re9u1at10n 0f the pr0m0ter5 and tran5cnpt5 0f rp0h, the...

Re9u1at10n 0f the pr0m0ter5 and tran5cnpt5 0f rp0H, the E5cher1ch1a c011 heat 5h0ck re9u1at0ry 9ene Jame5 W. Er1ck50n, 1 V1Ck1 Vau9hn, 2 W1111am A. Wa1ter, ~ Freder1ck C. Ne1dhardt, 2 and Car01 A. 6r055 ~

1Department 0f 8acter10109y, Un1ver51ty 0f W15c0n51n, Mad150n, W15c0n51n 53706 U5A; 2Department 0f M1cr0610109Y and 1mmun0109y, 7he Un1ver51ty 0f M1ch19an Med1ca1 5ch001, Ann Ar60r, M1ch19an 48109-0620 U5A

1n E5cher1ch1a c011 the pr0duct 0f the rp0H (htpR) 9ene, 0 ~2, d1rect5 RNA p01ymera5e t0 1n1t1ate tran5cr1pt10n fr0m heat 5h0ck pr0m0ter5 at a11 temperature5. 7ran5cr1pt10n 0f the heat 5h0ck 9ene5 15 1ncrea5ed when ce115 are exp05ed t0 h19h temperature5 6ecau5e 0f 1ncrea5ed tran5cr1pt10n 1n1t1at10n 6y cr32-RNA p01ymera5e. A5 a 5tep t0ward under5tand1n9 the re9u1at10n 0f the heat 5h0ck re5p0n5e we have exam1ned the tran5cr1pt10n 0f the rp0H 9ene. U51n9 51 mapP1n9, pr0m0ter c10n1n9, and 1n v1tr0 tran5cr1pt10n, we have 1dent1f1ed the pr0m0ter5 and the term1nat0r f0r the rp0H tran5cr1pt10n un1t. 7he rp0H tran5cr1pt5 are m0n0c15tr0n1c and 0r191nate fr0m at 1ea5t three pr0m0ter5. N0ne 0f the pr0m0ter5 15 rec09n12ed 6y cr32-RNA p01ymera5e. 7w0 are rec09n12ed 6y (r7°-RNA p01ymera5e and are act1ve at 60th 10w and h19h 9r0wth temperature5. We d0 n0t kn0w what f0rm 0f RNA p01ymera5e rec09n12e5 the th1rd pr0m0ter. 7ran5cr1pt5 fr0m th15 pr0m0ter are a6undant 0n1y at h19h temperature and are pre5ent after 5h1ft t0 the 1etha1 temperature 0f 50°C, even at t1me5 when there are n0 detecta61e tran5cr1pt5 fr0m the 0ther rp0H pr0m0ter5. 7he am0unt 0f rp0H mRNA 1ncrea5e5 f1vef01d 6y 8 m1n after 5h1ft fr0m 30 t0 43.5°C 6ut rp0H mRNA 5ynthe515 1ncrea5e5 6y 1e55 than tw0f01d, 1nd1cat1n9 that there 15 p05t-tran5cr1pt10na1 c0ntr01 0f the 1eve1 0f rp0H mRNA and pre5uma61y 0f cr 32.

[Key W0rd5:P05t-tran5cr1pt10na1 re9u1at10n; heat 5h0ck; 519ma-32; rp0H; htpR]

Rece1ved March 24, 1987; rev15ed ver510n accepted May 12, 1987.

When ce115, t155ue5, 0r even wh01e 0r9an15m5 are ex- p05ed t0 e1evated temperature5 they re5p0nd 6y 1n- crea51n9 the 5ynthe515 0f the heat 5h0ck pr0te1n5. 7h15 re5p0n5e t0 h19h temperature, termed the heat 5h0ck re- 5p0n5e, 15 51m11ar 1n 0r9an15m5 fr0m every 6101091ca1 k1n9d0m (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 1f ce115 are 5h1fted fr0m a 10w temperature t0 a h19her 0ne w1th1n the1r 9r0wth ran9e, the 1nduct10n 0f the heat 5h0ck pr0te1n5 15 tran51ent. 1f the ce115 are tran5ferred t0 temperature5 a60ve the1r 9r0wth ran9e, the heat 5h0ck pr0te1n5 are expre55ed at h19h 1eve15 f0r a5 10n9 a5 the ce115 5ynthe512e pr0te1n (Ne1dhardt et a1. 1984; L1nd4u15t 1986).

7he funct10n5 0f the heat 5h0ck pr0te1n5 are n0t we11 under5t00d. 1t 15 c1ear that 50me 0f the heat 5h0ck pr0- te1n5 are re4u1red f0r 5urv1va1 at h19h temperature and that expre5510n 0f the heat 5h0ck pr0te1n5 1ncrea5e5 the 1en9th 0f t1me ce115 5urv1ve at temperature5 t00 h19h f0r 9r0wth (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 5t1mu11 0ther than heat can 1nduce the 5ynthe515 0f var10u5 heat 5h0ck pr0te1n5 (Ne1dhardt et a1. 1984; L1nd4u15t 19861. 7he5e 5t1mu11 1nc1ude 5tarvat10n f0r nutr1ent5, v1ra1 1nfect10n, and exp05ure t0 ethan01, 0x1dant5, UV rad1a- t10n, am1n0 ac1d ana1095, 0r DNA-dama91n9 a9ent5. 1t

5eem5 that the heat 5h0ck pr0te1n5 he1p pr0tect ce115 fr0m a var1ety 0f adver5e c0nd1t10n5.

7he heat 5h0ck re5p0n5e 1n E5cher1ch1a c011151nduced rap1d1y. 7he 5ynthe515 0f the 20 0r 50 heat 5h0ck pr0te1n5 15 1ncrea5ed w1th1n 1 m1n after 5h1ft fr0m 30°C t0 a h19her 9r0wth temperature, 5uch a5 43°C (Ne1dhardt et a1. 1984). Max1mum rate5 0f heat 5h0ck pr0te1n 5yn- the515 are reached w1th1n 5 -10 m1n, and 6y a60ut 30 m1n after the 5h1ft the 5ynthe515 0f heat 5h0ck pr0te1n5 ha5 dec11ned t0 a new rate character15t1c 0f the h19her temperature (Ne1dhardt et a1. 1984). 7he 1ncrea5ed 5yn- the515 0f heat 5h0ck pr0te1n5 after 5h1ft t0 h19her temper- ature5 15 cau5ed 6y 1ncrea5ed tran5cr1pt10n 1n1t1at10n fr0m heat 5h0ck 9ene pr0m0ter5 (Yamam0r1 and Yura 1982; 7ay10r et a1. 1984; C0w1n9 et a1. 1985). At a11 tem- perature5 m05t 0f the heat 5h0ck 9ene tran5cr1pt5 c0me fr0m pr0m0ter5 that are rec09n12ed 6y the f0rm 0f RNA p01ymera5e (E0 "32) that c0nta1n5 the 32-kD cr-5u6un1t, 0 ~2 (C0w1n9 et a1. 1985; Y. 2h0u, J. Er1ck50n, 7. Yura, and C. 6r055, 1n preparat10n). 0 ~2 15 the pr0duct 0f the rp0H (htpR, h1n) 9ene (6r055man et a1. 1984; Land1ck et a1. 1984; Yura et a1. 1984). Mutat10n5 1n rp0H that reduce the am0unt 0r the act1v1ty 0f 0 ~2 e11m1nate the 1ncrea5ed 5ynthe515 0f heat 5h0ck pr0te1n5 after 5h1ft t0 h19h tem-

6ENE5 • DEVEL0PMEN7 1:419-432 • 1987 6y C01d 5pr1n9 Har60r La60rat0ry 155N 0890-9369/87 $1.00 419

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F19ure 1. 51 nuc1ea5e mapP1n9 0f the 5• end5 0f the rp0H tran5cr1pt5.7he 5chemat1c 0ut11ne5 the 5trate9y f0r mapp1n9 the 5• end5 0f the rp0H tran5cr1pt5.7he t0p p0rt10n 0f the 5chemat1c 15 a re5tr1ct10n map 0f the rp0H pr0m0ter re910n. Pr0m0ter5 P1, P3, and P4 are drawn a5 arr0w5 t0 1nd1cate the d1rect10n 0f tran5cr1pt10n. 7he p051t10n 0f the 5• end 0f mRNA-2 15 marked w1th an arr0whead. 8eneath the map the 5•-end-1a6e1ed [a2P]DNA fra9ment5 (a2p 1nd1cated 6y *) u5ed f0r mapp1n9 are 5h0wn a10n9 w1th the 065erved pattem5 0f pr0tect10n. (5011d 11ne5) Pr0tect10n; (da5hed 11ne5) 51 nuc1ea5e 5en51t1v1ty. 7he 1etter5 A, 8, and C refer t0 the aut0rad10- 9ram5 A, 8, and C 1n wh1ch th05e DNA fra9ment5 were u5ed. (A) A 1040-6p Nc01-5a11 fra9ment 5•-end-1a6e1ed at the Nc01 51te wa5 hy6r1d12ed {at 45°C) w1th 50 1~9 tRNA (1ane 1) 0r 30 ~29 1n v1v0 RNA 1501ated fr0m 5tra1n 5C122 20 m1n after a 5h1ft fr0m 30 t0 42°C (1ane 2). 7he pr0tected fra9ment5 are marked w1th num6ered arr0w5.1n th15 exper1ment mRNA-4 wa5 re501ved 1nt0 tw0 5pec1e5 wh1ch we ca11 4a and 46. DNA 512e marker5 were 5•-end-1a6e1ed 32P-Hpa11 fra9ment5 fr0m p8R322 (1ane 3). (8) An 880-6p C1a1-5a11 fra9ment 5•-end-1a6e1ed at the C1a1 51te wa5 hy6r1d12ed at 30°C t0 RNA 1501ated fr0m 5C 122 8 m1n after 5h1ft fr0m 30 t0 42°C t0 map the 5tart 51te5 0f mRNA-2, mRNA-3, mRNA-4a, and mRNA-46 6y c0mpar150n w1th a chem1ca1 c1eava9e 1adder 0f the 5ame fra9ment. 7he 5ym6015 u5ed f0r the DNA 5e4uence 1adder5 are Y (C + 7), C, R {6 + A), and 6. {C) A 760-6p H1nd111-5a11 fra9ment 5•-end-1a6e1ed at the H1nd111 51te wa5 u5ed (hy6r1d12at10n5 at 30°C) t0 map the 5tart 51te 0f mRNA-1 6y c0mpar150n w1th a chem1ca1 c1eava9e 1adder 0f the 5ame fra9ment. 1n v1tr0 RNA wa5 5ynthe512ed w1th E(r 7° (5ee 1e9end t0 F19. 4A) and 1n v1v0 RNA wa5 1501ated fr0m 5C122 8 m1n after 5h1ft fr0m 30 t0 42°C. DNA 5e4uence 1adder5 are 1a6e1ed a5 1n (8).

perature (Ne1dhardt and Van809e1en 1981; Yamam0r1 and Yura 1982; 706e et a1. 1984; 6r055man et a1. 1985). 7 h e dem0n5trat10n that heat 5h0ck 9ene5 have pr0- m0ter5 rec09n12ed 6y E0 -52 (6r055man et a1. 1984;

C0w1n9 et a1. 1985} 1ed t0 the 1dea that the heat 5h0ck re5p0n5e 15 c0ntr011ed 6y fact0r5 that affect the am0unt 0r the act1v1ty 0f E0 "32 (6r055man et a1. 1984}. 50me ev1- dence 5u99e5t5 that 1ncrea5e5 1n the am0unt 0f 0 -52 may

420 6ENE5 • DEVEL0PMEN7




7ran5cr1pt10n 0f rp0H

acc0unt f0r the 1nduct10n 0f heat 5h0ck mRNA 5yn- the515 (6r055man et a1. 1984; 1987; 6r055 et a1. 1987). C0n515tent w1th th15 1dea, 7111y et a1. (1986)have 5h0wn that rp0H mRNA 1eve15 1ncrea5e w1th k1net1c5 51m11ar t0 heat 5h0ck 9ene mRNA 1eve15.

8ecau5e 0f the centra1 r01e 0f 0 "32 1n the heat 5h0ck re5p0n5e, we exam1ned the tran5cr1pt10n 0f the rp0H 9ene dur1n9 5teady-5tate c0nd1t10n5 and after 5h1ft5 t0 h19her temperature5. We f0und that rp0H 15 the 0n1y 9ene 1n 1t5 tran5cr1pt10n un1t. 7here are at 1ea5t three rp0H pr0m0ter5, tw0 0f wh1ch are rec09n12ed 6y the m05t a6undant f0rm 0f RNA p01ymera5e, Ecr 7°. E0 a2 d0e5 n0t tran5cr16e the rp0H 9ene 1n v1tr0, and mutat10n5 1n rp0H d0 n0t decrea5e tran5cr1pt10n 0f rp0H 1n v1v0, 5h0w1n9 that rp0H 15 n0t a heat 5h0ck 9ene. 7he 1eve1 0f rp0H mRNA 1ncrea5e5 after 5h1ft t0 h19h 9r0wth temper- ature5 6ut the 5ynthe515 0f rp0H mRNA d0e5 n0t 1n- crea5e pr0p0rt10nate1y. We 5u99e5t that th15 ref1ect5 e1- ther a d1rect c0ntr01 0f the 5ta6111ty 0f the rp0H mRNA5 0r 1nd1rect 5ta61112at10n, due t0 1ncrea5ed tran51at10n 0f the rp0H mRNA5 at h19h temperature.

R e 5 u 1 t 5

7he 10cat10n5 0f the 5• end5 0f rp0H mRNA

51 mapp1n9 wa5 u5ed t0 10cate the 5• end5 0f rp0H mRNA. 70ta1 ce11u1ar RNA wa5 1501ated fr0m 5tra1n 5C122 20 m1n after 5h1ft fr0m 30 t0 42°C, and the 5• end5 0f the rp0H mRNA 5pec1e5 were mapped a5 de- 5cr16ed 1n Mater1a15 and meth0d5 and 1n the 1e9end t0 F19ure 1. Pr0tected fra9ment5 c0rre5p0nd1n9 t0 f0ur mRNA 5pec1e5 were 5een: mRNA-1 (5• end near -220), mRNA-2 (5• end near -130), mRNA-3 (5• end near - 90}, and mRNA-4 (5• end near - 80) (F19. 1A). 0n 50me 9e15 mRNA-4 wa5 re501ved 1nt0 tw0 5pec1e5, wh1ch we ca11 mRNA-4a and mRNA-46. When RNA wa5 1501ated fr0m 5tra1n5 MC4100, M61655, CA611042, P90A5c, 0r the1r der1vat1ve5, 51m11ar re5u1t5 were 5een, w1th the d1f- ference that mRNA-2 wa5 pre5ent 0n1y 1n 5tra1n 5C122 (data n0t 5h0wn). 7ran5cr1pt5 1ar9er than mRNA-1 c0u1d have 6een d15t1n9u15hed fr0m fu11-1en9th D N A - D N A hy6r1d5 51nce the re910n 0f the pr06e h0m01090u5 t0 the 6acter1a1 DNA wa5 276 6p 5h0rter than the fu11-1en9th pr06e. 0verexp05ure 0f aut0rad109ram5 50met1me5 re- vea1ed a 6and that c0u1d have ar15en fr0m pr0tect10n 6y tran5cr1pt5 1n1t1at1n9 m0re than 400 6a5e5 up5tream 0f mRNA-1. 1f 5uch up5tream tran5cr1pt5 ex15t they ac- c0unt f0r 1e55 than 1% 0f the rp0H mRNA.

8etter e5t1mate5 0f the 10cat10n5 0f the 5• end5 0f the f0ur rp0H mRNA5 were 06ta1ned 6y c0mpar1n9 the m1- 9rat10n 0f the 51-re515tant fra9ment5 w1th the m19rat10n 0f the pr0duct5 0f DNA 5e4uenc1n9 react10n5 d0ne 0n the 5ame pr06e5 u5ed f0r 51 mapp1n9. 7he 5• end5 0f mRNA-2, mRNA-3, mRNA-4a, and mRNA-46 were de- term1ned t0 6e at p051t10n5 - 132 0r - 133, - 88, - 82 0r - 83, and - 78, 6y c0mpar150n w1th the 5e4uence 1adder 0f the pr06e 1a6e1ed at the C1a1 51te (F19. 18). 7he 5• end 0f mRNA-1 wa5 determ1ned t0 6e at p051t10n -221 6y c0mpar150n w1th the 5e4uence 1adder 0f the pr06e 1a- 6e1ed at the H1nd111 51te (F19. 1C). 7he re5u1t5 0f the5e

exper1ment5 are 5ummar12ed 1n F19ure 1 w1th the p051- t10n5 0f the 5• end5 0f the mRNA5 and the pr0m0ter5 (5ee the f0110w1n9 5ect10n5) 1nd1cated.

7he DNA 5e4uence 1mmed1ate1y up5tream 0f the rp0H 9ene 15 5h0wn 1n F19ure 2 .7he p051t10n5 0f the 5• end5 0f the mRNA5 are 1nd1cated w1th arr0w5.7he 5tart 51te 0f mRNA-1 15 10cated w1th1n what 15 pr06a61y the tran5cr1pt10n term1nat0r 0f the ft5YEX 0per0n (6111 et a1. 1986), wh1ch 15 10cated 1mmed1ate1y up5tream 0f rp0H. 7here are 5e4uence5 that re5em61e Ea 7° pr0m0ter5 up- 5tream 0f the 5• end5 0f mRNA-1, mRNA-3, and mRNA-4 (under11ned 1n F19. 2), 6ut there 15 n0 5e4uence that 100k5 11ke an E0 ~2 pr0m0ter (C0w1n9 et a1. 1985).

7he rp0H tran5cr1pt10n Em1t

8a5ed 0n the DNA 5e4uence 3• t0 rp0H, Land1ck et a1. (1984) and Yura et a1. (1984) pr0p05ed the ex15tence 0f a p-1ndependent term1nat0r 1mmed1ate1y f0110w1n9 the rp0H 9ene. 7he 3• end 0f the rp0H mRNA5 wa5 f0und at the pred1cted 51te (F19. 3, 1ane 3) 6y 51 mapP1n9 1n v1v0 RNA u51n9 a pr06e 3•-end-1a6e1ed at the 855H11 51te 1n rp0H (F195.3 and 9, 6e10w). We never 065erved a 1ar9er 5pec1e5 0f rp0H mRNA, 5u99e5t1n9 that the maj0r1ty 0f tran5cr1pt5 term1nated at th15 51te 1n v1v0. 7ran5cr1pt5 made 1n v1tr0 w1th E¢ 7° end at the 5ame 51te 1nd1cat1n9 that the 5e4uence funct10n5 a5 a p-1ndependent term1n- at0r (F19. 3, 1ane 2).

7he p051t10n5 0f the 5• and 3• end5 0f the rp0H mRNA5 and the DNA 5e4uence 0f the re910n 1nd1cate that rp0H 15 the 0n1y 9ene 1n 1t5 tran5cr1pt10n un1t. L1tt1e 0r n0 tran5cr1pt10n fr0m the ft5YEX 0per0n pr0ceed51nt0 the rp0H 9ene and the rp0H tran5cr1pt5 5t0p 6ef0re reach1n9 the 11vJ 9ene, wh1ch 15 ju5t d0wn5tream 0f rp0H (Land1ck et a1. 1984).

7he 0r191n5 0f the rp0H mRNA 5pec1e5

5evera1 appr0ache5 were u5ed t0 determ1ne wh1ch 0f the rp0H mRNA5 are 1n1t1ated fr0m un14ue pr0m0ter5 and what f0rm5 0f RNA p01ymera5e are re5p0n5161e f0r 1n1t1- at1n9 the tran5cr1pt5.7he ev1dence d15cu55ed 1n the f01- 10w1n9 para9raph5 1nd1cate5 that mRNA-1, mRNA-3, and mRNA-4 ar15e fr0m d15t1nct pr0m0ter5. We d0 n0t kn0w 1f mRNA-2 re5u1t5 fr0m 1n1t1at10n fr0m a un14ue pr0m0ter, P2, 0r fr0m RNA pr0ce551n9 0r de9radat10n.

7he pr0m0ter c10n1n9 p1a5m1d pK0 1 (McKenney et a1. 19811 wa5 u5ed t0 c0nf1rm that the DNA 5e4uence5 up- 5tream 0f the 5• end5 0f the rp0H mRNA5 c0nta1n pr0- m0ter5. DNA fra9ment5 fr0m the re910n pre5umed t0 c0nta1n the rp0H pr0m0ter5 were 1n5erted 1n fr0nt 0f the 9a1K 9ene 0f pK01, and the 9a1act0k1na5e act1v1ty pr0- duced fr0m the re5u1t1n9 p1a5m1d5 wa5 mea5ured. 7he 342-6p Ec0RV fra9ment (5ee F19. 9, 6e10w) wh1ch wa5 th0u9ht t0 c0nta1n a11 the rp0H pr0m0ter5, wa5 1n5erted 1nt0 pK01 1n 60th 0r1entat10n5, creat1n9 pJEK-1 and pJEK-9. 0f the tw0 p1a5m1d5, 0n1y pJEK-1 ha5 pr0m0ter act1v1ty (7a61e 1), dem0n5trat1n9 that the pr0m0ter5 are 0r1ented t0ward rp0H. 7he 342-6p Ec0RV fra9ment wa5 cut w1th H1nd111 and 60th the up5tream 235-6p fra9ment and the d0wn5tream 111-6p fra9ment were 119ated 1nt0 pK01, creat1n9 pJEK-11 and pJEK-5. 80th pJEK-11 and

6ENE5 • DEVEL0PMEN7 421




Er1ck50n et a1.

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F19ure 2. 7he DNA 5e4uence 0f the rp0H pr0m0ter re910n. 7he n0nc0d1n9 5trand 15 5h0wn. 7he 5e4uence c0nta1n5 the 10 term1na1 c0d0n5 0f the ft5X 9ene, the 1ntrac15tr0n1c re910n, and the f1r5t 7 c0d0n5 0f rp0H. 7he f1r5t 6a5e 0f the rp0H 1n1t1at10n c0d0n 15 num6ered 0 .7he pr0te1n pr0duct5 are 1nd1cated 6e10w the DNA 5e4uence. 7he 5tart 51te5 f0r mRNA5 1, 2, 3, 4a, and 46 are marked w1th arr0w5. P055161e Ecr m pr0m0ter -35 and - 10 5e4uence5 10cated up5tream 0f the 5tart5 0f mRNA-1, mRNA-3, and mRNA-4 are under11ned. An 1nverted repeat that 15 pr06a61y the ft5X tran5cr1pt10n term1nat0r (6111 et a1. 1986) 15 under11ned w1th head-t0-head arr0w5. Re5tr1ct10n en2yme c1eava9e 51te5 u5ed f0r c10n1n9 0r 51 mapp1n9 are 1nd1cated. 7he 5e4uence 5h0wn wa5 determ1ned 6y Land1ck et a1. (1984) and Yura et a1. (1984) t0 p051t10n - 192.7he 5e4uence fr0m - 127 t0 -278 wa5 determ1ned 6y 6111 et a1. (1986) and c0nf1rmed 6y u5.

pJEK-5 had pr0m0ter act1v1ty (7a61e 1). 7he5e re5uh5, c0m61ned w1th th05e 0f the 51 mapp1n9 exper1ment5, dem0n5trate the ex15tence 0f at 1ea5t tw0 rp0H pr0- m0ter5; at 1ea5t 0ne re5p0n5161e f0r mRNA-1 and/0r mRNA-2 and at 1ea5t 0ne re5p0n5161e f0r mRNA-3 and/ 0r mRNA-4.

7he rp0H 9ene wa5 tran5cr16ed 1n v1tr0 w1th Ea 7° and the pr0duct5 were 1dent1f1ed u51n9 51 mapp1n9. 1n the pre5ence 0f pure E¢ 7° mRNA-1 and mRNA-4 were 5yn- the512ed (F195. 4A and 1C), mean1n9 that pr0m0ter5 f0r mRNA-1 (P1), and f0r mRNA-4 (P4), ex15t and are rec09- n12ed 6y E~ 7°. 1n 50me exper1ment5 (data n0t 5h0wn) mRNA-4a and mRNA-46 were re501ved, 1nd1cat1n9 that they 60th can 6e 5ynthe512ed 6y E¢ 2°, 6ut we d0 n0t kn0w 1f mRNA-4a and mRNA-46 ar15e fr0m d1fferent pr0m0ter5. 80th the re1at1ve and a6501ute am0unt5 0f mRNA-1 and mRNA-4 c0u1d 6e var1ed 6y chan91n9 the NaC1 c0ncentrat10n 0r 6y u51n9 11near 0r 5uperc011ed DNA, 6ut P1 wa5 a1way5 the 5tr0n9er pr0m0ter (data n0t 5h0wn).

mRNA-3 wa5 n0t 5ynthe512ed 1n v1tr0 w1th e1ther pure E¢ 7° 0r E ~ 2. 70 5ee 1f mRNA-3 0r191nated fr0m a 5pec1f1c pr0m0ter, we a5ked 1f 1t c0u1d 6e 5ynthe512ed 1n v1v0 1n the a65ence 0f any up5tream pr0m0ter5. We u5ed

a p1a5m1d, p7FN97, that carr1e5 the rp0H 9ene preceded 6y P4 and the pre5umed P3 6ut wh1ch 15 m1551n9 the up5tream pr0m0ter(5) P1 and P 2 . 7 0 e11m1nate any tran- 5cr1pt5 fr0m p1a5m1d 5e4uence5 that c0u1d 6e 5u65trate5 f0r RNA pr0ce551n9, a 5tr0n9 tran5cr1pt10n term1nat0r wa5 1n5erted ju5t up5tream 0f P3 (at the H1nd111 51te) 1n p7FN97. P0tent1a1 chr0m050ma1 precur50r5 0f mRNA-3 were e11m1nated 6y u51n9 a 5tra1n, PM107, that ha5 an 15/ e1ement 10cated 6etween the rp0H pr0m0ter5 and the 5tructura1 9ene (R. Ca1endar, per5. c0mm.). We 150- 1ated RNA fr0m PM107 carry1n9 p7FN97, 51 mapped the rp0H tran5cr1pt5, and f0und that mRNA-3 wa5 pre5ent (F19.48). 51nce n0 p1a5m1d 0r chr0m050ma1 tran- 5cr1pt ex15ted that c0u1d 6e c1eaved t0 y1e1d mRNA-3, there mu5t 6e a mRNA-3-5pec1f1c pr0m0ter, P3.

E0 "32 d0e5 n0t rec09n12e the rp0H pr0m0ter5

N0 5pec1f1c rp0H tran5cr1pt5 were made when pure E0 ~2 wa5 u5ed 1n 1n v1tr0 tran5cr1pt10n react10n5 (data n0t 5h0wn). 70 c0nf1rm that E0 ~2 d1d n0t rec09n12e the rp0H pr0m0ter5 1n v1v0 we u5ed a mutan t (CA69301 rp0H120:: kan) that 1ack5 0 ~2 (6r055 et a1. 1987; Y. 2h0u, J. Er1ck50n, 7. Yura, and C. 6r055, 1n prep.). RNA wa5 1501ated fr0m the rp0H120 : : kan mutant , and fr0m

422 6 E N E 5 • D E V E L 0 P M E N 7





2

•14 E E

~11D 911D -,.-~

. c

~ m

A

6 6 7 A A 7 6 C 6 C 7 6 C 6 C 6 C 6

11e 91u a1a end A [ - ~ A77 6 ~ 6C6 7 ~ 777CC6C7A77~6CA6A6A [ T ~ T T T T T G G G C C T C T

1 2 3

P ~ 855H2

1

4 5 ~ 1006p :

F19ure 3. 1dent1f1cat10n 0f the rp0H term1nat0r. 7he 5chemat1c 5h0w5 the 5trate9y f0r mapp1n9 the rp0H term1nat0r. 7he 5ym6015 are a5 1n F19. 1. 7he rp0H term1nat0r 15 repre5ented a5 a 5tem-and-100p 5tructure. 7he pr06e, 3•-end-1a6e1ed at the 855H11 51te, wa5 hy6r1d12ed at 50°C t0 tRNA (1ane 1), RNA made 1n v1tr0 w1th E~ 7° (1ane 2), 20 ~L9 1n v1v0 RNA fr0m 5C122 (1ane5 3 and 5). DNA 512e 5tandard5 (1ane 4). DNA 512e 5tandard5 m1xed w1th a 5amp1e 1dent1ca1 t0 that 1n 1ane 3 (1ane 5). 7he pr06a61e 10cat10n5 0f the 3• end5 are 1nd1cated 6y a 60x ar0und the 5e4uence (Land1ck et a1. 1984; Yura et a1. 1984) wh1ch a150 5h0w5 the pred1cted 5ec0ndary 5tructure f0r the term1nat0r.

1t5 rp0H + parent, and 51 mapped. 7ran5cr1pt5 1n1t1at1n9 f r0m pr0m0ter5 P1, P3, and P4 were 065erved 1n 60th 5tra1n5 (F19. 5), 5h0w1n9 tha t 0 a2 wa5 n0t re4u1red f0r the1r 5ynthe515. C0m61ned w1th the 1na6111ty 0f E ~ 2 t0 5ynthe512e rp0H m R N A 1n v1tr0, the5e 1n v1v0 re5u1t5 1nd1Cate tha t 0 -32 15 n0t nece55ary f0r the 5ynthe515 0f any rp0H m R N A 1n the5e 5tra1n5.

7he re1at1ve am0unt5 0f the rp0H mRNA5 vary w1th temperature

7 h e exper1ment5 0f 7111y et a1. (1986) 5h0wed tha t the am0Unt 0f rp0H m R N A var1e5 w1th the 9r0wth tempera-

ture, 6u t they d1d n0t d15t1n9u15h the 1nd1v1dua1 m R N A 5pec1e5. We u5ed 51 mapp1n9 t0 5ee 1f the am0unt5 0f the rp0H mRNA5 chan9ed at d1fferent temperature5. RNA made at 30°C wa5 c0mpared w1th RNA made after 5h1ft t0 43.5°C, at wh1Ch tempera tu re the hea t 5h0ck re5p0n5e 15 tran51ent, 0r w1th RNA made after 5h1ft t0 the 1etha1 t empera tu re 0f 50°C, at wh1ch tempera ture hea t 5h0ck pr0te1n5 are expre55ed at h19h rate5 f0r a5 10n9 a5 the ce115 m a k e pr0te1n (Ne1dhardt et a1. 1984).

7 h e pred0m1nant rp0H m R N A 5pec1e5 at 30°C were the (~7°-c0ntr011ed tran5cr1pt5 mRNA-1 and mRNA-4, t09ether acc0unt1n9 f0r a60ut 90% 0f the t0ta1 rp0H

7a61e 1. 6a1act0k1na5e act1v1ty fr0m rp0H pr0m0ter-9a1K fu510n p1a5m1d5

6a1act0k1na5e P1a5m1d Pr0m0ter5 act1v1ty at 30°C *

Re1at1ve p1a5m1d c0py num6er at 30°C 6

pK01 - - 5.8 1.0 pJEK-1 P1 ••P2•• P3 P4 220 0.9 pJEK-9 4P 3P ••2P•• 1P c 17 N7 a pJEK-5 P3 P4 145 0.83 pJEK-11 P1 ••P2•• 155 0.85

P1a5m1d pJEK-9 wa5 carr1ed 1n 5tra1n CA61573.7he 0ther p1a5m1d5 were 1n 5tra1n CA6 1831. a 6a1act0k1na5e act1v1ty e4ua15 nm01e5 0f 9a1act05e ph05ph0ry1ated per m1nute per m111111ter 0f ce115 per 0D650 un1t. 6 Re1at1ve 8-1actama5e act1v1ty. c pJEK-9 ha5 the 5ame 1n5ert a5 pJEK-1 6ut 1n the 0pp051te 0r1entat10n. a (N7) N0t te5ted.





Er1ck50n et a1.

mRNA. After 5h1ft t0 43.5°C, the am0unt5 0f a11 the mRNA5 1ncrea5ed (F19. 6A), w1th mRNA-2 and mRNA-3 1ncrea51n9 the m05t. 7he 1ncrea5e 1n the 1eve1 0f mRNA-3 wa5 de1ayed re1at1ve t0 the 1ncrea5e 0f the 0ther rp0H mRNA5. 7h15 de1ay 1mp11e5 that the 1eve1 0f mRNA-3 15 c0ntr011ed 1n a d1fferent way than the 1eve1 0f the 0ther rp0H mRNA5.

7he re1at1ve am0unt5 0f the rp0H m R N A 5pec1e5 at 50°C were d1fferent than at e1ther 43.5°C 0r 30°C (F19. 68). 7he ~F°-c0ntr011ed pr0m0ter5 P1 and P4 appeared t0 5hut 0ff 9radua11y after 5h1ft t0 50°C 6ut mRNA-3 per- 515ted at a h19h 1eve1 f0r at 1ea5t 85 m1n 1data n0t 5h0wn). Near1y 1dent1ca1 re5u1t5 (data n0t 5h0wn) were 06ta1ned when RNA wa5 1501ated fr0m a 5tra1n carrY1n9 a temperature-5en51t1ve mutat10n, rp0D800, that cau5e5 (r 70 t0 6e de9raded rap1d1y after 5h1ft t0 h19h temperature (6r055man et a1. 1983). 1f the c0nt1nued pre5ence 0f

, • , , , 8. 51- = 03

pr06e ~ ~ ~ 1 pr06e ~ 1 1 -- -

,-.9e 1--•m

4 - - ~ ~ e

p7FN97 ~ P3P4

15 1

F19ure 4. 1dent1f1cat10n 0f the rp0H pr0m0ter5. 7he 5trate9y f0r 51 mapP1n9 wa5 a5 1n F19. 1A. {A) 1n v1tr0 tran5cr1pt10n 0f rp0H w1th Ecr 70. Pr06e wa5 hy6r1d12ed t0 20 ~t9 0f 1n v1v0 RNA prepared fr0m 5tra1n 5C 122 3 m1n after 5h1ft fr0m 30 t0 42°C 0r t0 RNA 5ynthe512ed 1n v1tr0 6y Etr 20 fr0m pKP11 1n 0.18 M NaC1, and 51 mapped. (8) mRNA-3 15 5ynthe512ed fr0m pr0- m0ter P3. 7he 5chemat1c d1a9ram5 the p1a5m1d and chr0m0- 50ma1 rp0H temp1ate5 1n 5tra1n PM107/p7FN97.7he th1ck 11ne repre5ent5 5e4uence5 fr0m pDP6442; the tran5cr1pt10n term1n- at0r 15 drawn a5 a 5tem-and-100p. 7he 15/ 1n5ert10n 1n the chr0- m050me 0f PM107 15 5h0wn 6y the hatched 6ar. 7he 15/ 1n5er- t10n wa5 acc0mpan1ed 6y a rearran9ement 0r de1et10n 0f the 5e4uence5 up5tream 0f rp0H. Pr06e wa5 hy6r1d12ed t0 the f01- 10w1n9 RNA5 and 51 mapped: 1n v1v0 RNA 1501ated 8 m1n after 5h1ft fr0m 30 t0 43.5°C fr0m 5tra1n5 5C122, PM107/p7FN97, PM107/pDP6442, and tRNA.


M 6 1 ~

2 eY rr

M

1

1

1

2--*

1

1

1 1 1 -

1 2 3 4 5

F19ure 5. 51 mapP1n9 0f 1n v1v0 rp0H tran5cr1pt5 1n the a6- 5ence 0f 0 a2. 5trate9y wa5 a5 1n F19. 1A. Pr06e wa5 hy6r1d12ed w1th tRNA (1ane 1), 1n v1v0 RNA fr0m 5tra1n5 M61655 rp0H + (1ane 2), and CA69301 rp0H120: : kan (1ane 3) 9r0wn at 16°C, 0r 5C122 (1ane 4) 20 m1n after 5h1ft fr0m 30 t0 43.5°C. DNA 512e 5tandard5 (1ane 5).

mRNA-3 ref1ected 1ncrea5ed tran5cr1pt10n 1n1t1at10n fr0m P3 at a t1me when tran5cr1pt10n fr0m the ~r7°-c0n- tr011ed pr0m0ter5 wa5 dec11n1n9, then th15 5u99e5t5 that a d1fferent f0rm 0f RNA p01ymera5e wa5 re5p0n5161e f0r tran5cr1pt10n fr0m P3.

At 43.5°C the am0unt 0f rp0H m R N A mcrea5e5 w1th0ut a 519n1f1cant 1ncrea5e m rp0H m R N A 5ynthe515

7he 1eve15 0f rp0H m R N A 1ncrea5e w1th k1net1c5 51m11ar t0 th05e 0f the heat 5h0ck 9ene mRNA5 after w11d-type ce115 are 5h1fted t0 h19h temperature (7111y et a1. 1986). 7he am0unt 0f rp0H m R N A 1ncrea5e5 f1ve- t0 e19htf01d after 5h1ft fr0m 30 t0 43.5°C a5 mea5ured u51n9 N0rthern 610t5, 0r 6y 51 mapP1n9 0f the 3• 0r 5• end5 0f the rp0H mRNA5 (7111y et a1. 1986 and F19. 6A). 7he 1ncrea5ed rp0H m R N A c0ncentrat10n c0u1d re5u1t e1ther fr0m 1n- crea5ed tran5cr1pt10n 1n1t1at10n 0r decrea5ed de9radat10n 0f the mRNA. We u5ed tw0 appr0ache5 t0 determ1ne whether there wa5 1ncrea5ed tran5cr1pt10n 1n1t1at10n fr0m the rp0H pr0m0ter5 after 5h1ft t0 h19h temperature.

7he f1r5t appr0ach wa5 t0 mea5ure the act1v1ty 0f the rp0H pr0m0ter5 6ef0re and after 5h1ft t0 h19h temperature u51n9 fu510n5 6etween the rp0H pr0m0ter5 and the 9a1K 0r 1ac2 9ene5. P1a5m1d pJEK-1 (7a61e 1) c0nta1n5





4 3 . 5 °

A. e" 9

5 0 °

p r 0 6 e ~ - - ~ " ~ "•" "••" p 7 0 ~ -11~ 0 aP

2 ; aD a1, 1D "w a

0 m 8 e 0 " 2 "••-)"

4

a 11

1 2 3 4 5 6 1 2 3 4 5

0

D

6

F19ure 6. 51 mapp1n9 0f the rp0H tran5cr1pt5 at 30°C and at t1me5 after 5h1ft t0 43.5 0r 50°C. Mapp1n9 5trate9y wa5 a5 1n F19. 1A. (A) 5h1ft fr0m 30 t0 43.5°c. Pr06e wa5 hy6r1d12ed w1th 30 ~9 0f 1n v1v0 RNA 1501ated fr0m 5tra1n 5C 122 at 30°C (1ane 1) 0r 4 m1n (1ane 2), 8 m1n {1ane 3), 12 m1n (1ane 4), and 20 m1n (1ane 5) after 5h1ft t0 43.5°C. DNA 512e marker5 {1ane 6). (8) 5h1ft fr0m 30 t0 50°c. 7he am0unt 0f 1n v1v0 RNA u5ed 1n each 1ane wa5 the am0unt 0f RNA 06ta1ned fr0m 2.5 m1 0f cu1ture, c0rrected f0r 5amp1e rec0very. Pr06e wa5 hy6r1d12ed t0 1n v1v0 RNA 1501ated fr0m 5C122 at 30°c (1ane 1), and 15 m1n (1ane 2), 30 m1n (1ane 3), 45 m1n (1ane 4), and 60 m1n (1ane 5) after 5h1ft t0 50°C. DNA 512e marker5 (1ane 6).

the rp0H pr0m0ter5 10cated 1n fr0nt 0f the 9a1K 9ene. 7ran5cr1pt10n 1n1t1at1n9 fr0m the rp0H pr0m0ter5 wa5 4uant1f1ed 6y determ1n1n9 the rate5 0f 5ynthe515 0f 9a- 1act0k1na5e at 30°C and after 5h1ft t0 43.5°C. Ce115 c0n- ta1n1n9 pJEK-1 were pu15e-1a6e1ed w1th [3H]1euc1ne and [3H]1y51ne and the pr0te1n5 5eparated 0n tw0-d1men- 510na1 9e15. 7he 5p0t5 c0rre5p0nd1n9 t0 9a1act0k1na5e and the heat 5h0ck pr0te1n 6r0EL were cut fr0m the 9e15 and c0unted (F19. 7A). 7ran5cr1pt10n fr0m the rp0H pr0- m0ter5 1ncrea5ed 5119ht1y, t0 appr0x1mate1y tw1ce the 30°C rate, 6y 20 m1n after 5h1ft t0 43.5°C. 1n c0ntra5t, the 5ynthe515 0f the heat 5h0ck pr0te1n 6r0EL 1ncrea5ed m0re than 12-f01d 6y 10 m1n after 5h1ft and then de- c11ned 6ef0re 9a1act0k1na5e 5ynthe515 1ncrea5ed 519n1f1- cant1y.

70 c0nf1rm that the re5u1t5 06ta1ned w1th the mu1t1- c0py p1a5m1d pJEK-1 m1m1cked the re5p0n5e 0f the rp0H pr0m0ter5 1n 51n91e-c0py, we c0n5tructed an 0per0n fu- 510n 6etween the rp0H pr0m0ter5 and the 1ac2 9ene 0f pha9e kR545, creat1n9 ~,JE2-30. We made ME2-30 1y- 509en5 0f 5tra1n CA611042 and exam1ned the 5ynthe515 0f ~-9a1act051da5e fr0m th15 51n91e-c0py fu510n 6y pu15e- 1a6e11n9 the ce115 w1th [355]meth10n1ne and 1mmun0pre- c1p1tat1n9 f3-9a1act051da5e. 7he re5u1t5 (F19. 78) are 1n 4ua11tat1ve a9reement w1th th05e 06ta1ned w1th the mu1t1c0py p1a5m1d pJEK-1 (F19. 7A). 7here 15 n0 519n1f1- cant 1ncrea5e 1n tran5cr1pt10n 1n1t1at10n fr0m the rp0H pr0m0ter5 after 5h1ft t0 h19h temperature.

7he 5ec0nd appr0ach wa5 t0 pu15e-1a6e1 ce115 w1th pH]ur1d1ne and mea5ure the rate 0f rp0H mRNA 5yn- the515. 7here wa5 n0t en0u9h rp0H mRNA 5ynthe515 1n w11d-type ce115 t0 mea5ure accurate1y, 50 the am0unt 0f rp0H mRNA wa5 1ncrea5ed appr0x1mate1y 14 t1me5 6y 1ntr0duc1n9 the mu1t1c0py rp0H + p1a5m1d pKP11. 51 mapp1n9 wa5 u5ed t0 5h0w that the re1at1ve ma9n1tude and the k1net1c5 0f the 1ncrea5e 1n the 1eve1 0f rp0H mRNA after heat 5h0ck wa5 51m11ar 1n der1vat1ve5 0f CA611042 w1th pKP11 0r w1th the c0ntr01 p1a5m1d p8R322.7he ma9n1tude 0f the heat 5h0ck re5p0n5e wa5 51m11ar 1n the tw0 5tra1n5 6ut the h19he5t 1eve1 0f heat 5h0ck 9ene (9r0E 0r dnaK) mRNA wa5 reached 50me- what ear11er 1n the 5tra1n w1th pKP11 (data n0t 5h0wn).

51nce the re1at1ve 1ncrea5e 1n rp0H mRNA 1eve1 wa5 n0rma1 1n the der1vat1ve 0f C A 6 11042 carrY1n9 pKP 11, the 5tra1n wa5 u5ed t0 4uant1tate the 5ynthe515 0f rp0H mRNA and 0f heat 5h0ck 9ene (dnaK) mRNA 6ef0re and after tran5fer t0 43.5°C. RNA 5ynthe515 wa5 mea5ured 6y hy6r1d121n9 3H-pu15e-1a6e1ed RNA t0 rp0H 0r t0 dnaK DNA 1mm061112ed 0n n1tr0ce11u105e f11ter5 and mea- 5ur1n9 the 3H c0unt5 hy6r1d12ed, mRNA 1eve15 were 4uant1tated 6y 51 mapp1n9 n0nrad10act1ve RNA 5amp1e5 fr0m the 5ame cu1ture5 that were u5ed f0r pu15e-1a- 6e11n9. 7he data are dep1cted 1n F19ure 8 .7he am0unt 0f rp0H mRNA 1ncrea5ed f1vef01d, a5 5een prev10u51y (7111y et a1. 1986 and F19. 6A), w1th a d15t1nct peak at 8 m1n after 5h1ft. 1n c0ntra5t, there wa5 a 9radua1 and much





Er1ck5tm et a1.

F19ure 7. rp0H pr0m0ter act1v1ty after 5h1ft fr0m 30°C t0 43.5°C. (A) 5ynthe515 0f 9a1act0k1na5e fr0m the rp0H pr0- m0ter5 0n p1a5m1d pJEK- 1 1n 5tra1n CA6 1831.7he re1at1ve rate5 0f 9a1act0k1na5e (0) 0r 6r0EL (•) 5ynthe515 are p10tted ver5u5 t1me after temperature 5h1ft. Rate5 0f 5yn- the515 were determ1ned 6y pu15e-1a6e11n9 and tw0-d1men- 510na1 9e1 ana1y515 a5 de5cr16ed 1n Mater1a15 and meth0d5. (8) 5ynthe515 0f 8-9a1act051da5e fr0m a 51n91e-c0py rp0H pr0m0ter-1ac2 fu510n. A >,JE2-30 1y509en 0f CA611042 wa5 pu15e-1a6e1ed w1th [555]meth10n1ne at 30°C (1ane5 1 and 2) and at 3 m1n (1ane 2), 6 m1n (1ane 3), 9 m1n (1ane 4), and 20 m1n (1ane 5) after 5h1ft t0 43.5°C. 5amp1e5 0f e4ua1 555 cpm were 1mmun0prec1p1tated w1th crude ant1-8-9a- 1act051da5e ant15erum and detected 6y aut0rad109raphy. [3- 6a1act051da5e 15 1nd1cated 6y the arr0w.

A.

7

6

~0 4

3

,

3 0 ° 43.,5 °

6" 1 113" 9"

0 4 8 12 16 20 1 2 3 4 5 6

71me (m1n) 0t 43.5 °

5ma11er 1ncrea5e 1n rp0H m R N A 5ynthe515.80th the 5yn- the515 and the 1eve1 0f dnaK m R N A 1ncrea5ed and de- crea5ed a5 expected 0f a heat 5h0ck 9ene mRNA.

7he re5u1t5 0f the RNA pu15e-1a6e11n9 exper1ment5 and the pr0m0ter fu510n exper1ment5 are c0n515tent. 7here 15 a 5ma11 1ncrea5e 1n the 5ynthe515 0f rp0H m R N A after 5h1ft t0 43.5°C, 6ut 1t 15 n0t en0u9h t0 ac- c0unt f0r the f1vef01d 1ncrea5e 1n the rp0H m R N A c0n- centrat10n.

D15cu5510n

5tructure 0f the rp0H tran5cr1pt10n un1t

7 h e rp0H 9ene 15 10cated 6etween the 11v] 9ene (Land1ck et a1. 1984) and the ft5YEX 0per0n at 76 m1n 0n the E. c011 chr0m050me (6111 et a1. 1986). 7 h e 5tructura1 fea- ture5 0f the rp0H tran5cr1pt10n un1t are d1a9rammed 1n F19ure 9. 1n m05t 0f the 5tra1n5 we exam1ned there are three m0n0c15tr0n1c rp0H m R N A 5pec1e5, mRNA-1, mRNA-3, and mRNA-4. Each 0f the mRNA5 0r191nate5 fr0m a pr0m0ter 10cated 1mmed1ate1y up5tream 0f the 5• end 0f the mRNA. 7he pr0m0ter5 P1 (f0r mRNA-1) and P4 (f0r mRNA-4) are rec09n12ed 6y Err 7°. We d1d n0t de- term1ne what f0rm 0f RNA p01ymera5e rec09n12e5 P3, the pr0m0ter f0r mRNA-3. 1n 5tra1n 5C122 there 15 a f0urth m R N A (mRNA-2), 6ut we have n0t e5ta6115hed whether mRNA-2 re5u1t5 fr0m pr0ce551n9 0r a 5pec1f1c 1n1t1at10n. 1f any 0ther rp0H mRNA5 ex15t they mu5t ac- c0unt f0r 1e55 than 1 -2% 0f rp0H tran5cr1pt10n under the c0nd1t10n5 (r1ch med1a) we u5ed. 7he rp0H tran-

5cr1pt5 5t0p at a p-1ndependent term1nat0r 10cated 2 0 - 4 0 6p d0wn5tream 0f the rp0H c0d1n9 5e4uence. 7ran- 5cr1pt5 1n1t1ated fr0m P1 and fr0m P3 and P4 and end1n9 at the rp0H term1nat0r 5h0u1d 6e -1.1 and -1 .0 k6 1n 1en9th. 7 h e c0nc1u510n that the rp0H tran5cr1pt5 are m0n0c15tr0n1c 15 c0n515tent w1th the re5u1t5 06ta1ned fr0m N0rthern 610t5 6y 7111y et a1. (1986)wh0 e5t1mated the 1en9th5 0f the rp0H tran5cr1pt5 t0 6e -1 .4 and -1 .2 k6. A1th0u9h the1r 512e e5t1mate5 are 1ar9er than th05e made here, the e5t1mate 0f 1.4 k6 15 t00 5ma11 t0 1nc1ude rp0H and e1ther the up5tream ft5YEX 0per0n 0r the d0wn5tream 11vJ 9ene. 7he d1fferent 512e e5t1mate5 are pr06a61y due t0 d1ff1cu1ty 1n e5t1mat1n9 the 1en9th 0f the RNA5 u51n9 N0rthern 610t5.

7he c105e 9enet1c 11nka9e 6etween rp0H and the ft5YEX 9ene5, wh1ch are 1nv01ved 1n ce11 d1v1510n, 15 w0rth n0t1n9 91ven the rep0rt5 that rp0H (75uch1d0 et a1. 1986) and at 1ea5t tw0 0f the heat 5h0ck 9ene5, dnaK (Paek and Wa1ker 1987) and 10n (Ad1er and Har1d9ree 1964; H0ward-F1ander5 et a1. 1964), are 1nv01ved 1n ce11 d1v1510n. De5p1te the rep0rted 11nk 6etween rp0H and ce11 d1v1510n, there 15 n0 ev1dence that the ft5YEX 0per0n and rp0H are c0tran5cr16ed. 1t 15 p055161e that expre5510n 0f the ft5YEX 0per0n 1nf1uence5 the expre5510n 0f rp0H 51nce the 3• end 0f the ft5X 9ene and the maj0r rp0H pr0m0ter (P1) 0ver1ap; h0wever, ev1dence 5u99e5t5 that there 15 n0 c15-act1n9 effect 0f ft5X tran5cr1pt10n 0n tran- 5cr1pt10n 0f rp0H under n0rma1 9r0wth c0nd1t10n5 0r dur1n9 heat 5h0ck. 7ran5cr1pt10n 0f rp0H ha5 6een 5epa- rated fr0m tran5cr1pt10n 0f ft5X u51n9 the rp0H + p1a5m1d pKP11, wh1ch 1ack5 the ft5YEX pr0m0ter5 and a11 6ut the car60xy-term1na1 th1rd 0f ft5X. 80th the re1at1ve





7ran5cr1pt10n 0[ rp0H

10

7

u~ r~

6

e-•

.•> 5 4 n-

rp0H

, ~ 7

6

6~ .~>

3 F / 3

, 0 • • 0 _ _ 0 _ • ~ - 0 . . . . . . .

1 - 1

t L

t t

1 1 t t

1 1 1

,. "•9. . -.

d n a K

0 ~ 0 ~ 0 4 8 12 16 20 0 4 8 12 16 20

71me (m1n) 0t 45.5 ° "r1me (m1n) 0f 45.5 °

F 1 9 u r e 8. 5ynthe515 and accumu1at10n 0f rp0H and dnaK mRNA. 7he re1at1ve 1ncrea5e5 1n 5ynthe515 (d0tted 11ne5) and 1eve1 (5011d 11ne5) 0frp0H mRNA (0, •) and dnaK mRNA (n E3) are p10tted a9a1n5t t1me after 5h1ft fr0m 30°C t0 43.5°C. RNA wa5 1501ated fr0m a cu1ture 0f CA611042/pKP11 9r0wn at 30°C and at t1me5 after 5h1ft t0 43.5°C. 7he mRNA 1eve15 were mea5ured 6y 4uant1tat1ve 51 mapp1n9 0f the 3• end 0f rp0H mRNA (7111y et a1. 1986 and F19. 3) and the 5• end5 0f dnaK mRNA (C0w1n9 et a1. 1985) a5 de5cr16ed 1n Mater1a15 and meth0d5.7he rate5 0f mRNA 5ynthe515 were determ1ned 6y pu15e-1a6e11n9 w1th [~H}ur1d1ne and hy6r1d121n9 [3H]RNA t0 f11ter-60und rp0H 0r dnaK DNA a5 de5cr16ed 1n Mater1a15 and meth0d5, rp0H- 0r dnaK-5pec1f1c RNA wa5 determ1ned 6y 5u6tract1n9 the 3H cpm 60und t0 n0n5pec1f1c DNA f11ter5 fr0m the 3H cpm 60und t0 the rp0H 0r dnaK f11ter5.

am0unt5 0f mRNA- 1, mRNA-2, and mRNA-4 (mRNA-3 15 underrepre5ented fr0m a11 mu1t1c0py p1a5m1d5 te5ted) and the re5p0n5e 0f the rp0H tran5cr1pt5 t0 temperature 5h1ft5 appear 1dent1ca1 whether they c0me fr0m pKP11 0r the chr0m050me (data n0t 5h0wn). 7h15 5u99e5t5 that tran5cr1pt10n 0f ft5X d0e5 n0t d1rect1y 1nf1uence tran- 5cr1pt10n 0f rp0H.

E03 2 d0e5 n0t tran5cr16e the rp0H 9ene

RNA p01ymera5e c0nta1n1n9 032 d0e5 n0t rec09n12e the rp0H pr0m0ter5 1n v1tr0, n0r d0e5 1t 1n v1v0, 51nce the 5• end5 0f 1n v1v0 rp0H m R N A are the 5ame whether 0r n0t funct10na1 032 15 pre5ent. 7he5e re5u1t5 mean that 032 15 n0t a heat 5h0ck pr0te1n, 6ecau5e 6y def1n1t10n {Ne1d- h a r d t e t a1. 1984) heat 5h0ck 9ene5 are tran5cr16ed 6y E03 2. 7he 5ame c0nc1u510n wa5 reached 6y 8100m et a1.

(1986) wh0 were una61e t0 dem0n5trate 5ynthe515 0f 0 -52 1n an 1n v1tr0 tran5cr1pt10n/tran51at10n 5y5tem c0n- ta1n1n9 E032; h0wever, the1r c0nc1u510n wa5 weakened 6y the fact that the rp0H temp1ate they u5ed, pFN97, d1d n0t c0nta1n a11 the rp0H pr0m0ter5. 50me ev1dence 5u9- 9e5t5 that the 1eve1 0f rp0H m R N A 15 h19her after 5h1ft t0 h19h temperature 1n rp0H mutant5 than 1n w11d-type ce115 (unpu6115hed data and F19. 3 0f 7111y et a1. 1986). 7h15 may mean that there 15 50me f0rm 0f aut0re9u1a- t10n 0f 0 "a2 5ynthe515, 6ut we fav0r the 1dea that the 1n- crea5ed 1eve1 0f rp0H m R N A 1n rp0H mutant5 ref1ect5 the ex15tence 0f a feed6ack mechan15m where6y the 5yn- the515 0f 0 -32 15 1ncrea5ed 1n re5p0n5e t0 a def1c1ency 1n heat 5h0ck pr0te1n 5ynthe515. 5uch a mechan15m may 1nv01ve tran51at10na1 derepre5510n 0f the rp0H mRNA5, a5 d15cu55ed 1ater.





Er1ck50n et a1.

,2006p. 1 •

E H E,C N 8 HC

P 1~

E I "~C 5X • ~ ~ ~ 11 1/.p0H

/ P1 2 P3 P4

~ 1006p 1 4

F19ure 9. D1a9ram 0f the rp0H tran5cr1pt10n un1t. (70p) 7he rp0H 9ene 15 5h0wn 6etween the ft5X 9ene (6111 et a1. 1986) and the 11v1 9ene (Land1ck et a1. 1984). Each 9ene 15 tran5cr16ed 1eft t0 r19ht a5 drawn 1n the f19ure. Pr0m0ter5 are 1nd1cated 6y a cap1ta1 1etter P and term1nat0r5 6y a 5ma11 1etter t. Re5tr1ct10n en2yme c1eava9e 51te5 u5ed f0r c10n1n9 0r 51 mapp1n9 are 5h0wn. 5ym6015 are: (E) Ec0RV; (H)H1nd111; (C) C1a1; (N) Nc01; (8) 855H11; (Hc) H1nc11. (80tt0m) Expanded v1ew 0f the ft5X-rp0H 1ntrac15tr0n1c re910n. 7he 5tart 51te5 0f the rp0H mRNA5 are 1nd1cated a5 arr0whead5. 8e10w the arr0whead5 the pr0m0ter re5p0n5161e f0r each tran5cr1pt (1f kn0wn) 15 1nd1cated. 7he wavy 11ne5 repre5ent the rp0H mRNA 5pec1e5.7he pre5umed tran5cr1pt10n term1nat0r 0f the ft5YEX 0per0n (6111 et a1. 1986) 0ver1ap5 the 5tart 51te 0f mRNA-1.

7he 1ncrea5e 1n rp0H mRNA c0ncentrat10n 15 ach1eved 6y p05t-tran51at10na1 c0ntr01

7111y et a1. (1986) rep0rted that the am0unt 0f rp0H m R N A 1ncrea5e5 after 5h1ft t0 h19h temperature and that th15 1ncrea5e 15 d1fferent fr0m the 1nduct10n 0f the heat 5h0ck 9ene mRNA5 6ecau5e 1t 0ccur5 1n rp0H mutant5. 7 h e 1ncrea5ed rp0H m R N A c0ncentrat10n c0u1d have re- 5u1ted fr0m 1ncrea5ed 5ynthe515 0r decrea5ed de9radat10n 0f rp0H mRNA. 7w0 mechan15m5 c0u1d 1ead t0 1n- crea5ed 5ynthe515 0f rp0H mRNA; 1ncrea5ed 1n1t1at10n 0f rp0H tran5cr1pt5, 0r an ant1term1nat10n c0ntr01 that w0u1d a110w c0mp1et10n 0f m0re rp0H m R N A after 5h1ft t0 h19h temperature. We u5ed three techn14ue5 t0 5h0w that the 1ncrea5e 1n rp0H m R N A 1eve15 cann0t 6e ex- p1a1ned 6y 1ncrea5ed 5ynthe515. (1) 51 mapp1n9 wa5 u5ed t0 5h0w that the 5• and 3• end5 0f the rp0H mRNA5 1n- crea5e t0 the 5ame extent; th15 ru1e5 0ut any type 0f an- t1term1nat10n c0ntr01 0f rp0H m R N A 5ynthe515. (2) Fu- 510n5 0f the rp0H pr0m0ter5 t0 e1ther the 9a1K 0r 1ac2 9ene5 were u5ed t0 dem0n5trate that the pr0m0ter5 are n0t heat 1nduc161e. (3) 7he 5ynthe515 0f rp0H mRNA wa5 mea5ured 6y pu15e-1a6e11n9 w1th [3H]ur1d1ne t0 5h0w that rp0H m R N A 5ynthe515 1ncrea5e5 0n1y 5119ht1y 1n the f1r5t few m1nute5 after 5h1ft t0 h19h 9r0wth tempera- ture5. 1t 5eem5 11ke1y that chan9e5 1n the 5ta6111ty 0f the mRNA5 acc0unt5 f0r the 1ncrea5ed 1eve1 0f rp0H m R N A 065erved 1mmed1ate1y after 5h1ft t0 43.5°C 51nce rp0H m R N A 5ynthe515 15 n0t 519n1f1cant1y 1ncrea5ed dur1n9 heat 5h0ck. 1t 15 p055161e t0 1ma91ne c0nd1t10n5 where6y a 5ma11 1ncrea5e 1n the 5ynthe515 0f rp0H m R N A c0u1d 1ead t0 a 1ar9er 1ncrea5e 1n the rp0H m R N A 1eve1. F0r examp1e, 1f rp0H m R N A were de9raded at 0r near the max1mum rate at 30°C, a 5ma11 1ncrea5e 1n 5ynthe515 0f rp0H m R N A c0u1d 5aturate the de9radat10n 5y5tem thu5

decrea51n9 the rate 0f de9radat10n and 9reat1y e1evat1n9 m R N A 1eve15.7he fact that the am0unt 0f rp0H m R N A can 6e 1ncrea5ed m0re than 10 t1me5, 6y the 1ntr0duc- t10n 0f a mu1t1c0py rp0H + p1a5m1d, w1th0ut chan91n9 the re1at1ve 1ncrea5e 1n rp0H m R N A 1eve15 6ef0re and after temperature 5h1ft ar9ue5 a9a1n5t 5uch a m0de1.

1mp11cat10n5 f0r the re9u1at10n 0f the heat 5h0ck re5p0n5e

Recent exper1ment5 have dem0n5trated that the c0ncen- trat10n 0f ~2 tran51ent1y 1ncrea5e5 after 5h1ft t0 h19h temperature (6r055 et a1. 1987; Le51ey et a1. 1987; D. 5trau5, W. Wa1ter, and C. 6r055, 1n prep.). 0 u r re5u1t5 5u99e5t that the 1ncrea5e 1n rp0H m R N A 1eve1 (and pre- 5uma61y 032 5ynthe515) 0ccur5 6y a p05t-tran5cr1pt10na1 mechan15m. We pr0p05e that the 1nduct10n 0f 032 5yn- the515 0ccur5 6y p05t-tran5cr1pt10na1 derepre5510n and that the 5hut-0ff 0f 032 5ynthe515 15 the rever5e: p05t- tran5cr1pt10na1 repre5510n. 7h15 pr0p05a1 15 c0n515tent w1th the exper1ment5 0f 6r055man et a1. (1987) that 1m- p11cate p05t-tran5cr1pt10na1 c0ntr01 1n the 5hut-0ff 0f 032 5ynthe515. Repre5510n 0r derepre5510n 0f 0 32 5ynthe515 c0u1d 0ccur e1ther thr0u9h d1rect c0ntr01 0f m R N A 5ta- 6111ty 0r 6y re9u1at10n 0f tran51at10n.

5pec1f1c c0ntr01 0f m R N A 5ta6111ty 1n re5p0n5e t0 en- v1r0nmenta1 c0nd1t10n5 15 unu5ua1 1n enter1c 6acter1a. 7he c0ntr01 0f the 5ta6111ty 0f K1e651e11a pneum0n1ae n1f m R N A 1n re5p0n5e t0 0xY9en, amm0n1a, 0r temperature (C0111n5 et a1. 1986) and the 9r0wth rate dependence 0f E. c011 0mpA m R N A ha1f-11fe (N11550n et a1. 1984) are tw0 examp1e5.1f the rp0H m R N A c0ncentrat10n 15 ra15ed 6y 1ncrea51n9 the 5ta6111ty 0f the mRNA5 at h19h temperature, 1t c0u1d 6e the re5u1t 0f a 5pec1f1c fact0r that 5ta61- 112e5 the mRNA5, 0r 1t c0u1d re5u1t fr0m 1ncrea5ed

428 6 E N E 5 • D E V E L 0 P M E N 7





tran51at10n 0f the rp0H mRNA5 1ead1n9 t0 1ncrea5ed 5ta- 6111ty, a5 1n the ca5e 0f r-pr0te1n mRNA5 (51n9er and N0- mura 1985; C01e and N0mura 1986). We fav0r the 5ec0nd p05516111ty, that 1ncrea5ed ¢32 5ynthe515 1ead5 t0 h19her 1eve15 0f rp0H mRNA, 6ecau5e the 5ynthe515 0f 022 peak5 6ef0re the h19he5t 1eve15 0f rp0H m R N A are reached (6r055 et a1. 1987; D. 5trau5, W. Wa1ter, and C. 6r055, 1n prep.).

15 rp0H P3 rec09n12ed 6y a n e w f0rm 0f R N A p01ymera5e•

0 f the f0ur rp0H tran5cr1pt5 mRNA-3 5tand5 0ut 6e- cau5e 0f 1t5 un14ue re5p0n5e t0 temperature. At 30°C mRNA-3 acc0unt5 f0r 1e55 than 3% 0f the rp0H tran- 5cr1pt5. After 5h1ft t0 42 0r 43.5°C the am0unt 0f mRNA-3 1ncrea5e5 m0re than 10-f01d (eventua11y am0unt1n9 t0 a60ut 15% 0f the t0ta1 rp0H mRNA), a1- th0u9h the 1ncrea5e 15 de1ayed re1at1ve t0 that 0f the 0ther rp0H mRNA5 and the heat 5h0ck 9ene mRNA5. 7 h e de1ay may mean that act1vat10n 0f P3 re4u1re5 a fact0r pr0duced 1n re5p0n5e t0 h19h temperature d1f- ferent fr0m the fact0r that 1nduce5 the heat 5h0ck re- 5p0n5e. At 50°C mRNA-3 acc0unt5 f0r near1y a11 0f the rp0H mRNA.

1t 15 p055161e that P3 15 rec09n12ed 6y an unde5cr16ed f0rm 0f RNA p01ymera5e. Ne1ther Ecr 7° n0r E¢ 32 tran- 5cr16e fr0m P3 1n v1tr0; h0wever, pre11m1nary exper1- ment5 u51n9 extract5 prepared fr0m ce115 5h1fted t0 50°C f0r 30 m1n 5u99e5t that mRNA-3 can 6e 5ynthe512ed 1n v1tr0 {data n0t 5h0wn). 8ecau5e tran5cr1pt5 were n0t 1n1- t1ated fr0m the cF°-c0ntr011ed pr0m0ter5 P1 and P4 1n the5e extract5, we 5u5pect that a f0rm 0f RNA p01y- mera5e 0ther than Ecr 7° 15 1nv01ved 1n the 5ynthe515 0f mRNA-3. 7he f1nd1n9 that mRNA-3 accumu1ate5 1n v1v0 under c0nd1t10n5 where the am0unt 0f cr 7° 5h0u1d have 6een very 10w (the rp0D800 mutan t at 50°C) 5up- p0rt5 th15 1dea. 1t 15 un11ke1y that E0 "a2 15 the 0ther f0rm 0f RNA p01ymera5e 51nce mRNA-3 15 made 1n ce115 1ack1n9 0 ~2 {F19. 5).

7he exc1u51ve pre5ence 0f mRNA-3 at 50°C may re- f1ect the ex15tence 0f an emer9ency 5y5tem t0 pr010n9 5urv1va1 at very h19h temperature. 1t 15 kn0wn that 1n- duct10n 0f the heat 5h0ck pr0te1n5 pr0v1de5 pr0tect10n fr0m the 1etha1 effect5 0f h19h temperature5 (Yamam0r1 and Yura 1982). 1f the a6111ty t0 5ynthe512e heat 5h0ck

pr0te1n5 15 nece55ary f0r 5urv1va1, 1t can 6e 1nferred that c0nt1nua1 5ynthe515 0f 0 "32 w0u1d a150 6e re4u1red 51nce 0 ~2 15 un5ta61e at 50°C (A. 6r055man and W. Wa1ter, un- pu61.). 51nce tran5cr1pt10n fr0m the cr7°-c0ntr011ed pr0- m0ter5 P1 and P4 appear5 t0 6e 5hut 0ff at 50°C (F19. 68), E. c011 may have ev01ved a mechan15m t0 en5ure c0n- t1nued tran5cr1pt10n 0f rp0H fr0m a heat-re515tant pr0- m0ter, P3, rec09n12ed 6y an0ther f0rm 0f RNA p01y- mera5e. 7h15 w0u1d a110w c0nt1nued expre5510n 0f 0 -32 and m19ht 1ncrea5e the chance 0f a ce11 5urv1v1n9 unt11 the temperature wa5 decrea5ed. An attract1ve feature 0f th15 1dea 15 that 1t pr0v1de5 a te5ta61e exp1anat10n 0f the near1y exc1u51ve 5ynthe515 0f heat 5h0ck pr0te1n5 at 50°C. 1f cr 7° 15 1nact1vated at 50°C 6ut 0ther ~ fact0r5 1n- c1ud1n9 0 ~2 are n0t, the 0n1y pr0te1n5 made w111 6e th05e wh05e 9ene5 are rec09n12ed 6y f0rm5 0f RNA p01y- mera5e 0ther than Ecr 2°. 7he 1dent1f1cat10n 0f pr0te1n5 5ynthe512ed at 50°C may 1ead t0 the 1dent1f1cat10n 0f 9ene5 that are rec09n12ed 6y a1ternate f0rm5 0f RNA p01ymera5e.

Mater1a15 and meth0d5

8acter1a, pha9e, and med1a

7he 6acter1a1 5tra1n5 are 115ted 1n 7a61e 2. Pha9e hc160dnaK(Am)dnaJ + wa5 06ta1ned fr0m C. 6e0r90p0u105 (Un1ver51ty 0f Utah). hrp0H + (Y. 2h0u, J. Er1ck50n, 7. Yura, and C. 6r055, 1n prep.) 15 a der1vat1ve 0f Char0n 25 that c0nta1n5 the 6acter1a1 DNA fr0m pKP11, hR545 (511havy and 8eckw1th 1985) wa5 06ta1ned fr0m R. 51m0n5 (Un1ver51ty 0f Ca11f0rn1a, L05 An- 9e1e5}. hc1857 5am7 DNA wa5 purcha5ed fr0m Pr0me9a-810- tech, (Mad150n, W15c0n51n}. M9 (M111er 1972) 0r M0P5 (Ne1d- hardtet a1. 1974) med1a were 5upp1emented w1th 91uc05e (0.2% wt/v01J, v1tam1n5, and 20 am1n0 ac1d5, except 1n pu15e-1a6e11n9 exper1ment5 where the am1n0 ac1d 0r nuc1e051de u5ed f0r 1a- 6e11n9 wa5 0m1tted. When Amp r p1a5m1d5 were pre5ent, amp1- c1111n (100 ~9/m1) wa5 1nc1uded 1n the cu1ture med1um.

Rea9ent5

7he K1en0w fra9ment 0f DNA p01ymera5e 1, 74 DNA 119a5e, and 74 p01ynuc1e0t1de k1na5e were purcha5ed fr0m New En- 91and 810La65 (8ever1y, Ma55achu5ett5). Re5tr1ct10n end0nu- c1ea5e5 were fr0m New En91and 810La65 0r Pr0me9a-810tech. Ca1f 1nte5t1ne a1ka11ne ph05phata5e, 51 nuc1ea5e, RNa5e A, and RNa5e 71 were fr0m 80ehr1n9er-Mannhe1m 810chem1ca15 {1n- d1anap0115, 1nd1ana). Ant15erum a9a1n5t f1-9a1act051da5e wa5

7a61e 2. 8acter1a1 5tra1n5

De519nat10n 6en0type 50urce

5C122

C A 6 1 8 3 1

C A 6 1 5 7 3

PM107 M61655 CA69301 CA6 11042 MC1061 MC4100

1ac2(Am) trp{Am) ph0(Am) ma1(Arn) 5upC(75) rp5L re1 5C122 9a1K2 nadA50 : : 7n10 th1-1 thr-1 1eu-6 1acY1 t0nA21 5upE44 9a1K2 recA56 rp0H107p : : 15/ rp0D285 1ac24 ar9675 th1 pr0t0tr0ph1c E. c011 K12 M61655 rp0H120 : : kan 2h921 : : 7n10 F• 1ac1 ° 1ac2 : : 7n5 pr0A + 8+ /A{9pt-1ac15 trp(Am} th1 ara 9a1K2 5upC(75) rp5L A1acX74 9a1K 9a1U A(ara-1e0}7697 araD139 h5dR rp5L A(ar9F-1ac)205 araD139 rp5L150 th1A1 re1A1 f165301 de0C1 pt5F25 r65R

C00per and Ruett1n9er (1975) 1a6 c011ect10n 1a6 c011ect10n 6r055man et a1. (1985) C 6 5 C a 1a6 c011ect10n fr0m C5H 26, M111er (1972) M. Ca5ada6an M. Ca5ada6an

a C65C E. c011 6enet1c 5t0ck Center, Ya1e Un1ver51ty.





Er1ck50n et a1.

pr0v1ded 6y 80r15 R0tman (8r0wn Un1ver51ty). N1tr0ce11u105e f11ter5 were fr0m 5ch1e1cher • 5chue11, 1nc. (Keene, New Hamp5h1re) and yea5t tRNA wa5 fr0m 519ma Chem1ca1 C0. (5t. L0u15, M1550ur1). N1tr0cef1n wa5 fr0m 88L M1cr0610109y 5y5tem5 (C0ckey5v111e, Mary1and). L-[355]Meth10n1ne {>800 C1/ mm01e), c-[4,5-5H]1euc1ne (52 C1/mm01e), L[4,5-5H]1y51ne (46 C1/mm01e), [5,6-5H]ur1d1ne (50 C1/mm01e), [~-52p]A7P (3000 C1/mm01e), [~-52P]dC7P (3000 C1/mm01e), and [a-52p]d67P (3000 C1/mm01e) were fr0m Amer5ham C0rp. (Ar11n9t0n He19ht5, 1111n015). D-[1A4C]6a1act05e (40-50 mC1/mm01e)wa5 purcha5ed fr0m Re5earch Pr0duct5 1nternat10na1 C0rp. (M0unt Pr05pect, 1111n015).

P1a5m1d5

7he Amp r, rp0H + p1a5m1d5 pFN97 and pKP11 have 6een de- 5cr16ed (Ne1dhardt et a1. 1983; Paek and Wa1ker 1986). pKP11 c0nta1n5 620 6p 0f E. c011 DNA up5tream 0f rp0H, the rp0H, 11vJ, and 11vL 9ene5, and part 0f the 11vK 9ene 1n5erted 1nt0 8amH1 51te 0f p8R322 (8011var et a1. 1977). 5tandard techn14ue5 (Man1at15 et a1. 1982) were u5ed f0r re5tr1ct10n en2yme d19e5- t10n5, 5ma11-5ca1e 1501at10n 0f p1a5m1d DNA, a9ar05e 9e1 e1ec- tr0ph0re515, DNA 119at10n5, and tran5f0rmat10n5. When nece5- 5ary, 0verhan91n9 5• end5 were f111ed 1n w1th dN7P5 u51n9 the K1en0w fra9ment 0f DNA p01ymera5e 1. 7he 5tructure5 0f a11 p1a5m1d5 were checked 6y re5tr1ct10n mapp1n9, p7FN97 wa5 created 6y 1n5ert1n9 a 61unt-ended H1nd111 t0 H1nc11 fra9ment c0nta1n1n9 the rp0H 9ene fr0m pFN97 1nt0 the Ec0R1 51te 0f pDP6442 (06ta1ned fr0m D. P01aye5 and J. Dah16er9, Un1ver51ty 0f W15c0n51n). pDP6442 c0nta1n5 the tran5cr1pt10n term1nat0r fr0m the 5pf 9ene up5tream 0f the Ec0R1 51te. 7he 5pf term1n- at0r ha5 6een 5h0wn t0 funct10n w1th 9reater than 99% eff1- c1ency 1n v1tr0 (J0yce and 6r1nd1ey 1982). 7w0 pr0m0ter c10n1n9 vect0r5 were u5ed, pK01 {McKenney et a1. 1981), car- ty1n9 the 9a1K 9ene enc0d1n9 9a1act0k1na5e, and pR5415 (511- havy and 8eckw1th 1985, 06ta1ned fr0m R. 51m0n5), carry1n9 the 1ac2 9ene enc0d1n9 [3-9a1act051da5e. A11 rp0H pr0m0ter fra9- ment5 0r191nated fr0m pKP11. 7he fu510n5 6etween the rp0H pr0m0ter5 and the 9a1K 9ene 0f pK01 are de5cr16ed 1n Re5u1t5 and 1n 7a61e 1. 7he DNA fra9ment5 were made 61unt-ended and 1n5erted 1nt0 the 5ma1 51te 0f pK01. pJE2-30, a fu510n 0f the rp0H pr0m0ter5 and the 1ac2 9ene, wa5 made 6y 1n5ert1n9 a 61unt-ended 880-6p 5a11-C1a1 DNA fra9ment c0nta1n1n9 the rp0H pr0m0ter5 1nt0 the 5ma1 51te 0f pR5415.

C0n5truct10n 0f a 51n91e-c0py fu510n 0f the rp0H pr0m0ter5 and the 1ac2 9ene

7he pha9e kR545 wa5 c0n5tructed t0 a110w tran5fer 0f 9ene fu- 510n5 fr0m p1a5m1d5 t0 the pha9e 6y h0m01090u5 rec0m61na- t10n (511havy and 8eckw1th 1985). 5tra1n MC1061 carry1n9 pJE2-30 wa5 1nfected w1th kR545 and a rec0m61nant Lac • pha9e carry1n9 the rp0H pr0m0ter-1ac2 fu510n wa5 1501ated and named kJE2-30, kJE2-30 1y509en5 0f 5tra1n CA611042 were pu- r1f1ed fr0m the center5 0f p1a4ue5 and the num6er 0f pr0pha9e/ ce11 wa5 determ1ned 6y the re1at1ve am0unt5 0f [3-9a1act051da5e pr0duced.

6a1act0k1na5e and ~-1actama5e a55ay5

Ce115 were 1y5ed a5 de5cr16ed 6y McKenney et a1. (1981), except that 50d1um de0xych01ate (0.3% wt/v01) wa5 1nc1uded 1n the 1y515 6uffer. 6a1act0k1na5e act1v1ty wa5 determ1ned 6y mea- 5ur1n9 the c0nver510n 0f 9a1act05e t0 9a1act05e-1-ph05phate (McKenney et a1. 1981}. [3-Lactama5e act1v1ty wa5 a55ayed 6y mea5ur1n9 the hydr01y515 0f n1tr0cef1n (70m12awa 1985).

Pr0te1n 1a6e11n9 and mea5urement5 0f pr0te1n 5ynthe515

Ce115 9r0w1n9 1n M9 91uc05e med1um were pu15e-1a6e1ed 6y tran5ferr1n9 0.5-m1 5amp1e5 t0 prewarmed tu6e5 c0nta1n1n9 e1- ther 10 ~C1 0f [355]meth10n1ne 0r 50 ~C1 0f pH]1euc1ne and 50 ~C1 0f pH]1y51ne. After a 1-m1n pu15e, exce55 n0nrad10act1ve meth10n1ne, 0r 1euc1ne and 1y51ne, wa5 added. F0r 4uant1tat1ve exper1ment5, pu15e-1a6e1ed 5H-5amp1e5 were m1xed w1th a11- 4u0t5 0f 555-1a6e1ed extract5 fr0m the 5ame 5tra1n t0 c0rrect f0r 5amp1e rec0very, prepared f0r tw0-d1men510na1 9e1 e1ectr0ph0- re515 (0•Farre11 1975), and 4uant1tated a5 de5cr16ed 1n 6r055man et a1. (1987). F0r 1mmun0prec1p1tat10n5, 555-1a6e1ed 5amp1e5 were ch111ed 0n 1ce, centr1fu9ed, and re5u5pended 1n 2% (wt/ v01) 5D5, 0.05 M 7r15 hydr0ch10r1de (7r15-HC1) (pH 7.5) and 6011ed f0r 2 m1n. 7he 5amp1e5 were 1mmun0prec1p1tated (McKn19ht and Fr1ed 1981) u51n9 ant15erum a9a1n5t ~-9a1act051- da5e. 7he 1mmun0prec1p1tate5 were exam1ned 6y 0ne-d1men- 510na1 5D5-p01yacry1am1de 9e1 e1ectr0ph0re515 (Laemm11 1970).

RNA preparat10n

A m0d1f1cat10n 0f the meth0d 0f J1nk5-R06ert50n et a1. (1983) wa5 u5ed t0 prepare RNA fr0m ce115 9r0w1n9 1n M0P5 91uc05e med1um. Ce11 5amp1e5 0f 5 0r 10 m1 were tran5ferred 1nt0 an e4ua1 v01ume 0f h0t (95-100°C) 1y515 med1um [1% (wt/v01) 5D5, 0.008 M ED7A, 0.1 M NaC1] and 1ncu6ated at 95-100°C f0r 1.5 m1n. An e4ua1 v01ume 0f water-5aturated phen01 wa5 added and 5amp1e5 were 5haken f0r 5 m1n at 64°C. After centr1f- u9at10n t0 5eparate the pha5e5, the phen01 extract10n wa5 re- peated. An add1t10na1 extract10n wa5 d0ne at r00m temperature w1th an e4ua1 v01ume 0f phen01/ch10r0f0rm/150-amy1 a1c0h01 (25 : 24 : 1) 6uffered w1th 7r15-HC1 (pH 7.9). 7he 5amp1e5 were prec1p1tated tw1ce w1th ethan01 and re5u5pended 1n 0.01 M 7r15-HC1 (pH 7.5), 0.001 M ED7A and 5t0red at -80°C. 7he RNA c0ncentrat10n wa5 ca1cu1ated 6y a55um1n9 that a 501ut10n 0f 40 ~9 RNA/m1 ha5 a UV a650r6ance 0f 1.0 at 260 nm. When [5H]RNA wa5 made, ce115 (1.0 m1) were tran5ferred t0 a prewarmed tu6e c0nta1n1n9 50 ~C1 0f pH]ur1d1ne; 30 5ec 1ater 1.0 m1 0f h0t (95-100°C) 1y515 med1um c0nta1n1n9 0.20 m9 0f yea5t tRNA wa5 added. 7he rema1nder 0f the pr0cedure wa5 a5 de- 5cr16ed, except that the RNA wa5 re5u5pended 1n f11ter hy6r1d- 12at10n 6uffer [0.1 M 7r15-HC1 (pH 7.5), 0.75 M NaC1, 0.002 M ED7A, 0.5% (wt/v01} 5D5, 50% (v01/v01) de10n12ed f0rmam1de]. 5H 1nc0rp0rat10n wa5 mea5ured 6y tr1ch10r0acet1c ac1d prec1p1- tat10n.

1n v1tr0 tran5cr1pt10n

E97° wa5 rec0n5t1tuted fr0m pure c0re RNA p01ymera5e and cr 7° (L0we et a1. 1979). E0 ~2 wa5 pur1f1ed u51n9 the 1n1t1a1 5tep5 0f 6r055man et a1. (1984) and chr0mat09raphy 0n 51n91e-5tranded DNA a9ar05e and 5ephacry1 5400 (L0we et a1. 1979). E0 ~2 wa5 5eparated fr0m c0re RNA p01ymera5e and re51dua1 E~ 7° 6y three pa55a9e5 thr0u9h a 810-Rex 70 c01umn (6r055man et a1. 1984). React10n c0nd1t10n5 were th05e 0f 6r055man et a1. (1984) except that pKP11 wa5 the DNA temp1ate, a11 f0ur N7P5 were pre5ent at 0.2 mM, and the NaC1 c0ncentrat10n var1ed 6etween 0.05 M and 0.2 M. RNA wa5 pur1f1ed fr0m 1n v1tr0 react10n5 6y extract10n w1th water-5aturated phen01 f0110wed 6y ethan01 prec1p1tat10n, and 5t0red 1n 0.01 M 7r15-HC1 (pH 7.5), 0.001 M ED7A at - 80°C.

Pur1f1cat10n 0f p1a5m1d and pha9e DNA

P1a5m1d5 were amp11f1ed [Man1at15 1982) and pur1f1ed u51n9 the p01yethy1ene 91yc01 prec1p1tat10n meth0d 0f Humphr1e5 et a1. (1975). k pha9e and k DNA5 were 1501ated a5 de5cr16ed 6y Dunn and 81attner 11987).





7ran5cr1pt10n 0f 11701-1

DNA end-1a6e11n9 and 1501at10n 0f 1a6e1ed fra9ment5

P1a5m1d DNA5 were cut w1th a re5tr1ct10n en2yme, deph05- ph0ry1ated u51n9 ca1f 1nte5t1ne a1ka11ne ph05phata5e, and 1a- 6e1ed at the 5• end5 w1th [~1-32p]A7P u51n9 74 p01ynuc1e0t1de k1na5e (Maxam and 6116ert 1980) 0r DNA5 were 1a6e1ed at 3• end5 u51n9 the K1en0w fra9ment 0f DNA p01ymera5e 1 and the appr0pr1ate [a-32P]dN7P (Man1at15 et a1. 1982). 7he end-1a6e1ed DNA5 were cut w1th a 5ec0nd re5tr1ct10n en2yme and e1ectr0e- 1uted fr0m p01yacry1am1de 9e15 u51n9 an 181 M0de1 UEA e1ec- tr0e1uter (1nternat10na1 810techn01091e5, 1nc., New Haven, C0n- nect1cut) acc0rd1n9 t0 the d1rect10n5 0f the manufacturer. A11 5•-end-1a6e1ed rp0H pr06e5 0r191nated fr0m pKP11. 7he fra9- ment5 were 5•-end-1a6e1ed at the 51te5 1nd1cated 1n the f19ure5 and recut w1th 5a11. 7he 3•-end-1a6e1ed pr06e5 came fr0m e1ther pKP11 0r pFN97. 7hey were 3•-end-1a6e1ed at the 855H11 51te and recut w1th H1nc11.

51 mapp1n9

RNA-DNA hy6r1d12at10n c0nd1t10n5 were th05e 0f 8erk and 5harp {1978). A11 5amp1e5 c0nta1ned exce55 DNA. RNA 5amp1e5 {adju5ted t0 50 ~t9 0f RNA per 5amp1e w1th tRNA) were hy6r1d- 12ed t0 0.1-0.5 1a9 0f 1a6e1ed p1a5m1d 0r the m01ar e4u1va1ent 0f pur1f1ed re5tr1ct10n fra9ment f0r 14-16 hr at 45°C. 51 nuc1ea5e {170 un1t5 per react10n) react10n c0nd1t10n5 were th05e 0f 8erk and 5harp {1978), except that react10n5 were f0r 1 hr at 37°C. 51-re515tant DNA fra9ment5 were e1ectr0ph0re5ed 1n 6% {wt/ v01) p01yacry1am1de 9e15 c0nta1n1n9 50% {wt/v01) urea {Maxam and 6116ert 1980). 7he exper1ment5 were 4uant1tated 6y mea- 5ur1n9 the rad10act1v1ty 1n DNA fra9ment5 cut fr0m the 9e15.

DNA 5e4uenc1n9

7he meth0d 0f Maxam and 6116ert {1980) wa5 u5ed.

F11ter hy6r1d12at10n5

hc1857 5am7, krp0H +, 0r M60dnaK(Am)dnaJ + DNA wa5 dena- tured and 60und t0 n1tr0ce11u105e f11ter5 (8.2 cm d1ameter) {C00per 1972). 5even-m1111meter-d1ameter c1rc1e5 c0nta1n1n9 a60ut 5 1a9 0f DNA were prepared fr0m the 1ar9e f11ter5 w1th a hand punch and 6aked at 80°C f0r 3 hr 1n a vacuum 0ven. [3H]RNA wa5 hy6r1d12ed t0 f11ter-60und DNA f0r 14-16 hr at 38°C 1n 1.0 m1 0f f11ter hy6r1d12at10n 6uffer [0.1 M 7r15-HC1 (pH 7.51, 0.75 M NaC1, 0.002 M ED7A, 0.5% (wt/v01) 5D5, 50% (v01/v01) de10n12ed f0rmam1de] c0nta1n1n9 0.2 m9 0f yea5t tRNA {m0d1f1ed fr0m C0urt et a1. 1980). Hy6r1d12at10n5 c0n- ta1ned 850,000 cpm [3H]RNA and 0ne rp0H-5pec1f1c, 0ne dnaK- 5pec1f1c, and 0ne n0n5pec1f1c {h) f11ter. F11ter5 were wa5hed w1th 500 m1 0f 2 x 55C [0.3 M NaC1, 0.03 M 50d1um c1trate (pH 7.0)], treated w1th RNa5e A (0.5 m9) and RNa5e 71 (500 un1t5) f0r 30 m1n 1n 500 m1 0f 2 x 55C, r1n5ed w1th 500 m1 2 x 55C, wa5hed tw1ce w1th 95% ethan01, dr1ed, and c0unted 1n 5 m1 0f 5c1nt111a- t10n f1u1d. A11 hy6r1d12at10n5 were d0ne w1th exce55 DNA.

A c k n 0 w 1 e d 9 m e n t 5

We thank D. C0w1n9, A. 6r055man, D. 5trau5, and E. W1115 f0r c0mment5 0n the manu5cr1pt. We are 9ratefu1 t0 R. Ca1endar, C. 6e0r90p0u105, D. P01aye5, R. 51m0n5, 6. Wa1ker, and Y. 2h0u f0r pr0v1d1n9 5tra1n5, pha9e, p1a5m1d5, and unpu6115hed 1nf0rmat10n. 7h15 re5earch wa5 5upp0rted 6y Nat10na1 1n5t1- tute5 0f Hea1th 9rant5 6M-36278 and A1-19635 t0 C.A.6. and 6M-17892 t0 F.C.N.

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10.1101/gad.1.5.419Access the most recent version at doi: 1:1987, Genes Dev.

J W Erickson, V Vaughn, W A Walter, et al. Escherichia coli heat shock regulatory gene.Regulation of the promoters and transcripts of rpoH, the

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