trm2 A NA N N F G AE DPPR GR E K VS T Q I SI T N A VI K Q L QL E Y E R N A IV K FS ............E P.P ET M CD SF N L Hs_Trm2 A NA N N F G AE DPPR GE R Q LS E R L TL R Q V IL A S I AI R D V D E V HC D VP .....VS...... LAS HL A LH KV L R

Sc_trm2 Y SCN G D FPQ H E KII I SQ KE E S Q IR T V I RIYNP.A VH ARDVEYFL T N... AH IES GF F H V S C MK .......Sp_Trm2 Y SCN G D FPQ H E RIV I TQ SQ E R K IR S I T KVYSP.Y VH ARDVGFLL . K... SY IDE GF L H V S L LT V......Hs_Trm2 Y SCN G D FPQ H E RLL V AA RA S I R AV T L L RVAKNLR PR MGNFVDLC P NRVK PF PVK AV L P C M I FE EHPNGTG

Sc_trm2 .............................Sp_Trm2 ............................SHs_Trm2 VLGPHSPPAQPTPGPPDNTLQETGTFPSS

Bs_RumA E NA N N F DP R G I EL T A EA E LVV K L RT DAKR GN E AVG ETVIPKW............YE GITADT P CDEA L VELp_RumA E NA N N F DP R G I KS T V NL V VLI S I KQ RAYM HI D YAA DDVMEVR............SL NTSFSK P AL.E V DSEc_RumA E NA N N F DP R G I RL Q V NL A VLL A V QQ KGQQ GL T YHE EEDVTKQ............PW KNGFDK A AA.G M IK

330 340 350 360 370

Bs_RumA P R VYVSCNP TLARD L GY DMFPHT H E LM V V VT EV V V K K .K G LR .E.......DG R QPV N CC I LKE......Lp_RumA P R VYVSCNP TLARD L GY DMFPHT H E LI I I VL TA V A Q D .E I TD VN.......QK I GVM A SI F KG.......Ec_RumA P R VYVSCNP TLARD L GY DMFPHT H E LL I A TI RL L V S E .I A SE L........KA A AML G SM F RVK......

380 390 400 410 420 430 ........

Bs_RumA .............................Lp_RumA .............................Ec_RumA .............................

α6 β18 η1 β19 α7

β20 α8 β21 β22

▼ ◆

Motif IV

Motif VI Motif VIII

Figure S1: Structure-based amino acid sequence alignments of several

members of the PabTrmU54/TrmA/Trm2/RumA family.

Alignment of protein sequences of TrmU54 (Pa, Pyrococcus abyssi; Pf,

Pyrococcus furiosus; Tk, Thermococcus kodakarensis), TrmA (Ec, Escherichia

coli; Hi, Haemophilus influenzae; P p Pseudomonas putida), Trm2 (Sp,

Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Hs, Homo

sapiens), and RumA (Bs, Bacillus subtilis: Lp; Legionella pneumophila, Ec,

Escherichia coli) was performed with T-Coffee (1) and rendered with ESPRIPT

(2). The secondary structure elements of PabTrmU54 and RumA are shown

respectively above and below the alignment. For PabTrmU54, the secondary

structure elements are colored red, green and blue for the TRAM, central and

catalytic domains, respectively. Contrary to TrmA, PabTrmU54 contains the

four conserved cysteines (indicated as stars) that form the [Fe4S4] cluster in

RumA and possesses an N-terminal domain similar to that of RumA. The

catalytic nucleophilic cysteine and the catalytic base are indicated as

triangles. Contrary to TrmA, PabTrmU54 contains the four conserved cysteines

(indicated as stars) that form the [Fe4S4] cluster in RumA and possesses an

N-terminal domain similar to that of RumA. The catalytic nucleophilic cysteine

and the catalytic base are indicated as full triangle and full diamond,

respectively.

1. Notredame, C., Higgins, D.G. and Heringa, J. (2000) T-Coffee: A novel method forfast and accurate multiple sequence alignment. J Mol Biol, 302, 205-217.

2. Gouet, P., Courcelle, E., Stuart, D.I. and Metoz, F. (1999) ESPript: analysis ofmultiple sequence alignments in PostScript. Bioinformatics, 15, 305-308.