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Е.А. Николайчик РЕГУЛЯЦИЯ МЕТАБОЛИЗМА курс лекций Минск 2002

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2002

1. . ................................................ 1 1.1 ...................................................................................... 2 lac ....................................................................................................................................................................... 2 1.2 ? .............................................................................................................. 3 .......................................................................................................... 3 1.3 - ........................................................... 4 1.4 ............................................................................................................................................. 6 1.5 ....................................................................................................................................... 7 . ................................................................................................................................................................. 8 1.6 . .......................................................................... 9 2. .......................................................................... 10 ...................................................................................................................................... 13 bgl .............................................................................................................. 14 3. - - .................................. 14

- . . ............................................... 16 rpoS ................................................................................................................................................................................. 16 rpoE ................................................................................................................................................................................ 174. , ............................................ 19 ....................................................................................................................................... 19 - .................................................................................................................................... 21 5. ......................................................................................................... 24 6. ............................................................................................... 26 ................................................................................................................................ 27 .................................................................................. 29 . ........................................................................................................................ 30 HPt- ................................................................................................................................................................. 30 ...................................................................................................................................................... 30 ...................................................................................................................................................... 30 ...................................................................................................... 30 ..................................................................................................................................... 30 . .............................................................................................................................................. 31 .............................................................................................................................................. 31 . ........................................................................................................................ 31 .................................................................................................................... 32 ...................................................................................................................... 32 ....................................................................................................................................... 33 . ................................................................................................................................... 33 ................................................................................................................. 34 ..................................................................................................................................... 34 7. ................................................................................................................ 34 7.1 ....................................... 34 .................................................................................................. 35 7.2 ? ................................................................ 36 7.3 .................................................................................................................. 37

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....................................................................................................................................... 37 ? ...................................... 38 ...................... 39 . ....................................................................................... 39 8. .................................................................................................... 41 9. . ..................................................... 43 : ............................ 43 . .................................................................................................................... 43 . ..................................................................................... 44 . ................................................................................................................. 44 SoxRS ............................................................................................................................................................. 44 OxyR ............................................................................................................................................................... 45 . ...................................................................................................................................... 45 FNR .................................................................................................................................... 45 ArcB ................................................................................................................................................................................ 46 10. .................................... 47 CAULOBACTER CRESCENTUS. ........................................................................ 59 11. BACILLUS SUBTILIS. ........................................................................ 62 ........................................................................................................................................ 62 ? ....................................................................................... 63 ...................................................... 64 12. . QUORUM SENSING..................................... 66 . ............................................................................................................................................................ 67 Erwinia ............................................................................................... 68 - . - ( ). ................................................................................................................................................................. 69 13. ..................................................................................................... 69 . ................................................................................................................. 71 (GSP). .................................................................................................... 72 Sec . ............................................................................................................................................................... 72 GSP ................................................................................................................. 74 GSP ............................................................................................................................................................ 75 III ............................................................................................................................... 76 1.1. III . ......................................................... 76 1.2. III . ............................................................................................. 77 1.3. , III ................. 78 . ....................................................................................................................................... 79 IV ............................................................................................................................... 80 14. .......................................................................................................... 82 15. SOS ..................................................................................................................... 83 SOS- ........................................................................................................................................................... 84 16. ........................................................................ 84 , ................................................................ 86 ...................................................................................................................................................................... 86 III .................................................................................................................................................................... 86

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............................................................................................................................... 86 II ..................................................................................................................................................................... 87 . ............................................................................................ 87 - ...................................................................................................................... 87 . ............................................................................................................... 88

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1. . , , , , , , . , , , , , , - . , . , , , . , . , , - . , . - , . , , , . , , . , , . , , .., . , , , - . ( , ), , . , ( ), ( ). - (, , ). , ( , , , ., - ). . : ( ): I- ( ) - cell cycle; II : ) (+ ) ) ( ) III : ) (rpoS) ) (cold shock) IV : - ; ()

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A priori , . , (, , ). . 1.1 , Escherichia coli. 1961 . "". , , ( , .. , ), , . , . , ( ) , . . : 1. - , - . - TTGACA - 17 bp - TATAAT. ( , - ) . 2. Operator - . - 20 bp. . , , , , , . () . . (, ) , , . "" , . 3. . , . , , . 4. . . , -, , . - , . - . , , -, , . - (-) . . . ., : ( ) ( ) lac lacZ - lacY -

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lacA - - - 1000 . - - lacI, - -. . lacI . , . - . (, ) . , . . , lac- . lac- CAP (Catabolite Activator Protein, CRP) (). . CAP , . CAP-cAMP , . , , , ( .. ) . , , CAP-cAMP, . 1.2 ? , , - . . - - , "" . , . ( , ). - , ( ) "" . 1. . . (). , , , . . 2. . - . -, .., . , N- "", , . .., , - , . - , . . - ,

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, . . , , . ( ). . ? - - , . : - , ; ( ) . E. coli , , , ( ). cI . . , - . - ( , ) . Lac 10 4000 JH Miller'a Gal LacI, - ( LacI) Gal HU . ?

1.3 - , -. 10 35 2 4 -. , . - - -. , , 10 , 2 - -. , -, . - , , ( ). . -. -; , .

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, . RNAP (a) (. 1.1.) a NTD b/ b' . 70 - - a CTD - (CTD) -35 -10 . 35 . , (b) a NTD . b/ b' CTD. 70 s AA a CTD , . N- -35 -10 - , CTD (c) , . a CTD a NTD b/ b' , , , -. s 70 AA Region 4 , 61 ( lac ), -35 -10 71, -81, -91. CTD ? (d) , a NTD b/ b' , , s 70 AA Region 4 a CTD . ? -35 -10 CTD - (cI) , cI- . 1.1. - cI cI. cI CTD, , , . , 70- - , 4. cI . , 35 PRM. ( 70 ). , - -. , ( ) -. N- - , 35 . DnaA PR ' -. , -.

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, 35 , CTD . CTD . , , FNR ( ). - -, CTD. , FNR -. - , 35. , N- - -. CTD. , . MerR Tn501. , 10 35 . , -, 10 35 . SoxR ( , ). , . . . , , ( ) . . 4 : ( ). , ( ); , -. MalT malK : - MalT -. 35 , upstream, CTD. . Promoter occlusion 1.4 ara E. coli. lac . lac -, LacI , , . ara ( araC) , . 6 (araC). , araBAD, araC. araC araBAD, 147 . , araE araF , araCBAD, .. . , , .. araC. . , () , , , , . , araC , . , , () ,

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. araC Crep Cind. C araC araB. , , , araC araBAD. , - . Crep araC, Crep . , araC , . () , araBAD. araC , , , araBAD. .., lac . Crep araC , LacI lac . , , . - . , , , - Cind. Cind , , , , . Cind - , - , -, . ? , ara . . (, -72...50 lac -50...-23 gal ), -. . ara -107...-78. - . , , . . . , - . , gal lac -, ara . , . - , , - , , -35 . , , . , , , . 1.5 (lac ara), , (). , . , , - . , - , () . .

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5 , , . (1 .190). , - . , -. trp trpR. -TrpR . ( - TrpR vs trpR). 70 . ( 1000- lac ). - -, , -, , . . , . , - . , , , . , . , , , . . - , "" , . . , , 162 .. trpE . . - GC , 8 U ( , ). ~90% ( 600- ). RBS, , 13 . ? 9 10 . , , . , , . 1 2, 3 4. , . 1 2, 2 3. 4 , . , , . , , , . - 1 2, , , 2, 2-3, . - -, 2-3. , . , , 1-2 2-3 , , 3-4.

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- , , , . - thr thr ile, a ilv - ile, leu val. , , , - - . 1.6 . , . . , - . . , , . , . , , , , . , , , , , , . - . : (), . "", , . - pN pQ. . . , N cro "" "" PL PR. N cro tL1 tR1. pN - tL1 tR1 . . , Rho, , -, . , , , C G, . Rho - - NusG. pN , , . , - , - . , pN , - , . nutL nutR ( . N utilisation) . , : " ?". pN nut , - , . , 17 .. ( ). , boxB, pN . , boxA, . pN , , , BoxB , -, . , .

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, pN, , , NusA NusB (N utilisation substance). NusA -, , , NusB , boxA. NusB . , NusG, - , Rho -.

2. : ( , ) - , . , , . , . , , . . , , , , , , . , , Treponema pallidum, , , , . : (.) + (.) --> + (.) -P (.)

, - (). - -14.7 / (, ), - -3 /. , . , -, , , -, , ( ). , - , ( , ). , . , , , .

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- I (EI), HPr (histidine protein) II (EII). EII - ( EIIA, EIIB, EIIC EIID), , . I HPr - 64-85 9-10 kDa, , . , II , ( ), . EI, HPr, EIIA EIIB. E. coli .2.1. (.2.1.()). EI , His-189. EI HPr His-15. P-HPr ( ) EIIA, (His-90), Cys-241 EIIBC, -6-. ? , . EIIC. C II - , , - . ( EIIB) . EIIB , . IIB Cys ( ). . , . , , - , . , . , , . , "" . . , , pts , , , , .

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pts , : ptsH ( HPr), ptsI (EI), crr (EIIAGlc). -. , - . - (P0 P1) ptsH, (P2) - crr ( ptsI). P0 P1 . P0 -, P1 - . , , , ptsH. P2 crr. EIIAGlc HPr EI. pts EIICBGlc (c P0 ). , pts - EIICBGlc (?) - , , . , EI HPr, . Enterobacteriaceae , , , , , N-, , , . , ( ) - , , , , , , . ? , - , . IIAGlc . IIAGlc P-HPr IICBGlc - . IIAGlc P-IIAGlc . IIAGlc , , , , . . . , IIAGlc . , . , IIAGlc , - ? , - (EI HPr) , "" (.2.1.(b)). "" , IIAGlc HPr , IIAGlc. - - IIAGlc, - , - P-IIAGlc. EIIAGlc ? EIIAGlc - . , , , - , , . EIIAGlc , . EIIAGlc , , . - , PEIIAGlc - (.2.2.).

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, , , E. coli . , . - B. subtilis (. 2.3.). , HPr. , HPr B. subtilis .2.2. Escherichia coli His-15 I Ser-46 ( , --) - HPr- ( ptsK). Ser-46 . : , CcpA (catabolite control protein) , , , HPr . CcpA , , cre (catabolite responsive element). CcpA LacI- . cre , xyl, gut ( ), hut ( ), lev ( ) . CcpA cre , Ser-46 HPr. CcpA HPr-S46-P. .2.3. Bacillus subtilis - . LevR, LicT LacT, , - -. ( BglG E.coli), (PRD). HPr EII. HPr-H15-P PRD , . HPr Ser-46 HPr- . ( HPr-), HPr , PRD .

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bgl . bgl E. coli () : bglG, (), bglF, - bglB, , -. bglG bglF. BglF - . BglF , BglG, , BglG. bgl , . BglG , . BglG ( ) . BglG . - BglF BglG; BglF BglG, . . sac genes of B. subtilis 3. - - - - ('2) () , (. 3.1). - ( 10-20). -, , -, (. 3.1). , , ( , , ..). - , . - , , , . ( 38). , -, rpo (RNA polymerase), - ( RpoS). - E. coli 70 kDa 70 RpoD. -, , 54 kDa, 54 RpoN. , ,

.3.1.

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-. - 70. - , , , , ( , , ). 54 , . 3.1. - Escherichia coli RpoD RpoS RpoH RpoF RpoE FecI RpoN , 613 330 284 239 202 173 477 ~1000 ~100 ~40 ~ ~5 ~5 ~15

- E. coli 70 kDa 70 RpoD. -, , 54 kDa, 54 RpoN. , , (. 3.2). - 70. - , , , , ( , , ). 54 , . , -, , , -. RpoD RpoS ( - ) , , .3.2. - , -. , . -

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() - . - , , .

- . . -, . - SPO1 B. subtilis. . . - (55 B. subtilis). , 28, 33 34. 28, gp28, -. -, - . 33 34, gp33 gp34, gp28, , . , - . rpoS E. coli - s, rpoS. - 70. , , , - in vivo. S s , . - , , s .. . , , . , pH, . , , ( - ) ( ). E. coli s, rpoS . - - 70 ( rpoD). , , , . s 30% 70, s ( 100) s- . , , . s , . s , s . , , , , . , , , lac-. s - s- . s , . - .

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, s , rpoS. . - Hfq (HF-I). , rpoS . s , . , , , pH, , - s . s ClpXP RssB. s , 70. RssB - ClpXP . RssB s /, - . -, s? , . ftsQAZ . KatE, KatG Dps, . , , , Es E70, . , , Es , E70 , in vitro . ( Es 35 ) s- ? H-NS, Lrp, cAMP-CRP, IHF Fis. . s- . , , . s , 70 . rpoE , s, -, - 70. - 70 , "" 70. ( ) - E. coli, E. ECF (extracytoplasmic factors) -. - (, ). E , () , , , E ( o -, 32), - . E , . -, , , , - . RseA. E RseA. RseA RseB. , , RseB , RseA. RseA E,

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RseA E, - (. 3.3). : fkpA, ( ); degP - , ; rpoH - -, ; rpoE rseABC; . E , E. E RseA RseB, , . E . , P. aeruginosa E (AlgU) alg, , . , E. coli, - .3.3. RpoE. , MucA. AlgU , , , . RseA MucA , - . - -, -. E , - , 28 ( ) F ( B. subtilis). - , Salmonella typhimutium. ( ) 28. 28 - FlgM. , FlgM , 28. 28 , - , . , FlgM, FlgM , - . : 1. D. Missiakas and S. Raina. The extracytoplasmic function sigma factors: role and regulation. Mol Microbiol 1998 28(6):1059-1066

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4. , . , , ( - E. coli). .. , (DnaK, GroEL) - (Lon, ClpAP). ( ) . - ( ) . . , . -: . , . ( , ), . , , . Hsp70 Hsp60 ( heat shock protein + . ) . Hsp60 E. coli GroEL, GroES, 55 (. 4.1.). GroES-GroEL 26S

. 4.1. GroELS

. 4.2. GroE

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, . E. coli Hsp70 DnaK, DnaJ GrpE (. 4.3). E. coli , SecB, ClpB IbpAB, , Hsp70 Hsp60 . ? , , (. 4.4). , (. 4.5). , , DnaK-DnaJ-GrpE, . , , "" . 4.3. DnaK , . , GroES-GroEL. , . . . , SecB . , , . , DnaK 4-5 , ; . 36 , . . GroES-GroEL DnaK - , . , - ClpAP, ClpXP, HslVU - , - - . , , . HflB (FtsH), , . , , , . ( , .. - .) - , . , ClpAP , . , , . :

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, . , - , . -. , 5 . "" . "" . , - P- (, , -) A- ( ). , ssrA, -, , , 10 , . . 4.4. , - 11 , ( Tsr, - HflB ClpXP ). , "", , , . - , (26S ), , , , ( ). , , . , () . . 4.5. ( ). , .

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, . , , , , . , 5-10 . E.coli 4 - . ClpAP ClpXP. , , (. 4.6). ClpA ClpX . clpX clpP , clpA . . ClpYQ/HslUV HflB(FtsH) . 4.6. - : Zn - , , RpoH, SecY ( ). - , E. coli. RpoH- . Lon. N , SulA, RcsA . , Lon E. coli. , , . . . , , Lon. . 32 (. 4.7). , E . , ,

. 4.7. Escherichia coli

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.. - E rpoH, 32, (, 50C). RpoH, , , , . E. coli, 30 42, 32 ( ) , . 32 32 rpoH. 32 DnaK-DnaJ-GrpE, - , 32. , , HSP ( , , , 32). DnaK-DnaJ , . 32 , , - HflB (FtsH). (HslVU, ClpAP Lon), , 32. DnaK-DnaJ , . , , 32 . -, 32 . 32 -, . 32 , . 5' rpoH , . , , - . rpoH , E, DnaA cAMP-CRP. E. coli , . , (32 - ). DegP (HtrA) (- ) FkpA, . , . , E , RseA RseB. RseA - E , E . RseB RseA E. , RseB , RseA, E. , - ( ""), RseA, E. . E. coli, . B. subtilis . ( CIRCE/HrcA) DnaKJ-GrpE GroEL-GroES, HrcA, dnaK. - CIRCE. , , DnaKJ-GrpE 32 E. coli, GroEL-GroES. HrcA, HrcA

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groE dnaK. , GroE, DnaK . HrcA CIRCE- , -, , , . o (50-100) , B, . - - ( ). , . : 1. Susan Gottesman. Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 1996. 30:465506 2. Susan Gottesman. Regulation by proteolysis: developmental switches. Current Opinion in Microbiology 1999, 2:142147 5. : 30C , .. . , , : 10C , , : , . , : , , . : CspA ( , , , ). ( ), CspA, CsdA, Rbf . . . CspA CspE , , . . 5.1. ,

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(cold shock proteins, Csp). Csp E. coli . , 37C , - , , , ( 3', . A. tumefaciens VirD2 - , , VirD2, - . , , . , , . , Tra RP4 RecA,

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- VirE2 (SSB ) , VirF. , VirE2 VirE1. VirE1 VirE2 in vivo in vitro, Syc Yersinia. IV Tra , , . , Ptl B. pertussis PT, A/B 5 , S1-S5. S1, S2-S5 1:1:2:1. Tra PT . Sec , Ptl. . . IV ( Tra ) , , , , . , , : , . ; ; . II-IV . , III IV -. . ( ) , . , - . II ( PT B. pertussis). , , PT B. pertussis, II . , CTX V. cholerae II , II . , .13.6. I-III "" , IgA Neisseria gonorrhoeae : 1. A. Economou. Following the leader: bacterial protein export through the Sec pathway. Trends in Microbiology 1999 VOL. 7 NO. 8 p. 315-320 2. D. M. Anderson and O. Schneewind. Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more. Current Opinion in Microbiology 1999, 2:1824

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2. S. Lory. Secretion of proteins and assembly of bacterial surface organelles: shared pathways of extracellular protein targeting. Current Opinion in Microbiology 1998, 1:2735 ( 4 ) 4. L. W. Cheng and O. Schneewind. Type III machines of Gram-negative bacteria: delivering the goods. Trends in Microbiology 214-220 Vol. 8 No. 5 May 2000 5. C. J. Hueck. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiology And Molecular Biology Reviews, 1998, p. 379433 Vol. 62, No. 2 14. , ( ) , , EF-Ts, EF-Tu EF-G, - -, . 510% . , , . , relA. , - (ppGpp) (pppGpp). . ppGpp - , . RelA (p)ppGpp - . - , , , . , . , , . , , . . , , 105 , , . (p)ppGpp, , , . (p)ppGpp , E. coli, , . , , - . A- , . csgA, a, C - 17 . C-, , - , ( csgA) . C, , FruA. FruA - frz, devTRS csgA. , Che E. coli , . devTRS .

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15. SOS , . . , , , , . "SOS-" SOS- E. coli RecA LexA, SOS-. RecA. RecA LexA. LexA. SOS- . SOS- . , , , , RecA. RecA . LexA RecA-. LexA , LexA . , LexA , UmuD, MucA CI. RecA (- RecA ). SOS- . "" LexA , RecA. LexA ( 5'-taCTGTatatatatACAGta-3'). "" SOS- ( 30): / SOS- lexA ; SOS- recA ruvAB recN (, ) uvrA () uvrB () uvrD (SOS-) umuDC sulA SOS- , LexA. SOS- , . , . , SOS-. , UvrA , , , 20-30 SOS- . RecA ( , 10 ), RuvAB RecN , 30 . "" ( umuDC), 30-40 .

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E. coli, 50 . , "" SOS- ( ) ( ), , ( ) . , umuDC, , . , , SulA- "" . SOS- , SOS- . , SOS- . UmuDC- , , ( ) , , . ( ). , - . SOS- , RecA UmuD UmuC, UmuD ( LexA) UmuD', UmuC (UmuD'2UmuC), ( UmuD'2UmuC - V). UmuD'2UmuC RecA, -, , (, , SOS- ). : 1. W.H. Koch, R. Woodgate. DNA Repair in Prokaryotes and Lower Eukaryotes. DNA Damage and Repair.vol.1: Edited by: Nickoloff and MF Hoekstra 2. G.C. Walker. The SOS Response of Escherichia coli. The Bible. 1996 p 1400-1416 17. . , , . , ( ), . . , . , 20 20 . , , , ( ) . , , , (. 17.1). 3' . , . , E. coli 500 REP (repetitive extragenic palindromes) , 3' . 5' .

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5' REP ( RBS -). , , 3' 35-50 5' , . , .17.1. , , Protected 3 end . . 3 . 5 , , , Endonuclease . - , , 5 3 . , , , , Exonuclease (., ssrA E. coli). , , , 5 3 . E. coli - . , II .17.2. (, PNPase), 3'->5'. -, . II , (a) 53pathway PAB PAB PAB AAAA AAAA AAAA 3 . 5Gppp , Deadenylation and decapping . 5 3 . 3' ( - ) , (b) 3 pathway 5 PAB PAB PAB . 5Gppp AAAA AAAA AAAA 3 " " Deadenylation , 3' , . E ( E, RNAse E), 5Gppp 3 5S , . .17.3. , .

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, 5'->3' 3'->5'. 5' () ()- (PAB) 3' , . 5'->3' , XRN1. 3'->5' . , E. coli 5'->3' . 5' , 5'->3' , , , . 5' , , , . 5' , . , 3'->5' ( 3' ) . , E. coli. rne. (1061 ). . . - . N- S1. . . (A/G)AUU(A/U), "" AU . , 5' , . , . , 5' . , , . 40- rne- . III III ( rnc) E. coli , . - , . , .. . III , III. rnc B. subtilis, E. coli. III - 25.4 . - . rnc ( , III ). III . pnp. 77.1 . . , S1. . , , .

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II rnb. , 3' , , , . 72.3 . S1 . II , , . . E. coli . , - RhlB (. 17.4). . ( ) ( ) PPK DnaK GroEL; . . N- . , () (U) . C- , RhlB, PNP . , , N- , C- , . , -, . 3'->5' . , PNP , . . mtEXO, , II E. coli, Saccharomyces cerevisiae. S. cerevisiae, .17.4. - , , 3'->5' , II PNP E. coli. Rpr4p , . - - RhlB - . RhlB DEAD- . - -, 8 , (D), (E), (A) (D). - , , . in vitro RhlB . , . , RhlB - . RhlB ""

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, . RhlB C- . mtEXO , RhlB. . ()- I (PAP I) E. coli pcnB (plasmid copy number), ColEI . PAP I I , II , ColEI. I, 2 , 5 5' . ( I-5) 10 pcnB ( PAP I ). E. coli (A)- (PAP II). (A)- . PAP I E. coli (A). , E. coli . , , . () , . , I, "", "" 3' . 3' . 3' ()- , (). , ()- , , PAB (poly(A)-binding). () . : 1. M. Grunberg-Manago. Messenger rna stability and its role in control of gene expression in bacteria and phages Annu. Rev. Genet. 1999. 33:193227

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