???????? DNA

1
??? ????????
2

• 10.1 ????
• ???????? DNA
• ???mRNA?????
• ?????mRNA???
• ?????????
• ???,?????
• ????
• 1958?Crick??????????????????????

3
10.2
DNA?????DNA??????????

• DNA??????????,?????????????
• Meselson???????? ???DNA???????????

4
?? DNA????

• ????,?????????(replication fork)
• ????,??????????E.coliDNA??

??5?3????
5
(No Transcript)
6
???????(origin)

• ??DNA?????????(ori),???DNA??????ori???????
  ??????????DNA????????,???,?????????A?T??,?????????
  ??????DNA????????,????????????????

7
?? DNA????

• ?????(??)( Cairns model )
• ????DNA??????

8
???DNA?,????????,??????
9
2.??DNA????? Rolling Circle Mechanism for
Replicating Circular DNA ????DNA,??F??(F
factor)?????????? DNA???
10

• 3. ???DNA????
• ??????,?DNA?????????,????????????,???????????
  DNA???,????DNA??

11
4. ???DNA???? D?????
12
??DNA???????

• ???DNA??????????,?????????????????????
  ????????DNA????????5?3???
• ???????????????

13
1968?,??(Okazaki)???(??????????????????
?)???DNA???,????????,?????????????,?3?5?????????
?????,????DNA????(leading strand),?5?3??????????
????,???????????????,?????,????DNA?????????(laggin
g strand)?????????? ???????,????DNA??????
?????
14
(No Transcript)
15
?? DNA????

• DNA????????DNA??????????
• 1. DNA??????????
• ????dNTP(dATP?dGTP?dCTP?dTTP)
• ??DNA??
• ????????????,???dNTP?5'??????????3
  '????3 '- 5'?????,??????5 '?3',??????? ?
• ??????????????

16
DNA???????? ?????????,????????????????????? ????
?????DNA?,??????????,??????,?????????(processitivi
ty)?
17
(1)????DNA???????, ?DNA??? I?II?III?
I II III
??? 1 4 10
M.W 103 000 88 000 830 000
???? v v v
?????? ? ?? ??
????(N/s) 1620 40 2501000
5'-3 '??? v v
3'-5 '??? v v v
?? ??????????? ?????? ???????? DNA??????
18
DNA??? I (Kornberg ?) (DNA Polymerase I, Pol I)
???? ???109 kD ?DNA?????,???dNMP?,?????,?????
?3-5???????,???-OH??????????? ?5-3???????,??
???????DNA???? ??????? Klenow ??Pol I
????????????????,?????C??DNA????,???????3-5?????
???
19
3-5??????????-OH???????????? ???DNA?????????
20
5-3???????,?????????DNA????? ?DNA Pol I??????
21
Pol I ???????? DNA???,?????????????,???????5-3?
??????,???????????????,?DNA??????3????,?????????
?(nick translation) 5-3???????????DNA
5??RNA???????????
22
DNA???III (DNA Polymerase III, DNA Pol III)

• E. coli??DNA????DNA Pol III

?? ???(kD) ?? ????
a 130 dnaE
e 27.5 dnaQ
? 10
t 71 dnaX
? 47.5 dnaX
d 35
33
? 15
? 12
ß 40.6 dnaN
Pol III
Pol III
Pol III
Pol III??
23
(No Transcript)
24
DNA???????? ???? ??(thumb) ??(finger) ??(palm)
DNA?????????????????
25
(2)????DNA??? ????,?DNA???a?b?g? d
a b g d
MW 11012000 45000 60000 122000
??? 48? ??? ?????? ???
3?5????? ? ? ? ?
? ? ??????,???????? DNA???? ???DNA??? ??????,????????
26
2. ???(primase) ???DNA???,????????DNA???RNA
,??????1060????
27

• ????????,?????6??????????(primosome)??????????
• dnaB??????ATP?Mg2?????6?dnaC????????????i-????
  ???????DNA??n?n?n?????????????????????????
• n???DNA???????????????????????DNA?5?3??????????
  ??????????ATP??n?ATP????DnaB????DNA????????

28
??????????
????? MW ??? ? ?
??? 60000 1 ????
DnaB 300000 6 ?????????ATPase
DnaC 25000 1 ???
n ???
n' ATPase?????
n' ' 11000 1 ???
i ??????
?????6?DnaC
29
3. DNA??? (ligase) ???????????DNA???3 '-
5'??????????
30
DNA???
???????,?5??RNA???DNA Pol I???DNA????????,??????(
nick)?DNA???(DNA ligase)???
31
4. DNA????(helicase)(???) ??DNA??????,??????,?
2??ATP, ???DNA??????,????????
??E.coli???????,I?II?III?rep???
I?II?III???????5'-3 '????,rep?????3 ' -5'?????
32
5. ??????(single strand binding protein SSB)
MW74000,??? ?????????????
????????????,????????????,??DNA?
??SSB???DNA????????,????????????,???????????SSB,??
SSB????,??????? ??????,?????????????,??????,?
??????????
33
????(helicase) ???????????Rep??
??????(single-strand binding protein,SSB) ?????DNA
,???????
34

• 6. DNA?????(topoisomerase)
• ?????? I??????II

?????I ??????????,?????-??,????110000,
????,????DNA???,??????,????,??????????,????????,??
?????
35
?????II E.coli?,MW1400000????,A2B2,???????????
390000???,???DNA???????,?????,????????,??????????A
TP???
36
?????????? ?DNA?????????????????????????Topo
II ( gyrase )???
(-) ()
37
7. ???(telomerase)
??????RNA(????????) ??????,???????,???????? ???
?????RNA?????,????DNA3-?????DNA
???????????,3-??????????I????,????3-?????????????
?????????,???????
38

• 5TTGGGGTTGGGGTTGGGGTTGGGGTTG
• 3 AACCCC
• 5TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
• 3 AACCCCAACCCC
• 5TTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG
• 3 AACCCCAACCCCAACCCC

3---AACCCCAAC---5 ???
3---AACCCCAAC---5 ???
3---AACCCCAAC---5 ???
39
??DNA????
40
???????????????
41

• ????,???????1?DNA??????,????????????DNA
  ??????????????DNA?????DNA?????????,??????,????????
  ?????????????????????DNA???????????,??????????,??
  ????????????????????????????,PriA?DnaB??????????

42
????
43
???(replisome)
???? DnaB gyrase Pol III DnaG PriA SSB
?? ???? ??? ??? ??? ?????? ??????
44
Replisome model
45
???????
DNA Pol I,ligase
46
10.3 DNA?????

• ????

DNA???????,?DNA????????????????DNA????????
??,??DNA??????,??????????????
??DNA??????????????????????,???????
47

• ?DNA????????(??260nm??)???,???DNA???????????????,?
  ????,??TT????

48

• ?????????????,??????????
• ?DNA???,????????????,???????,??????????,??????????
  ?????

49
????

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

50
???????(Excision repair)
??DNA???????????,????????,???????????????
1. UvrABC?????
51

• ??????(Recombination repair)
• ??????,???????????????????????recA,recBCD
  ??

52

53
??SOS?? ???????,?DNA???????????????
???,???????????????E.coil???,??SOS??????????LexA??
???,???DNA???????RecA????????,??LexA??,?SOS???????
,????????????????
54
(No Transcript)
55
10.4 ???(???)
?? ?RNA?????DNA???? ????
????????RNA?????? ????????dNTP???,???????
????RNA???,????DNA? ??????????,??RNADNA???
???RNA? ???????DNA?????????DNA,???????? ???
?
56
10.5 RNA?????
???? ???DNA?????RNA???
??????DNA????????RNA???
???
???
????DNA???????RNA???????
???
???
57

• ??RNA???
• ???2??a??,ß?ß'?s???????????465 000?
  ???????????
• ??????RNA????,s????????????????,????a2,ß,ß'??
  ,????????

58
???RNA????? a2ßß'?s465 kD ß?ß'??
????,?????? a????????????????? s???????,??
??????? ????
59
RNA????????? (???????s??)
60

• s???RNA?????????????
• ????????,??????s??????s??????RNA????????????
  ??

61
?? ????

• 1???? (promoter)
• ???RNA???????????????DNA???
• ????
• ? Pribnow?( Pribnow box)??-10?,??? TATAAT
• ?Sexfama?(Sexfama box)??-35?,???TTGACA

62
??
63
(No Transcript)
64

• ???????
• 1??????????
• 2?????10 bp?,???6 bp?????,??-10 ?(??Pribnow
  Box),?????
• T80 A95 T45 A60 A50 T96
• 3?????35 bp?,??? 6 bp????,??-35 ?,?????
• T82 T84 G78 A65 C54 A45
• 4?90?????,-10?-35?????16-18bp????????????????,??
  ?????

65
(No Transcript)
66

• 2. ???? (????????)
• ?????????????????
• ??????????,RNA?????????????DNA???
• ??????,RNA???????? RNA??????????????????

67
?????
??-???????????? ????????????? ??????????,?????
????,??????? ????????,????9????,??????????,?????
?RNA ?????,???s???
68
RNA??????DNA??,????????
????
69
??
??
??
70
RNA?????

• ???(terminator)??RNA???????DNA??????????,???????
  
• RNA??????????????RNA???
• ???RNA????????????????????(???,antitermination)???
  ??????????

71
(1)???????????(intrinsic terminators )
???RNA??????? 1?????????????????????????G-C? ?
??????????? 2?????6????U???A-U???????????
72
(No Transcript)
73
(No Transcript)
74
(2)????????
????,????46kD ???RNA?ATP??? ?E.coli???????RNA?,
??RNA???
????????C??G???
75

• ?????RNA??
• ? RNA???
• RNA??????????
• ?????????????,???RNA-DNA????

76
(No Transcript)
77
(No Transcript)
78
3?????RNA???

• (1)?????RNA???

RNA???I?? rRNA RNA???II?? mRNA RNA???III??
tRNA? ????????,??????????????,???????????????(?)
79
?? I II III
???? ?? ?? a-?????? ?? rRNA?? ??? ?? 12? hnRNA (mRNA??) ???? ?? tRNA?5SrRNA ????
80
(2)????????
1)RNA???I ???????? ??????? ?????(core
promoter) -4520, ??????(upstream control
element,UCE) -180-107
81
2)RNA???II???
???????????,?????A? TATA box??RNA???II???????-
25 bp?????7????,??????????A-T???,??????G-C? CAAT
box????-75?????????? ???(enhancer)???????????,?
?????????????????
TATA box
T82A97T93A85 A83
CAAT box
GG CAATCT
82
3)RNA???III????
??5S RNA?tRNA?snRNA(small nuclear RNA)??
5S RNA?tRNA?????????????
83
????????III????
snRNA????????????????
84
(3)???(enhancer)

• ????????,DNA??????????????????????????
  ????,????????????,???????????,??????????????????,?
  ?????????????????RNA????????????,????RNA??????????
  ???

85

• (4)????(transcription factor)
• RNA????????????????????????,????????????????,?????
  ??????

86
(No Transcript)
87
(No Transcript)
88
(No Transcript)
89
10.6 RNA?????
?????????????????????,???????????????mRNA??
??????????????,??????????????????????RNA???????RNA
(heterogeneous nuclear RNA, hnRNA). hnRNA
?????????,?????????mRNA,????????
90
?????RNA?????mRNA???,??????,??????RNA(heterologous
nuclear RNA,hnRNA)? hnRNA?????????????,??hnRNP??
????????
91
??
RNA???????????
????
??
??
92

• ??????
• ???????,???-???????????????????,?????????????????
  ????,???????
• ?????????????????
• ?????tRNA???????
• ????tRNA?,RNA??????????(transesterification)?????

93
?? ?RNA???
(?)?RNA????? ????????????????,????????????,??(??)
?GT,??(??)?AG???????GT-AG????RNA???GU-AG?
???????????(donor site)??5',????????(acceptor
site)??3'?
94
???5 '?????3 ' ???????
???????? ??????????
95
???????????
?????
?????5?6
?????4?5?6?7
?????3
mRNA
96
??(lariat)??
??????,????5 ??????????? 5????????3???A
??2-OH ?????????? A ??????????(branch
site) ???,?3????,??????,????????
97
????A??2'-OH??5'??
?????3'-OH??3'??
???????????(transesterification)??
98
(?)???(spliceosome)
??????????RNA,??snRNA(???RNA,small nuclear
RNA),???100300(??????)??100gt1000(yeast). ???????
????????snRNPs. scRNA(????RNA,small cytoplasmic
RNA),?????????scRNPs. snoRNA(???RNA,small
nucleolus RNA)??rRNA???
99
snRNP??????????(spliceosome)? ?????snRNA????,????
?snRNA?U1, U2, U4, U5, U6???snRNP???snRNA?????,???
??????8???? ?U6 ?,???????PuAU3-6GPu? snRNA?????
?????????????????RNA??snRNA???????????????
100
U1 snRNA???????5'????,??????????
101
???????????
E ???
A ???
B1 ???
B2 ???
C1 ???
C2 ???
102
spliceosome ???lariat????
5 of Intron ???lariat ?????
3 of intron ???lariat ??,exons????
Cut-ligate
103
U6?U4?U2??????????
??U4 ?U2 ?????U6????????????????,?? U6/U4 ? U6/U2
?????
104
?? ?????RNA
??(ribozyme)?????????RNA? ??P?????,?????????RNA?
RNA????tRNA???????????????,?????RNA???? ?????RNA
?????????????????,?????????????? I ?? II
????????????mRNA????????
105
?????(Group I )
????????????????rRNA??????????????????????T4?????
???????????(self-splicing / autosplicing)????
35S RNA????????,????????,????????
?????????????????(GNP)GNP?????3'-OH?
106
GNP?3-OH?????5?,??G-???,?????? ??????3-OH???
??????5? ??????3-OH????5?15????
???????????????
107
(No Transcript)
108
??L19 RNA????????(C5)??
109
I ???????????
??9????(????),??P4?P7???? P3?P4?P6?P7????????,
?????????????? P1???????,??IGS(internal guide
sequence)?
110
??(group II)??????
(?RNA??????)
II ?????domain5?domain6???,?????RNA???????U6-U2?U2
-???????????
(II ????)
111
????????????
????????????
?RNA????II?????????? ??3??????A?2'-OH
?????5'??,????(lariat)????
112
????tRNA???

• ???????????????????,?????????????????tRN
  A??????,????????,????????????????

113
tRNA ?????? (????)
114
pP
115
?? RNA?????
(?)mRNA?5'???
1???
5' 5' Gppp pppApNpNp...
? 5' 5' GpppApNpNp... pp p
????????,?5 '-???????,??????????,??GpppN???,?
?G?????? ???G?????5'??,????????(cap)??
116
????G?7???????????0(cap 0),??m7GpppX
2????
???????2'-OH???,?????1(cap 1),??m7GpppXm
???????2-OH???,?????2(cap 2),??m7GpppXmpYm?
117
(?)RNA?3'????
mRNA 3'?????
Pol I ?pol III ??????????
Pol II????????
118
Pol II???????,3-OH??????????????poly(A)???? ???A
AUAAA?????????poly(A)???????
119
????????????????(?CFI?CFII??)??RNA ????(CPSF)??A
AUAAA?? ????CstF,????????????G-U????
poly(A)???(PAP)??poly(A)??
120
?? RNA ??
RNA ??(RNA editing)???RNA???????????? ????????,??
mRNA?????????,???????????????????,????????????????
121
????B(apo-lipoprotein B, apoB)??????????????? C
U??????????,???????
122
???????II???????????,?????(frameshift)????????????
???4?U???
123
T. brucei ?coxIII gene ????????RNA ??
??????U,????????U?
124
P
? ? U ? ? ?
U-OH
U
U
U
U
OH
U
U
U
U
U
U
U-OH
U
U
U
125
1. rRNA?? (1)??????? rRNA?????
(2)????????
126
2. tRNA??? (1)??
127
(2) ?? ?3'?CCA??????,?????tRNA?????????tRNA?3'?
?CCA,???????????
????????????????????
128
10.7 ?????
?? ???????
1.mRNA ???????????????? ( 1)??????( 5'?),
????( 3'?)
????(??),??????????
????mRNA ??5'??????,3'?????
129
????mRNA???????????????,??????mRNA?
????mRNA???????????????,??????mRNA?
130
(2) ????(genetic code) mRNA?????????????????????
????????
?????
mRNA 5'-?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? mRNA 3'-??
mRNA 5'-?? U C A G mRNA 3'-??
U ?? ?? ? ? ? ? ? ? ? ? ?? ?? ?? ?? ?? ? U C A G
C ? ? ? ? ? ? ? ? ? ? ???? ???? ? ? ? ? U C A G
A ?? ?? ?? ??(??) ? ? ? ? ???? ???? ? ? ? ? ? ? U C A G
G ? ? ? ? ? ? ? ? ?? ?? ? ? ? ? ? ? U C A G
131
??????????????,61 ? ???????????????,?????? UAA
UAG UGA ?????????????????? AUG(GUG)
132
(1)???????????5'-3',???????? ????(frame shift
mutation)??????? ????????????????????????????????
?????????
133
(2)???(degeneracy)??????????????????
??????????? ACU?ACC GUA ?GUG
CCC?CCU ? ? ? ?
? ?
134
(3)???(wobble)????????????????????,???????????????
?????????
????(5'???) ???(3'???)
A C G U I U G U C G A U C A
135
????????? ???????????????????,???????????????,??
???????tRNA?????????? ????????
??????????????,?????????????
136
(4)??????????????????? ???????,???????????????
(??????)????(????)??????????
?????????????????
??? ???? ???
AGA AGG UGA AUU ??? ??? ???? ???? ???? ???? ??? ????
137
2????
???????
???????
(1)?????
138
(2) ????
139
(1) ?????
???? ???????
T?C?
??? (???)
??????tRNA??????mRNA ???????????????????
????
140
???? tRNA????????(Met) ?
??tRNAiMet
????tRNA??????????(fMet)?
??tRNAfMet
141
(2)??????
??-tRNA??????????????????????tRNA

142
(No Transcript)
143
????????????
1. ???????
(1)????
????? AUG SD(Shine-Dalgasrno)??????????(5' ?
??)10??????,??16SrRNA3' ?????????
144
(2)???? ????
145
70S?????
146
(1)?? ?????-tRNA??A??????????Tu?Ts?????GTP???
2. ??
Tu-Ts ??
??A?
147
(No Transcript)
148
(2)??
149
(3)??
150
(No Transcript)
151
3. ??
????????????????
152
(No Transcript)
153
(No Transcript)
154
???
???
155
??????
156
?? ?????
????????????????????-????????????????????????
????????????????????????????????
??????????,????????(chaperone)???????????
???????????????????,?????????????????????????,????
????????
157
??? ????(heat shock protein)70
????????????????????????? ????(chaperonin)?????
?????????????????????????
158
????(heat shock protein)70 ?? Hsp40(DnaJ)????????
??,??Hsp70(DnaK)?????? Hsp70??ATP,?????? GrpE?
?ADP,????????????
159
????(chaperonin) ?? Hsp60 ?14 ???????7
???,???????????
160
???(????)?Hsp10(E.coli??GroES)?????7??????GroES??
????????????
??
??
161
???ATP?????? GroES????,??????????
GroEL????????GroES??? ???????????,???????
162
10.8 ??????

• ??????????? ???
• (1)??

????
????
????
RNA???????
????????,??????
163
(2)????
??????? ?????
???? ???
???? ??
164
Lac operon expression
165
(No Transcript)
166
? ??????--------??????
???? ??
???? ???
167
Ribosome pass through Trp codon
168
Ribosome pausing at Trp codon
169
2. ??RNA
???mRNA????????RNA ???????mRNA???????mRNA???
????
?????
170
10.9 ????????
DNA???????
DNA????
???????(polymerase chain reaction, PCR)
??????
??????? ?????
171
(No Transcript)
172
(No Transcript)
173

• DNA???????
• (1)???????
• (2)????
• (3)??DNA?????
• (4)??DNA????
• (5)??????????

174
(No Transcript)
175
PCR???????
176
??????

• RFLP(????????)
• ???????????DNA????????????????,???????
  ????????????DNA??????,?????DNA????DNA????????,??
  ?????????????????????????????????????????????,??
  ????????????????DNA?????

177

• RAPD(??????DNA)
• ?PCR?????????????????(9-10bp)??????????,????
  ????????????,?????????

178

• AFLP(?????????)
• ?????????????????????,??????????????,????PC
  R?RFLP?????????????,???????????

179
???????
180
?????