E scherich ia coli trna L eu. L i Yong L iu W en W ang En2D uo

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1 14 6 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy D ec 1998 trna L eu :,, 34, : , : (, ) (, ) trna L eu trna L 2 eu 2tRNA 2tRNA,, 2tRNA K m M g 2+, :,, 2tRNA, Prepara tion, Pur if ica tion and Properties of E scherich ia coli trna L eu T u H ua2m in M eng J ian2x in Yang Yan2Sheng (D ep artm ent of Chem istry, Z hong shan U niversity, Guang z hou ) L i Yong L iu W en W ang En2D uo (S tate2key L aboratory of M olecular B iology, S hanghai Institute of B iochem istry,a cadem ia S inica, S hanghai ) Abstract T he b io logical activity of a nucleic acid is in stinctively associated w ith its th ree2di2 m en sional structu re M etal ion s affect the secondary and tertiary structu res, stab ilities, and confo rm ational states of RNA and DNA T he in teraction of rare earth ion s (R E 3+ ) w ith trna had been studied by the spectro scopy m ethodṡ T he resu lts show ed that the structu ral ro le of M g 2+ cou ld be sub stitu ted by R E 3+, bu t th is sub stitu tion induced a confo rm ational change of trna that m igh t affect the b io logical function of trna In o rder to study the b io2 logical effect of R E 3+, leucine isoaccep to r tran sfer ribonucleic acid of and trna L 2 eu from E coli w as p repared and purified u sing an overexp ressed strain A detailed steady2state k inetic study of the am inoacylation of the and M g 2+ 2free from E coli w ith differen t R E 3+ by the purified E coli L eucyl2trna syn thetase (L eur S) w as carried ou ṫ T he b inding of to the L eur S and the catalytic efficiency w as affected by the participation of the R E 3+ T he apparen t K m value fo r native and M g 2+ 2free w as m arkedly changed T hese resu lts indicated that M g 2+ requ irem en t of the am inoacylation reaction cat2 697

2 alyzed by L eur S from E coli cou ld be rep laced by R E 3+, bu t the affin ities fo r trna L eu 1 w ith L eur S w as differen ṫ Key words: L eucine tran sfer RNA,Am inoacylation,l eucyl2trna syn thetase, R are earth ion (tran sfer ribonucleic acid, trna ), 2tRNA (am inoacyl2trna syn thetase, aar S) [ 1 ] trna, [ 2, 3 ], trna, trna,,, NM R [ 4 ] CD [ 5 ] UV [ 6 ] [ 7 ] ( rare earth ion, R E 3+ ) trna, R E 3+ M g 2+, trna, R E 3+ trna M g 2+, trna, 405, trna L eu [ 8 ], R E 3+ trna,,, : M T 102 (ara 139, ( ara, leu ) 7697, lacx74, glau, galk, stra, h sdr ) trna ; pt rc99b Am ann pkk23322 ( F ig 1), trp glac L acz rrnb T 1 T 2, pt rc99b lac I q, pu C18 lac lac I q, IPT G M T 102 pt rc99b ( In stitu te de B io logiem o lecu larir et cellu lair du, CN R S) Gangloff F ig 1 P lasm id schem atic of pt rc99b : O xo id ; A T P, D T T, Chelex2100, 2Β2D 2 ( IPT G), BD 2 Sigm a ; L a (A c) 3g115H 2O (> 9919% ) Am resco ; N dc l3g6h 2O (991999% ) EuC l3g6h 2O (9919% ) A ldrich ; A pp lied B io system s DNA grna : D EA E2Sepharo se CL 24B Pharm acia ; T 4 DNA Boeh ringer M annheim ; DNA [ 14 C ]2L 2 Am ersham ; : LB (L u ria2bertan i ) : (bacto2 698

3 tryp tone) 10 g; (bacto2yeast ex tract) 5 g; N ac l 10 g;, N aoh ph 710, 1 L, 2 YT : (bacto 2t ryp tone) 16 g; (bacto2yeast ex tract) 10 g; N ac l 5 g; LB IPT G: 8 m l 2 g IPT G, 10 m l, 0122 Λm, 1 m l : L 1523 ( ), (B ioengineering A G) ;, So rvall RC5C So rvall RC3C, D u Pon t ;, Cen trifuge 5402, Eppendo rf ; L S29800, Beckm an trna L eu : trna L eu, pt rc99b 2L eu1 pt rc99b 2L eu2; M T 102 M T 2L eu1 M T 2L eu2, [ 8 ] tr 2 NA L 2 eu F ig 2 F ig 2 Sequences of the trna L eu 1 and trna L eu 2, arranged in the classical cloverleaf structure trna trna L eu M T Λggm l LB,, 37 A ,,, 37, ph 7, A , IPT G 015 mm o lgl, 8 h,,,,, trna 4, ; T E, ph mm o lgl T ris2hc l 20 mm o lgl M g (A c) 2,,, 15 m in ;,, 2 95%, 1g20 50 m o lgl N ac l N aa c,

4 2 h ; ; ph m o lgl T ris2hc l, m in, ; 2 95%,,, trna trna L eu 1 trna L eu 2 trna N ac l 013 m o lgl N ac l, trna ; 017 m o lgl N ac l trna, M T 2L eu1 M T 2L eu2 trna 014 m o lgl N ac l A [ 20 mm o lgl KA c2ha c (ph 410), 10 mm o lgl M gc l2 0101% Β2M E 0102% N an 3 ] D EA E2Sepharo se CL 24B (2 cm 20 cm ), m o lgl N ac l,, m o lgl N ac l A, ; 115 m o lgl N ac l,, 014 m o lgl N ac l B [100 mm o lgl T ris2hc l (ph 715) 10 mm o lgl M gc l2, 0101% Β2M E 0102% N an 3 ] BD 2 (1 cm 15 cm ), m o lgl N ac l B,,, - 20 trna Chelex trna L eu L eur S ( gm g) [ 9 ], A 260 nm trna, 50 Λl, L eur S 4 nm o lgl, : mm o lgl, 1 mm o lgl, trna Leu 1 trna Leu 2 trna trna aar S, ( ) trna trna, trna trna, trna pt rc99b 2L eu1 pt rc99b 2L eu2 6 EcoR g P stg : 11AA T TC GCGAA GGT GG CGGAA T T GGT A GA CG CG; 21CTA GC T TCA G GT GT T A GT GT CCT TA CGGA C GT G ( ) ; 31GGGGT TCAA G TCCCC CCCCT CGCA C CA CT G CA ; 41GT GGT GCGA G GGGGG GGA CG T GAA C CCCCA CGTCC G; 51TAA GG A CA CT AA CA C CT GAA GCTA G CGCGT CT; 61A CCAA T TCCG CCA CC T TCGC G trna L 2 eu : 11AA T TC GCCGA GGT GG T GGAA T T GGT A GA CA CG; 21CTA CC T T GA G GT GGT A GT GC CCAA T A GGGC T TA ( ) ; 31CGGGT TCAA G TCCCG TCCTC GGTA C CA CT G CA ; 41GT GGT A CCGA GGA CG GGA CT T GAA C CCGTA A GCCC T; 51A T T GG GCA CT A CCA C CTCAA GGTA G CGT GT CT; 61A CCAA T TCCA CCA CC TCGGC G 6, pt rc992l eu1 pt rc992l eu2 700

5 212 trna Leu m l trna A 2 rg, trna ; ph (ph 7 8), trna ; IPT G 015 mm o lgl,, 100 Λgg m l, ph YT, A mm o lgl IPT G,,,,M T 2L eu1 M T 2L eu2 trna pm o l ( A 260 ) 1039 pm o l ( A 260 ), M T 2L eu1 trna 72% ; trna L 2 eu M T 2 L eu2 trna 65% trna 85 pm o l ( A 260 ) SD S2 PA GE M T 102 M T 2L eu1,m T 2L eu2 trna F ig 3, M T 2L eu1 M T 2L eu2 213 trna Leu 1 trna Leu 2 pt rc99b 2L eu1 pt rc99b 2L eu2, M T 102,, M T 2L eu1 M T 2L eu2 trna pm o l( A 260 ) pm o l( A 260 ),,, trna L 1 eu trna L 2 eu trna L 1 eu, D EA E2 Sepharo se, pm o l( A 260 ) ; BD 2, trna L eu (1 600 pm o l ( A 260 ) ) trna L 2 eu, F ig 3 SD S2PA GE of to tal trna transfo rm ants of trna 11To tal trna of ho st strain; 21To tal trna of M T 2L eu1; 31To tal trna of M T 2L eu2 from overexp ress F ig 4 T he p rocess of trna L eu 1 purification analysis by SD S2PA GE 11To tal trna of ho st strain; 21To tal trna of M T 2L eu1; 31tRNA after purification using D EA E2Sepharo se CL 2 4B co lum n; 41tRNA after purification using D EA E2Sepharo se CL 2 4B and BD 2Cellulo se co lum n 701

6 , trna A 260, 1 A Λg trna, trna [ 9 ] T ab le 1, SD S2PA GE F ig 4,, trna trna aar S X2, Table 1 Purification of trna L eu 1 in transfo rm ant M T 2L eu1 P rocess To tal trna gm g Specific activitygpmo l trna L eu 1 g% trna L eu 1 gm g Yieldg% C rude extract D EA E2Sepharo se BD 2Cellulo se [10 ], 37, 100 mm o lgl T ris2hc l (ph 718), 1 mm o lgl M gc l2 1 mm o lgl L nc l3 (L n = L a, Ce, N d, Eu, T b), 30 mm o lgl KC l, 1 m o lgl A T P, 011 mm o lgl ED TA, 015 mm o lgl D T T 011 mm o lgl [C 14 ]2, K m K cat, W oo lf L inew eaver2burk,, F ig 5, F ig 6 K m K cat T ab le 2 F ig 5 D eterm ination of K m, trna different k ind of m etal ions fo r native trna L eu 1 at M g 2+ ; L a 3+ ; Ce 3+ ; Eu 3+ F ig 6 D eterm ination of K m, trna fo r M g 2+ 2free trna L 1 eu at different k ind of RE 3+ Ce 2+ ; N d 3+ ; Eu 3+ ; T b 3+ R E 3+ M g 2+, R E 3+ M g 2+, trna, K m K cat N d 3+ Eu 3+, R E 3+ M g 2+, L a 3+ Ce 3+ T b 3+ M g 2+ ; N d 3+ Eu 3+ M g 2+, 3 (A T P, trna ), 3 M ga T P 2- A T P, A T P A T P, A T P, 702

7 Table 2 K inetic constants of trna L eu 1 and M g 2+ 2free trna L eu 1 at the participance of R E 3+ RE 3+ K m gλmo lgl - 1 K cat gs - 1 K catgk m gs - 1 gλmo l - 1 gl K 3 m gλmo lgl - 1 K 3 cat gs - 1 K cat 3 gk m 3 gs - 1 gλmo l - 1 gl L a Ce N d Eu T b M g Sp T he apparent steady state k inetic constants fo r M g 2+ 2free trna L 1 eu T he m easured conditions w ere 1 mmo lgl A T P, 011 mmo lgl leucine, 1 mmo lgl RE 3+ tration of trna L eu 1 in the range of mmo lgl o r M g 2+, and the varied concen2 trna, R E 3+ trna M g 2+,, R E 3+ M g 2+, trna, trna trna trna, K m, (K catgk m ) M g 2+, trna trna, References 1 Sak s M E, Samp son J R, A belson J N T he transfer RNA identity p roblem: A search fo r rules S cience, 1994, 263: Cherayil J D Iso lation, fractionation, and characterization of transfer RNA In: Cherayil J D ed T ransf er RN A s and O th2 er S oluble RN A ṡ N ew Yo rk: CRC P ress, Boca Raton, 1990: , : (W ang D e Bao, L iu W ang2yi ed T ransf er R ibonucleic A cid s S tructu re, F unction, and S y nthesiṡ H angzhou: Zhejiang Science and T echno logy P ress), 1995: T u H ua2m in, W u Yi2J ie, Yang Yan2Sheng, Gong M eng2l ian, Zhang Hong2J ie Effect of lanthanum ions on trna Phe structure: Im ino p ro ton NM R spectro scopy Ch in S ci B u ll, 1998, 43 (6) : ,,, trna, (T u H ua2m in,m eng J ian2x in, Yang Yan2Sheng, Gong M eng2l ian Effect of lanthanum (g ) ions on C ircular dichro ism spectrum of yeast trna A cta S ci N atu r U niv S uny atseni), 1998, 37 (1) : 5 8 6,,, trna (T u H ua2m in, Yang Yan2 Sheng, L i Yong,W ang En2D uo Effect of rare earth ions on the m elting curve and m elting temperature of som e trna Ch in J S p ectro L ab), 1997, 14 (6) : M eng J X, T u H M, Gong M L, Yang Y S, Zhou J Y, Zhang H J Study on the binding of cerium (g ) ion and sperm ine to trna Phe S p ectro A cta (part A ), 1997, 53A (10) : L i Yong,W ang En2D uo,w ang Ying2L ai O verp roduction and purification of E scherich ia coli trna L eu 1, S cience in China (Series C), 1998, 41 (3) : in p ress 9,, 2tRNA (L i Tong,W ang En2D uo,w ang Ying2L ai The overp roduction of L eucyl2trna synthetase in E coli and its purification A cta B iochim B iop hy s S in), 1997, 29 (6) : L in S X, Sh i J P, Cheng X D,W ang Y L A rginyl2trna synthetase from E scherich ia coli purification by affinity ch ro2 m atography, p roperties, and steady2state k inetics B iochem istry, 1988, 27 (17) :