8.1 ????? lactose operon

1
8 ???????????

• 8.1 ????? lactose operon
• 8.2 ??????
• 8.3 ?????????
• 8.4 ???RNA?????

2
8.1 ?????

• 8.1.1 Discovery of Operon
• 1961?, F. Jacob J.Monod??, ????????1965???????
  ????
• 1940?, Monod????????????????????,????????,??????,
  ???????????,?????????????????????
• ??????????,????????
• 1947?,???????????????????

3

• 1951?,Monod?Jacob??,??????
• Z?????ß-???????
• I??????????????
• SzilardI??????????,???????,?????,????????,???????
  ??
• Jacob??????????(????),?????????????,??????????

4
???????
????
5

• ????????????????????????????,??????,????,????,????
  ????
• ??????mRNA,?????
• ???? promotor,operator???????
• ??????????(???????)
• 
  ????????
• ??????,????????
• 
  ???????

6
8.1.2 ??????
B-?????
7
?????????????????!

• ????,??B-gal/cell
• ?????,5000?
• ?????,lac mRNA??
• ?????lac mRNA???
• ????????????????,?????????
• ???(35S-aa, ???)? E.coli???
• ???(?35S -aa, ????)
• ?
  ß-gal(?35S)

8
8.1.3 ????

• 1 ?????
• lacI, 1045bp,??Pi
• P, 82bp,-821
• O, 35bp,-728
• lacZYA

9
???????? ???RNA pol,
off RNA pol???, on

• 2. ????

10

• 3 ????

?????????????,?????????????????,?????????
???
11
4 ???????
????
??lac???
???ß-????????
Allolctose ???? ???
12
gratuitous inducer ?????
?????

• ??????????,??????
• IPTG,???????
• TMG ,???????
• ONPG, O-??????

????????,??????
13
8.1.4 ?????????

• 1 ??????
• 38KD,4 ??,
• ????????IPTG??
• lacI ?????
• Pi ????,
• 5-10?/cell
• ?????
• ????(?lacO??)
• ????(??????)

14
????????

• ??,-NH2?,lacO???
• ???
• ???,-COOH,
• ??????

15
4???????????????
16
(No Transcript)
17

• ?????????--lacO???

???,11 ????,6bp
18
3 ?????RNA pol?????

• ?????RNA pol????DNA??
• RNA pol ??????????? 1.9X107
  ??????, 2.5X109
• ????RNA pol????. ???????, ???????, ???????????.
• ????????RNA pol????????

??????????????????
19
8.1.5 lac operon????1 ???????

• ??,?lacGlu????
• E.coli???G,??G
• ???,????lac
• ?????lac mRNA???
• ????????????????
• ???????????lac????,???????

20

• ????lac????????????
• ?????????cAMP? CAP??????
• cAMP?????

• CAP, catabolite activator protein
• ?crp??
• CRP, catabolite receptor protein

21
(No Transcript)
22
2 CAP????

• CAP????, 45KD ,?cAMP??
• ????22bp I -70 -50
• II -50 -40

23
???????,??????cAMP???????
????????
24
3 CAP????DNA?????

• DNA??
• ?????CAP???????????
• ???CAP???????RNA pol ??

25
4 CAP??????

• (1)CAP?????????????
• CAP?????????,????????
• CAP????????????,??????

26
(2)?????cAMPCAP??????, ???????????
cAMP-CAP??????,???II?????GC????????????,??-10?????
???,??????????????
27
(3) CAP???????

• CAP?????RNA pol a??
• ??RNA pol a???C???,???CAP?????
• ???DNA,?????

28
8.1.6 lac operon ?????

• lac operon??????????????????(coordinate
  regulation)??????????????,CAP????????CAP????,????
  ???P?????????
• CAP?????,??cAMP-CAP??????cAMP??
• ????????????,?????????????,??cAMP-CAP?????????????
  ?????????
• ?????????????????,?????????

29

• 2. A????????
• ??B-??????????,??????????????????!
• ????????????????????????????,????????????,??,
  ???????????????,???. ??lacA???????????,???????????
  
• 3. lac??????, 10.50.2
• ????????,????????????. ?????????
  ??????????
• ????? ?lac mRNA????,a???z??????????

30
Summary
31
(No Transcript)
32
Summary of lac operon regulation
Glucose cAMP Lactose Transcription of lac mRNA
High Low Present low rate of expression
High Low Absent essentially none
Low High Absent essentially none
Low High Present high rate of expression
33
8.2 Trp operon
???????????, ???????????????????,??????,????????,?
???????
34
8.2.1 ????

• trpR, ????
• P,-4018
• O, -211
• L, 1162
• ????

• t, A??36bp, ???p
• t, t??250bp,??p

35
sne298
36
8.2.2 Trp operon ?????

• 1 Trp R ???

37
????trp ? ???????
trp O ????

• SNE299

38

• 2 ?????????
• trpO -21 1,??????
• trpP -40 18
• ??????trpO ????RNA pol???????????
• SNE299

39
3 ????
????

• ????????,
• ???Trp?, mRNA????,???????,???trpE??????

40
8.2.3 ???? attenuation

• 1 ???,???,a
• ??RNA,140bp

a
41

• ???,
• ???,a

42
??????,??4???????,?????????
43
2 ???14aa

S312
44
(No Transcript)
45
Lack of trp
3 ??????
Lack of aminoacyl tRNA
Ribosome pause at trp codons , occluding
sequence 1
Form 23 hairpin anti-terminator
Transcription into trpE and beyond
46
High trp
Trp is inserted at the trp codons
Translate to the end of leader message
Ribosome occlude sequence 2
Terminate transcription at (34 form terminator)
47
???????????
summary

• ????,10th and 11th codons encode trp residues
  (rare AA)
• ??,??????2?Trp ?????,????, ??4??????

The leader peptide is to determiner trp
availability and to regulate transcription
termination
14amino-acid (encoded by 27-68 of the leader RNA
48
8.2.4 ????????????

• ????
• ???????????R?R-??70?
• ????
• trp???,??10?RNA pol????
• -
  trp
• ????? ----? ??????
• ?----
• trp

trp????????????
49
Negativerepressible operon ????????????
??
?????????? ???Trp ???,???????????????,????mRN
A???
50
????? O ??
???? trp?,RNApol??, ??L.S.???,????
?????????? ?trp????,????????????,????,??????
trp ???????????????????,??????????Trp???
51
8.2.5 ???????????????

• ??tRNA????????,????
• ??????????????,??tRNA ?????????????
• ??????,????????
• ???? ???trp?,???
• ???trp?,???LS
• ???? ??

?????????????
?????????????
52
(No Transcript)
53
8.2.6 ???????????

• 1 ?????
• ???????????????,????????????????
• ???????????????

54
(inducible operon)
Add signal mol. ? Operon on
(repressible operon)
Add signal mol. ? Operon off
55
2 ?????

• ??????????????,?????????????????
• ????

56
8.3 ?????????

• ????????????????,?????????????????
• ?? ??????
• ????????,?????
• ??
• ??????(????,E.coli,T4 phage)
• T7????????RNA pol???
• ???????????

57
??????
????????????????,?????????????????????????????,???
?????????????????????????????,??????????RNA???????
????

• 8.3.1???????SPO1??????????
• SPO1,?????
• ???????????????
• ???????,?SPO1?????????????

58
gp28 ??? 55
??,?2? ? ? 55,??gp28
59
??, gp28 ??? 55??gp33, gp34
60
??,gp33, gp34?? gp28, ? 55
61
??????????????????????????????
????????????, ?????????????????
62
8.3.2 E.coli ????????

• ????(???,???)
• ?????, ?70
• ???, ? 32 ,32 kD
• rpoH
• ???????????

S304
63
?????????????????
64
Q ?70,????????,?????!?
????????,??????????,????? ?????,42?20???,????????
A ?70??? Phs (??????), ?????????????????.

• ?E.coli???,???????
• ?????,??????????????

65
8.4 ???RNA?????

• 8.4.1 ??mRNA???RNA
• mRNA-interfering complementary RNA
• 1983?,Mizuno, Simon??????RNA???????,
  ???????,RNA???,???????? RNA???????? ?!
• ??RNA,??mRNA??
• ?????S-D, AUG, ??N????
• ?RNA???????,???????
• ???????,???????

66
?, E coli ????? ompF ? ???? ????,cII??????????
Tn10?????????
67
8.4.2 E coli ? ompF ???????
Env,?????????????
ompC?ompF?????, ??????????
ompC
???,ompC??,ompF?? ???,ompC??,ompF??
68
(No Transcript)
69
RNAi ??

• 1998?2?,Andrew Fire????RNA??????????,????????????
  ,????????RNA?????,????????????????????????RNA??
• 1984?,????????
• 1988?, ?????(PG ????RNA)

70
???????????

• ACC, 1-??????????, ????????
• PG,??????
• ???????????

71
???

1. ????????????????????????????????????RNAi
2. ?????????????????
3. ???/???????,??????????,????