Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy. E, m RNA T IR

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1 ISSN CN gQ Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy 17 (1): m RNA 5 3,, (, ) 21 bp B 50 PCNA 2L ac Z m RNA SD 11 bp H ind g, B 50il B 50i2 m RNA,B 50il (T IR ), T IR ; B 50i2 T IR 5 ; D 13 B 50 SD 6 7 ; T IR SD AU G Β2 m RNA T IR E, m RNA T IR, T IR 5, m RNA T IR SD AU G, T IR 5, PCNA 2L ac Z PCNA 2L ac Z Β2,,,, (T IR ), E. coli Q 756 Effect of Secondary Structure in D ifferen t Position of the 5 End of m RNA on the Tran sla tion In itia tion in E scherich ia coli 3 X IA Yu2lei, CH EN N ong2an,lu Chang2de 3 3 (S hanghai Institute of B iochem istry, Chinese A cademy of S ciences, S hanghai , China) Abstract A 21 bp fragm en t w as in serted in to the H ind g site, 11 bp up stream of the Sh ineg D algarno (SD ) sequence, of the p lasm id B 50 w h ich w as a clone fo r the exp ression of the hum an PCNA 2L ac Z gene. If the fragm en t w as in serted in fo rw ard direction, the p lasm id B 50il w as ob tained and com puter analysis revealed that a hairp in fo rm ed ahead of the tran slation in itiation region (T IR ) bu t no t affected the T IR. If it w as in serted inversely, B 50i2 w as ob tained, w ith a secondary structu re at the 5 part of the T IR. A no ther p lasm id,d 13,w as differen t from B 50 in the secondary structu re of the T IR, becau se of differen t 6 bp up stream and 7 bp dow n stream of the SD sequence. By the com parison of the Β2galacto sidase activities and the energies ( E ) requ ired fo r releasing the secondary structu re of the T IR calcu lated by the com puter, of the fou r p lasm ids, it indicates that the efficiency of tran slation in itiation is no t affected by the secondary structu re ahead of the T IR, bu t by that of the 5 part T IR, though less than that near SD and AU G. M eanw h ile, the exp ression of the L ac Z w as m easu red and com pared w ith that of the L ac Z. T he Β2galacto sidase activity w ith ho loenzym e w as a little b it h igher than the Α2pep tide of enzym e, bu t the ratio s am ong the differen t p lasm ids w ere the sam e. : , : (N o ) 3 Tel: (021) , Fax: (021) , E2m ail: lucd@ sunm. shcnc. ac. cn,, , Received:M arch 24, 2000; A ccep ted: June 9, 2000 Suppo rted by N ational N atural Science Foundation of China,N o Co rresponding autho r T el: (021) , Fax: (021) , E2m ail: lucd@ sunm. shcnc. ac. cn

2 30 17 Key words tran slation in itiation, secondary structu re, tran slation in itiation region, E. coli E. coli ; B 50i2 B 50 D 13, m RNA T IR 5 SD,, 1, m RNA (T IR ) fm et2 trna, 111 [ 1 3 ] SD [ 2 4 ] : 5 A GC TCA AAA GA G [ 2, 3, 5 SD ] T IR CTC TCC CA T 3, 5 A GC TA T GGG A GA GCT [ 3, 5 10 ], CT T T T G 3, PCR 70S m RNA, T 4 DNA, 2Β2D 2 [ 11, 12 fm et2trna ] ( IPT G), Β2D 2 (X2gal) P rom ega 2Β2D 2 AU G SD SD AU G (ON PG),,, T IR (PM SF), T IR, 112, E. coli BL 21 (D E3) : hsds g a l (Κcg ts857 [ ], m RNA 5 ind 1 S am 7 n in5 L acuv 52T 7 gene 1), ( 5 SD ), AU G S1 B 50 D 13 (PCNA ) [ 19, 20 ] PCNA bp (38 ) pt Z19R 114 bp L ac Z, SD SD L acz 5, [ bp, ] [ 7 ] pgem 2T V ecto r System s P rom ega T IR, pβgal2basic CLON T ECH, 113 T IR, 2,, T IR 5 B 50 B 50, B 50i1 B 50i2, SD 11 bp, 2 PCR,B 50il B 50i2,, B 50 B 50i1 B 50i2 5 U TR 2PCNA B 50il m RNA T IR pβ2gal Basic L ac Z, T,, T IR B 50i2, T IR 5, B B i1 B i2 B 50 B 50il B 50i2 D 13 B B i1 B i2 SD AU G B 50, 1 D 13, B 50 SD 6 7 D B 50 SD, SD, pβ2gal Basic, PCNA 2L ac Z BL 21 (D E3), Β2 A (48 mm o lgl N a2h PO 4, 22 mm o lg AU G, L KH 2PO 4, 5 mm o lgl N ac l, 1817 mm o lgl N H 4C l, T IR T IR 5 SD AU G 1 mm o lgl M gso 4, 20 ggm l V B1, 014%, 100 Β2 ggm l, 15 ggl ),, 4, A, h, m RNA m l 115 m l IPT G A B 50 B 50i1 B 50i2, ( IPT G 014 mm o lgl ), 37 m RNA T IR 5 2 h

3 1 : mrna Β-, F ig. 3 F ig. 3 :, M iller : B 50i1 B 50i2 B 50 21,, A 600, 011 m l 019 m l B 50 m RNA SD 11 bp 21 ( 60 mm o lgl N a2h PO 4, 40 mm o lgl N ah 2PO 4, 1 mm o lgl M gc l2 50 mm o lgl Β2 ), 50 l 40 m ggm l, 100 l 100 mm o lgl PM SF,, 10 m in, B 50 ; B 50i2 T IR 25 l 10% SD S,, 37 5 m in, 100 l 4 m ggm l ON PG, 37 B 50 D 13 B 50, 013 m l 1 m o lgl N a2co 3, SD AU G, A 420 Β2 213 m RNA 5 : (U ) = 1000 A 420g(t V [ 21, 22 A 600), t (m in), V RNA ], [ 7 (m l) ( ] T IR JM 109 (D E3), BL 21, Β2 ( T ab le 1) (D E3), JM 109 (D E3) B 50il T IR B 50, T IR,, T IR ) 2 bp D 13 B 50, SD 6 7,B 50i1 T IR, 5, SD, B 50i2 T IR B kj g m o l, D 13 B kj gm o l, B 50i1 B 50,B 50i2 B 50 87%,D B 50 68%,B 50i2 T IR B 50i1 B 50i2, D 13, D 13 DNA, Table 1 Energy required to release the secondary structure B 50 D 13 B 50i1 B 50i2 F ig. 1 of T IR and exp ression of fush ion p ro tein (as Β2galacto sidase activity) Recom binant E 1) gkj mo l - 1 2) Β2Gal activity g10 3 U Relative exp ression 3) (% ) B B50i B50i D ) E is the energy required to release the secondary structure of T IR ( ) determ ined by the computer p rogram. 2) The Β2galacto sidase activity is the average of at least three independent m easurem ents w ith th ree data fo r each recom binanṫ 3) Percentage of B50 Β2galacto sidase activity. F ig. 1 T he structures of the p lasm ids B50, D 13, B50i1 and B50i2, B 50i1 T IR D 13, 212 m RNA 5 D 13 B 50i2 T IR 5, B 50 B 50i1 B 50i2 D 13 D 13 SD AU G,m RNA 5 U TR T IR B 50 B 50i1 B 50i2 D 13 T IR 5 F ig. 2 RNA 214 B B i1 B i2 [ 21, 22 ], m RNA + 20 Β2 Α

4 32 17 F ig. 2 T he 5 sequences of four recom binants F ig. 3 T he secondary structure of 5 U TR of four recom binants Table 2 Β2galacto sidase exp ression 1) Recom binant Β2Gal activity 1) g10 3 U Relative exp ression 2) (% ) B B i B i The Β2galacto sidase activity is the average of at least three independent m easurem ents w ith th ree data fo r each recom binanṫ 2) Percentage of B Β2galacto sidase activity. 5 U TR L ac Z pβ2gal Basic, B B i1 B i2, F ig B B i1 B i2, B B i1 B i2 Β2, 2 B 50 B 50i1 B 50i2 B B i1 B i2 Β2 (T ab le 1 2),, L ac Z B B i1 B i2 L ac Z B 50 B 50i1 B 50i2,B B i1 B i B 50 B 50i1 B 50i , B 50 B 50i1 B 50i2, Β2

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