Εισαγωγή στη Μοριακή Βιολογία.pdf
TRANSCRIPT
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17 cm x 24 cm
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1 DNA (. ) 5
2
DNA (. ) 53
3 ! DNA (. ) 93
4 "! #$% % (. ) 137
5 "! $% % (. ) 173
6 tRNA & (. . ) 215
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'$ ! mRNA (. . ) 249
8 ('! 301
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1 DNA
, ,
: . DNA, . . , , , RNA , .
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DNA DNA
1869, 1944 . , ( DNA ). DNA -. DNA , "" . .
Griffith 1928 Ba-cillus pneumonococcus. , - . , . , "" , "" . Griffith "" . , - . "" , , "" .
Griffith - . 1944 Avery, McCloud McCarthy in vitro. "" , , , RNA, DNA, .. , "" "", DNA.
DNA , Hershey Chase 1952. 4. , . , -, 40
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. , DNA . () Mg35SO4 ( S). , (, ) , . , 4. 4 35S. 4 , MgSO4 , (Na2H32PO4) . DNA , , , DNA . . , , , , . , . , , . , .
DNA , .
DNA 1. DNA
.
2. DNA . , - DNA (. ):
-
8
: 0,00008 pg/ : 0,01 pg/ : 6,0 pg/
3. , , DNA ' ( ). - DNA (, , ..)
4. (base composition) DNA . 194953 Chargaff - 4 DNA . :
.
.
. DNA : = G = C. (+G)
= (C+T). ,
. - .
DNA ( C)
H DNA . : DNA; ;
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9
- , , : - DNA - (.. ). DNA C - ( 1.1). H C DNA (pg), (bp), daltons.
C : () () C .
1.1
1.1 C - . -
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10
. - , . . .., C. elegans 8x107 bp. , , 108 bp , 4x108 bp , 8x108 bp , 2x109 bp .
(, , ), . , .. , ( 109 1011 bp). DNA (). , .., 10 . , , , , DNA ;
: - 3 , 1000 bp ( 1 kb) 30.000 daltons (. -). - 4 5 , E. coli 3000 4000 , 1.000.000 . , DNA , .
DNA mRNA RNA DNA. RNA (rRNA, tRNA) DNA.
, DNA , , .
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11
: DNA . DNA, . , (operon). .
DNA - -. (split genes), - , , mRNA . (exons) - (introns , , ) . . mRNA 3' 5' , . , - 7000 , mRNA 1859 , 1158 .
, 10.000 (bp), 300.000 - . , . mRNA - . , .., 10.000. - , .
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. Drosophila melanogaster 5000 -. 2000 bp 107 bp, 10 -. , .
, , DNA. - DNA Britten Kohne 1968, DNA 300 - . DNA - . , DNA: , . 1.1 DNA (. coli), - , , . , , ( , rRNA). , DNA, . , : , .
1.2 . , 1.2 , , DNA . ,
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DNA - . . 1990, , .. E. coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Oryza sativa (), Caenorhabditis elegans, Drosophila melanogaster, Mus musculus Pan troglodytes. 1995 Haemophilus influenzae (1830 kb) Mycoplasma genitalium (580 kb). 2005. 2850 Mb 28 Mb 308 .
1.1 DNA. - (E. coli) (). Cot = - DNA x .
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1.2 DNA.
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15
1.2 "" DNA - .
(introns)
- , / (alternative splicing and exon shuffling). snRNAs snoRNAs, .
DNA (satellite DNA)
. - -.
- - DNA (mini- and micro-satellite DNA)
, -. .
hnRNA 75% - .
SINES (short interspersed elements)
300 bp, - . Alu ( ).
LINES (long interspersed elements)
SINES, ( 7.000 bp).
(pseudogenes) , . - , . ( mRNA DNA , processed pseudo-genes).
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. , .. , , . - - . , , 30.000 ' . -, , , - .
DNA DNA dAMP, dCMP, dGMP,
dTMP, 3', 5' (. 1.3). - , - . - 5' , 5'- 3' , 3'- . 5' 3' : - .
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1.3 RNA DNA ( ).
DNA: : (), (G), (C) (T). N: A, , , ( - , G, C T ). : , , , -. : dAMP, dGMP, dCMP dTMP, . ( "-" "d").
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DNA 5' 3' 3' 5'. , , 5' . , , 3' 5'. DNA ( ) 5' 3'.
DNA, , -, , DNA. , , , , , .
, Sanger, DNA 174. DNA: - . D 174 390 846. ' : 568 840. : . D . ( , read-ing frame).
ATG CGC GCT TCG ATA AAA ATG ATT GGC GTA TCC AAC CTG CAG AGT
:
Met Arg Ala Ser Ile Lys Met Ile Gly Val Ser Asn Leu Gln Ser
, ,
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19
:
AT GCG CGC TTC GAT AAA AAT GAT TGG CGT ATC CAA CCT GCA GAG T Ala Arg Phe Asp Lys Asn Asp Trp Arg Ile Gln Pro Ala Glu
X174 DNA :
G4 : , C. , DNA.
SV40 112 - . , , - . , SV40 DNA Weissmann, , , ' . DNA mRNA .
DNA DNA
. 1953 : King's College , Wilkins Franklin DNA Cavendish Cambridge, Watson Crick , .
A C D J F G H E B
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20
-. - , - : ( ) - . Watson - , - Chargaff. -NH2 A, G C -NH-CO - G T ( 1.4).
T A, G, C ( ). ' . "" - . . .., - RNA. , "" - . DNA.
DNA, , G C . G C.
- - . DNA : DNA
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21
, DNA DNA.
1.4 A-T () G-C ().
1.5. : 3' 5' . ( ) 10 - . 0.34 nm, 3.4 nm (34 ). - : , - ( 1.6). DNA (major and minor grooves). , DNA.
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22
1.5 DNA. - . .
, DNA, in vitro , . DNA 12 , DNA ( 1.5).
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23
1.6 DNA.
DNA , -
DNA. - , DNA -. DNA , . in vitro
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24
C GCGCGC... in vivo (). - in vivo ( , - DNA) .
DNA, , ( ). , DNA -, DNA (.., ) , "" . DNA .
DNA DNA
. DNA , DNA -, - . , , , - .
, - DNA 260 nm ( 1.7). - DNA 260 nm DNA, ( 1.8). - . -
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25
DNA - ( ) , .
1.7 (m), 50% . A - , -: . (.. RNA) - , . , , : - G C. G C , , .
, , DNA ( "" DNA). DNA.
DNA -. , -, (dimethyl sulfoxide DMSO) DNA -. , . , -.
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26
1.7 DNA.
1.8 () DNA, () DNA () .
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27
, - . , "", - , . : - DNA, - . , - . DNA .
DNA DNA
. 106 - 0,34 mm 2 nm. DNA ( 3'- 5'- ) . DNA .
. . , , . "-" DNA . DNA, , , - .
DNA (. ): . DNA. DNA (- ), -
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28
( 1.9). "" DNA DNA "" (cruciform structure). DNA "" DNA, , .
1.9 DNA, , "".
, DNA, -
, DNA "", (negative supercoiling) DNA. - DNA , .
DNA 4000 (.. ) , 400 , 10 - . DNA 380 . DNA .
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29
. A , - , . - (twist), , ' . , , DNA . (relaxed). - . .
- ( 1.10) .
1.10 DNA - . - ( 1). - ( 2 3). 2 3 I 3 30 min, .
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30
E. coli DNA ( ). - ATP. - DNA ( 1.11, 1.12). .
1.11 DNA DNA . - - . ( - ) - DNA. , - DNA.
, DNA
, . - DNA. 5'- , 3'- . .
DNA . - DNA (single strand binding proteins SSB). , ,
-
31
DNA RNA , DNA . , - DNA - . DNA -DNA ( 1.13). - DNA , .
1.12 DNA - - DNA (a) - (b).
1.13 DNA - -, DNA - DNA (. 1.11).
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32
' , -
DNA. DNA E. coli - 1300 m. 1 m 3 m. DNA . - DNA , -, RNA DNA. E. coli: () DNA () RNA.
RN ( RNA), - ( ) , - , - , DNA. DN - DNA - DNA, , -. DNA. - DNA (, scaffold)) 45 10 ( 1.14).
1.14 E. coli, 15 45 - DNA D DNA.
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33
DNA ' DNA. T DNA -. , . Y 5 (1, 2, 2, 3, 4). ( ). . DNA- -. ' , , , , -. DNA .
(%)
1 215 1,4 28,4
2 132 9,1 9,1
2 124 6,5 16,1
3 135 13,3 8,9
4 104 13,7 9,8
, , . 4, , 109 . - (.. c ) -. .
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34
DNA ( ), , , . ( ), , DNA, DNA, , . , .., RNA . HMG ( , high mobility group) , .
1.15 DNA.
DNA . DNA ( ). -
-
35
DNA . , , " " (zinc finger proteins). 1.15 DNA, n (- / ).
DNA - (), 2 m ( 5 mm ). 10-6 m. DNA . , .
DNA . . 1011 nm - DNA. DNA 200 . (core particles) 146 . - , - H2A, H2B, H3 H4, DNA 146 ( 1.75 , 1.16). DNA . DNA - DNA (linker) . 1 DNA ( 1.17).
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36
1.16 () 1,75 DNA . : () DNA 1,75 . (3/4)2, , 2/2 (Arents Moudrianakis) () DNA , DNA (Arents Moudrianakis).
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37
1.17 .
1.18 - - - - DNA.
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38
: ~30 nm ~30 nm ( 1.17). , , ~30 nm 11 mm ( 1.18, 1.19).
1.19 30 nm - 6 .
, - 20 80 kb DNA ( 1.20). 50-100 . - ( ) RNA, ' DNA. - . ' (.. - 60 kb).
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39
1.20 - ' (scaf-fold).
. - DNA. DNA (phased nucleosomes). , - , - .
, . , DNA.
() ( 30 nm)
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40
() 10 nm. D I. DN, DNA , Southern ( 2). DN , Southern ' . DN . DN , , - .
DNA DN , DNA. DNA . , -, DN DNA ( DN - ). DN DNA , - ( 1.21).
1.21 - .
DN I Southern
-
41
. , : , () (). ' , . .
( 5-) : - -. , - DNA DNA .
, DNA, . - . , , . - DNA ( 1.22). - :
1. - (chromatin remodeling complexes)
2. - . - , , , .
-
42
1.22 . () , DNA CAF-1 and ACF (ACF1ISWI), , NAP-1, H2A, H2B, H3 H4. , . (b) DNA ( A) (SWISNF) . (c) SWISNF, HAT A , . H (RNA pol-II).
-
43
1.23 . (a) (TF1) ISWI , (repositioning nucleosomes), DNA. (b) (TF2) SWI/SNF. SWI/SNF - . (c) (TF3) . ().
-
44
(SWISNF) DNA.
. in vivo SWISNF - .
- . , , , (ubiquitinate), - . - (histone acetyltransferases, HATs) . - HAT ,
- - . HAT ( 1.23). SWISNF, .
- (gene silencing) DNA. (histone deacetylases, HDACs) DNA (DNA methyl-transferases, DNMTs) . HDAC - . - DNMT .
, - . DNA, -
-
45
. - HAT, HDAC and DNMT, - . - DNA -. ( 1.24) , , , . - , bromodomain, - , - .
1.24 - .
-
46
- . , - . SWI/SNF - / DNA - DNA. -, , DNA - CpG /-. DNA cis - , - DNA.
: DNA RNA
. , - , . , . .
- . -. DNA , - . : () , () DNA, RNA, () -. ,
-
47
-. , , . .
, , .
DNA RNA. : RNA (.., , R17, ), RNA (), DNA (), DNA (- 13 174), DNA (SV40, -), DNA ( 4, ), DNA - (), DNA - ( pox). DNA - DNA ( DNA).
-. .., 4 300 -, 30 E. coli. - , SV40 13, . , DNA - DNA, DNA. DNA, . RNA ( -) RNA- RNA () .
RNA DNA - . RNA - RNA . RNA (.., ) -. - RNA (
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48
). , RNA (.., ) mRNA . , .
RNA , RNA 3' RNA, 5' RNA , 5' 3' , 5' . DNA , , . , DNA - .
, DNA - , DNA . . , DNA . (, temperate phages). - . .
( E. coli) - :
1. (1 - 7) - DNA.
2. , . . coli, DNA ( - ) ( ) .
3. DNA , 174 13. DNA ,
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49
. DNA.
4. RNA RNA DNA. 4 ( RNA - RNA, , ). , mR, mRNA .
,
mRNA , . mRNA (+) , () . , DNA - mRNA () . (+) mRNA () RNA DNA. , - . , mRNA 1.25, - , 1.3. , :
DNA ( ). DNA DNA, , mRNA, .
( : , SV40, , : pox, ) DNA.
(, parvo = ) DNA, , mRNA.
-
50
RNA ( -VI).
RNA. () RNA (+) mRNA.
IV RNA, mRNA. RNA , . - RNA () , (+) (. mRNA). IV : IV, - , IV, - ( Sindbis).
V (-) RNA. RNA mRNA . . V ( ) RNA. V, , -. RNA V - RNA . RNA - mRNA. 12-15 5' mRNA . " " - RNA (+) mRNA () RNA.
VI , - RNA ( ) DNA. , - RNA () DNA. (+) . DNA (integrase) DNA - RNA, mRNA . , .
-
51
- (HTLV) (HIV) (AIDS).
1.25 mRNA. To DNA RNA . mRNA (+) () DNA RNA.
-
52
1.3
(kb)
(DNA) A () () SV40 ()
36 150 5.2
. DNA mRNA.
mRNA . DNA. - .
() 200 .
. mRNA .
(DNA) 1 2 DNA.
(RNA) 1.2 4.0 10 RNA
. .
mRNA . - mRNA.
IV (RNA) (IV)
7 mRNA - , - .
RNA. mRNA . -.
Sindbis (IV) ,
10 mRNA, .
-. - -.
V (RNA) (V)
,
12 RNA mRNA .
-. -, - .
(V) ,
10 3.3 8 RNA. RNA mRNA.
-. -, - .
VI (RNA) ,
,
5 8 - RNA DNA. DNA DNA .
. . AIDS.
-
2 DNA
. 1970, DNA . DNA , . - RNA . - DNA . DNA, .
-
54
DNA:
1. .
2. , , , .. , - , - (, , .).
, , , (, ) . , , , , - .
- ( ) .
- . - - .
, -, , . -, , - DNA (DNA microarrays), - RNAi, (SAGE, serial analysis of gene expression).
DNA - ( ) -
-
55
( ) - , in vitro in vivo.
- (, ). DNA - (transgenic). T . , - , , - . , . -, , .
DNA :
DNA . . DNA. DNA.
DNA . ,
, DNA. . . DNA , - "" DNA. DNA , DNA . DNA -
-
56
. , "" DNA.
2.1 - - DNA.
DNA, . -
-
57
. (.. EcoRI E. coli).
DNA - ( 2.1). - , DNA . DNA (. .. EcoRI Hind III 2.1).
(ligation) DNA
- DNA.
DNA , , DNA ( ). (ligation) DNA - DNA ( 2.2).
A (. .. paI 2.1), DNA ' . DNA DNA (linkers) - . (terminal transferase) - 3' DNA. , .., poly(dG) (GGG...) 3' DNA poly(dC) (CCC...) 3' DNA (vector). , - , DNA .
-
58
DNA, . 2.1 ' .
2.2 DNA, - . .
-
59
2.1 DNA.
5' RNA DNA
DNA - -
DNA
DNA * DNA -
) cDNA ) nick translation ( - DNA)
DN I* - DNA
nick translation
3' DNA
- DNA
5' DNA
- DNA
Bal31 3' 5' DNA
- DNA
-
ATP DNA RNA
DNA 32P
-
DNA -
DNA,
S1 DNA
DNA
A 3' DNA
"" DNA
* H DN DNA . , DN DNA. ' DNA DNA , DNA, 5' - 3' ( DNA). nick translation.
-
60
DNA DNA -
- . DNA, ' DNA (.. DNA ). - , (restriction map) DNA, (restriction sites) . DNA (restriction-site mapping).
2.3 X DNA . (a) DNA ( ) DNA. . (b) DNA . -, . .
-
61
2.3 - DNA. , : , , .
DNA - DNA .
, . ' , . , 5' DNA [-32] . 2.4, - DNA ' DNA, . .
DNA . Southern, -, , . , , -, . , - - , .
DNA (.. ). (genetic markers). , RFLP (restriction fragment length polymorphism), - .
-
62
RFLP , DNA (DNA fingerprinting), RFLP (: , , ..).
2.4 DNA.
-
63
DNA
DNA , DNA (.. ). , , DNA, ( , smear).
DNA DNA. , - DNA . .. -, , - 40.000 E. coli. , , "" "" . - DNA (, , ) , DNA.
DNA , . 20-30 , , .. - , 1000 . DNA -, , .. . - DNA , .
DNA, , - DNA . - (cloning vectors), '
-
64
DNA . , (transformation), - , . . - , - : ( 2.5).
2.5 DNA . - . - .
- DNA E. coli.
-
65
pBR322, . , . , ' , - ( 2.6). , DNA , . DNA : , . , .
2.6 pBR322. , - .
-
66
"" -. DNA, : () , () (ori), () () DNA. (polylinker) - DNA - ( 2.7). DNA , - - . , . coli , -. E. coli x-gal, - - . , , DNA, - x-gal. , , , - DNA.
2.7 .
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67
, , - DNA. , - , - , - DNA.
' . , . coli - . (~3 x 109
) 600.000 ( 5 kb, ) . , - DNA, - .
DNA DNA- - , DNA 20 kb. , , , . - , DNA, , - , - .
49.000 60 . - ( ). , , -
-
68
( DNA - ) - . , ( ) DNA ( 2.8).
2.8 - - DNA - .
-
69
. - . , - , DNA . DNA Sau3A - 4 DNA- BamHI ( BamHI 6 DNA Sau3A, ). DNA ~20 kb ( 49 kb) - in vitro .
DNA, .. 45 kb, . . , cos , - . , ("cos" cohesive sites), DNA . - DNA - DNA. DNA .
. .. V 180 kb, - . DNA , - (Cs, yeast artificial chromosomes). : (1) - cis S. cerevisiae, (2) (3) DNA -
-
70
(pulse-field gel electrophoresis). -, DNA YAC . - DNA . , DNA (ARS). , , . DNA YAC -. , DNA , .. EcoRI, 500 kb.
mRNA: cDNA , -
. .., mRNA -. DNA DNA (cDNA) mRNA cDNA . cDNA DNA .
cDNA mRNA - - . oligo(dT), poly(A) mRNA (reverse transcriptase). ,
-
71
2.9 cDNA mRNA.
-
72
RNA , RNA DNA (cDNA complementary DNA). , cDNA-mRNA. - cDNA , mRNA DNA. mRNA cDNA. cDNA DNA. 3' cDNA (.. G) - . - oligo(dC) DNA . cDNA ( 2.9).
, : 1. E . ..,
, - DNA cDNA. - - DNA, DNA. mRNA .
2. .
3. -. , , .
-
73
DNA 1000C
pH (pH 13), DNA . , , . .. DNA 650C , ( -). (DNA:DNA, RNA:RNA, DNA:RNA).
, DNA - DNA DNA (probe). . , - , ( DNA, 1).
A cDNA
DNA, . - DNA , . . DNA RNA - DNA.
-
74
-, - mRNA . mRNA, - . mRNA ( ), cDNA .
T cDNA cDNA ,
mRNA cDNA. cDNA . cDNA . ( 2.10). . , , . - , DNA. , DNA . , . , .
cDNA . - .
-
75
2.10 E .
mRNA , -. , , . ' ,
-
76
. ( 6 7 ). , - , . - - - (-32P) 5' -.
A cDNA , - .
: () , ()
. -, .., , - , .
, . . 2.11 . cDNA poly(A)RNA - . . cDNA RNA . cDNA . - ,
-
77
.
2.11 - .
. , mRNA , . - , cDNA - mRNA -
-
78
( ). 2.12 - cDNA - ( mRNA -). mRNA cDNA . cDNA (, ) - , . cDNA cDNA .
2.12 - cDNA .
-
79
2.13 gt11 EcRI -. - cDNA, -, .
-
80
- cDNA
, - . , cDNA - cDNA - , . cDNA E. coli. -. , . .
. ( 2.13).
, ( cDNA ) (.., , , , ..). 5', , .., .
: "Southern" "Northern"
"Southern" (Southern blot) . , DNA - . -, NaCl/NaOH DNA , - NaCl/ ( 2.14).
-
81
2.14 DNA RNA Southern Northern.
. DNA. DNA '
-
82
. DNA . DNA . , DNA . (cDNA, RNA, ). , DNA - , ( 2.14). .
, :
1. (, , ) DNA.
2. DNA ( - , , chromosome walking, 2.15).
3. .
4. .
, RNA, - "Northern" (Northern blot). RNA mRNA . RNA RNA, (DNA cDNA) ( 2.14). :
1. mRNA.
2. -.
-
83
3. mRNA - (, .., mRNA ).
4. - (.., RNA - - , R).
, , , ( Western, Western blot). Western DNA-, RNA- -.
2.15 DNA (chromosome walking) .
-
84
H (PCR, polymerase chain reaction) DNA .
, DNA -, , , "" DNA : PCR - DNA. H DNA, .
PCR, DNA . - , 3' DNA 3' . - (, primers) in vitro DNA, DNA , .
2.16. -, DNA 50 60C. DNA, , - DNA. - - DNA, - DNA - (Taq Thermus aquaticus). Taq - 72C. , 95C . , - , .
-
85
. - .
2.16 PCR. - DNA.
-
86
PCR . DNA . , Southern, (.. AIDS), - (forensic science) . PCR - (.., ), - DNA ( -), , in vitro , .
DNA DNA, -
. - DNA ( Maxam Gilbert Sanger) - 1970.
Maxam Gilbert
, DNA, , ( 32P) . DNA 4 DNA. DNA. , DNA . , 4 DNA, . .., G 5, 11, 15, 22 , 5, 11, 15 22 (
-
87
, ). 4 - DNA . DNA ( 2.17).
2.17 Maxam Gilbert - DNA.
-
88
M Sanger
DNA , ( ) ( 2',3'-). - 3'-. , DNA, , 3'- . - , , .
- DNA . ddGTP , . - , , - dG, -, ddG, . , - C, A T. (ddNTP:dNTP) , - , - DNA. - . ' - DNA ( 2.18).
, ( ), ( 1), , , , , c, .. ,
-
89
2.18 DNA Sanger.
-
90
- . - 6 DNA (3 DNA), , ' .
DNA
, Sanger - DNA. , ( ). , , . , -, , , . , , . - -, -, , . ( 2.19).
, - , . , - 96 - 750 bp , - 800.000 bp . , - DNA
-
91
. , - .
2.19 - DNA. () . . () - DNA. - .
-
92
, - (shotgun sequencing method). - (.. 1, 5, 100 kb) . , , (. - ). - DNA - . - , - , . - - () . -, , contig contigs .
, DNA - . ( -) , -, GenBank , EMBL DDBJ . , (in silico) . , ( ). , - (- ).
-
3 DNA
DNA, , . ,
, DNA, . , , - .
-
94
T DNA : E. coli (4 x 106 ) - 40 min. 1700 . , , 1 106 . . DNA , : 1700 , 160 . DNA. , , (replicons). , DNA 3000 (65 x 106 ) .
: , .
, DNA (ORC, origin recognition complex, ). - . DNA, - .
( , replisome). DNA , - . DNA, - , . ( ) -, , .
-
95
- DNA, DNA -.
Watson Crick
. , , ( 3.1). - DNA . , ( 3.2). , . DNA 1958 Matthew Meselson Franklin Stahl ( 3.3).
3.1 DNA Watson Crick.
-
96
3.2 15- DNA 14 , DNA.
, E. coli -
' 15NH4Cl. DNA "" 15N 14. 15-DNA (buoyant density) 14-DNA (CsCl). 15N-DNA 14NH4Cl . ' 14NH4Cl 15-DNA , -, (15 14) , (15 14) (14 14) DNA ( 3.3). DNA , DNA . , 14 15. .
-
97
3.3 Meselson Stahl (1958).
DNA DNA
, " ", . DNA DNA, . DNA . DNA ( , 3.4).
DNA - . Prescott Kuempel (1972) E. coli. E. coli - 3- . -
-
98
3.4 SV40 ( ).
3- . - DNA DNA. . E. coli
-
99
. , . . .
DNA ( "" )
DNA. . - - ( 3.5). . . , DNA ( 1), ( ). . .
3.5 DNA - .
-
100
DNA (rolling circle)
DNA ( "" -) 3.6. ' , , - . 5' 3' . , DNA -, DNA 3' , - . 5' -. . , 3' . , . . . , , - - .
3.6 - .
DNA: - DNA , 3' 5' -
-
101
5' 3' . 3' 5'. - DNA 3' . DNA , . - - . 1968 Okazaki , , DNA . , DNA, , 500 3000 - ( Okazaki): () , DNA E. coli ( 8S 10S) DNA. -, (S) -. DNA , .
Okazaki DNA. - Okazaki : dTTP dUTP DNA . DNA.
Olivera Tye (1978) DNA -: ("" ) , ("" ) ( 3.7). , ( - , - ).
-
102
3.7 () - ().
, -
. - . "" - , .
-
103
, , . ' , , , 3-, . , . - , - .
, DNA (ori) - - DNA, , . in vitro -, : E. coli (oriC), Saccharomyces cerevisiae SV40. E. coli .
) E. coli. - oriC E. coli, 245 bp, - . - 9 bp 13 bp. , - .
) . To , , . 400 17 S. cerevisiae. - - 11 bp ( ) 11 bp. -
-
104
RS (autonomously replicating sequence).
, - (RC, origin recognition complex) ARS. ORC 6 6 . RC .
) SV40. M 64 bp SV40 in vivo in vitro. - SV40 ori .
. coli, SV40, -: () - () - - (ORC) , () - , - DNA .
-
. DNA - . , . - DNA, "" "-", -.
-
105
, 180 , . E. coli . , : , - , . (TER) (Tus), DnaB ( ).
, , . DNA: , - , ' . , , . , , .
-
106
,
DNA E. coli DNA.
DNA E. coli - E. coli - DNA (dna , 3.1). -, - ("quick stop", ) - ("slow stop", ). in vitro , .
E. coli in vitro . DNA 13, G4 174 ( ) - - Okazaki. , - , in vitro . - E. coli.
, - in vitro DNA SV40. - DNA.
-
107
3.1. E. coli.
dnaA , ori dnaB 5' 3', prepriming dnaC DnaB. Prepriming dnaT prepriming dnaG Primase, RNA priA prepriming complex,
3 5, SSB priB prepriming priC prepriming dnaE DNA dnaQ DNA , (3' 5' ) holE DNA , dnaX DNA , (),
DNA- dnaX DNA , , holA DNA , holB DNA ', holC DNA , holD DNA , , dnaN DNA , () DNA polA DNA polB DNA gyrA DNA gyrB DNA lig DNA SSB DNA
To SV40 DNA 5 kb . - SV40 DNA DNP . ( ), . , - , DNA SV40,
-
108
DNA .
DNA -
, . - , , - , - DNA. , DNA -, , , . , DNA : - :
5'- (5'-dNTP). - 5'-dNTP 3', 5' 3'- (primer) DNA RNA , - - - . , , - 5' 3'. , 4 , - -.
. DNA ( DNA, RNA DNA) - RNA RNA . -
...
1 N2 N3 N4 N1 N2 N3 N4
+
... + PP
-
109
, , RNA - , DNA - - . , DNA -, , , 3'- - 5'-. , .
DNA E. coli , -
, E. coli Kornberg 1958. , - , DNA ( ), in vitro . - DNA, -, DNA. , , in vivo in vitro . E. coli, - . , , - DNA , - Okazaki.
DNA , DNA ( ), in vitro, - (1970) E. coli. 10 ( "- ", 3.13, 3.2). 400 , 40 10 . , - 40 - , ' .
-
110
, in vivo. -, , - DNA , , DNA ( ). - DNA.
DNA - . DNA E. coli : 3' 5'. 5' 3'. DNA . DNA , .
3' 5' - DNA: . , DNA , . -, , , 3' . , , , - (). , , .
- , .. - . . - . - : . - , -. , ,
-
111
, DNA .
5' 3' DNA , , "nick translation" (" ") - DNA. , . - DNA DNA. , Okazaki RNA ( 3.7) - , - .
, 5' 3' , Okazaki RNA-DNA DNA. in vivo DNA 5' 3' - . - 5' 3' , DNA: - 5' DNA 3' . 3.7, - - . DNA.
Okazaki, 5' - DNA. , DNA 3' . DNA - 5' . - DNA ( 3.7).
-
112
DNA 5 DNA
(, , , ). , DNA E. coli, DNA , () . DNA DNA, DNA DNA . DNA 3.2.
3.2. DNA .
E. coli I II III
: 5' 3' + + +
: 3' 5' + + +
5' 3' + + -
: 5' 3' + + + + +
: 3' 5' - -* + + +
: + - - - -
: + + - + + - - + - -
* DN V. nick translation 3' .
RNA
, DNA 5' 3' . , DNA -
-
113
. DNA 5' 3' DNA , () (): "" , "" ( 3.5). , DNA DNA ( ).
, ( DNA) - ( DNA). E. coli, (DnaG) RNA (RNA primers), DNA . - E. coli dnaG, - , DNA - .
DNA in vitro, - ( ). - , DNA RNA . , - DNA DnaB. 174, , DnaB - (PriA, PriB, PriC, DnaT, DnaC) , , DNA SSB RNA ( 3.8).
RNA Okazaki . , - ( 3.9). DNA 5' 3' DnaB. - DNA. SSB (single-stranded DNA binding proteins) DNA .
-
114
- . DNA ( 3.14) (replisome).
3.8 - .
-
115
3.9 Okazaki.
RNA DNA . RNA , - RNA .
DNA, - - E. coli SV40. DNA DNA .
-
116
(oriC) E. coli.
oriC, . , in vitro DnaA, - DnaA-ATP 4 9 bp ( dnaA boxes) oriC - DnaA ( 3.10). H DnaA -. oriC: DnaA 13 bp . - , DnaB-DnaC, - . - DNA. DnaB DNA - . (DnaG) RNA.
SV40.
- SV40 . , DNA . - ("early palindrome"). - ( DnaA E. coli). SV40 - DNA . SSB, DNA . A ' DNA , , DNA , PCNA , SV40 DNA in vitro.
-
117
3.10 oriC E. coli.
in vitro - , ' SV40. DNA DnaA E. coli, .
-
118
, - . Alu - . ' 14 bp - GAGGC SV40. - DnaA .
DNA
: DNA - . , , DNA .
, , . - DNA. , , ( H - DNA ) ( RNA DNA -). , (L ), , 3.11. ' D loop (displacement loop, ).
DNA , , , - L . - , L . ' L . -
-
119
, L ( 3.11).
(. ) , DNA DNA.
- David Clayton 80. 2003 Ian Holt ( - , Okazaki) , , - .
DNA .
. , DNA -. DNA - 5' DNA .
5' (55 kDa kilo-Daltons). 5' DNA (- 55 kDa) (80 kDa) CTP . 80 kDa dCTP. 80 kDa DNA - . dCTP. 3'- CTP DNA ( 3.12). RNA, . , Okazaki. DNA 29 Bacillus subtilis Streptomyces rechi. - .
-
120
3.11 DNA .
-
121
3.12 .
DNA : E. coli
, - RNA , . , , , RNA. , , - . -, - 3' .
DNA - 10 - ( DNA ). ( -, 3.13) . , 10 DNA . ,
-
122
.
DNA 4 : (), , ( ), DNA, (21'111), DNA , (2), - ( 3.13).
3.13 (). DNA * E. coli ( ). (). * 3' . * DNA.
- (distributive) (processive). DNA 10 50 , , DNA , - DNA 500 kb. ' DNA - DNA "-
-
123
" - ( 3.13). DNA, - "" DNA . 6 , 5 , DNA, .
- DNA - DNA . A , - , ( 3.14). 3' DNA RNA . , Okazaki, RNA . - .
- Okazaki. , - -. - 40 min, RNA 1 2 sec 1 2 kb. DNA 1000 nt/sec, -.
Okazaki "-" - . DNA- . , DNA,
-
124
3.14 - E. coli. () (). DNA primosome - RNA .
-
125
. . DNA, . , 5' Okazaki. - DNA . - .
. in vitro DNA . - . - . , . Okazaki . , DNA.
, - - Dna DNA - ( 3.15). Dna { - } DNA -. , { - DnaB} DNA - .
, - (replisome) , DnaB -.
-
126
3.15 DnaB.
H ' E. coli
-, , DNA . - .
DNA . DNA - ( ), - , - - SV40.
-
127
, , ( , ) , - E. coli. SV40 - , Okazaki ( - , . coli). , , E. coli, DNA . , , , DNA , .
, PCNA (proliferating cell nuclear antigen) . - PCNA E. coli (RFC), , PCNA DNA. RNA nick translation DNA .
Okazaki,
DNA. , DNA . , . - , , - DNA.
DNA ( ), , , -, . -
-
128
. - . DNA ( - ) DNA -. (decatenation) (catenanes) ( 3.16).
- DNA
, - - -. , DNA 5' 3' -. DNA, 5' , - DNA . - -.
, , - DNA, 5' - DNA. - 3' DNA , 5' . - DNA RNA ( 3.17). - .
-
129
3.16 DNA ( ).
-
130
3.17 . - 3' . - T4G4.
-
131
-
, , . , - S ; : , (licensing), - (Pre-RC) . - G1.
S . pre-RC 4 , . pre-RC ORC. ORC (Cdc6 Cdt1). Mcm ( 3.18). (. ) (Cdk, Ddk). G1 S . pre-RC , .
; pre-RC . : pre-RC pre-RC. G1 pre-RC . S pre-RC
-
132
DNA . G1, . , pre-RC ( G1) , pre-RC, ( S, G2 S ). pre-RC DNA .
3.18 - .
-
133
A (174, G4, M13), DNA (SS, single stranded). DNA, - (RF, replicative form):
: , RF DNA, , RF DNA , "+" "" .
, - ' , RF DNA. , , DNA RF DNA . , ' , - ' . - - , .
G4, - , SSB dnaG. DNA G4 .
+
+
SS RF
-
134
RF DNA G4. RF, . SSB DNA, , ( 3.19).
3.19 DnaB DNA . 13 G4 DNA SSB, - ' . SSB 174 DNA, (: n, n', n'' = - PriA, PriB, PriC i=DnaT) - ' ( ).
174 (-)
. , SSB
-
135
DnaG, 6 E. coli (PriA, PriB, PriC, DnaT, DnaC, DnaB). 6 . (pas, primosome assembly site). pas - PriA, 3' 5' SSB . RNA , , - ATP, , - DNA, ( ) ' - ( 3.19).
DNA DNA , . - . () 174 E. coli E. coli ( Okazaki).
"+" RF DNA - . A . RF DNA ' "+" . - . 5' . ATP, . (+) 174 E. coli. "+" . , , ( -, amplification) rDNA Xenopus. 3.20 174 DNA.
-
136
3.20
174
DN
A.
:
"+"
:
-
4
DNA - .
RNA (transcription).
-
138
DNA RNA -
- DNA . - , , . - , RNA DNA. DNA RNA, . DNA - . - (-) , . DNA , - RNA , DNA. .
- -, . . , - -. - (regulation of gene expression) - . , - RNA , RNA DNA, .
-
139
-, . - 10.000 , 10.000.000. - , - . - DNA - , - DNA RNA .
: () () DNA () . , - - DNA. RNA DNA- RNA (DNA-dependent RNA polymer-ases).
DNA (transcription unit) - .
, . , RNA DNA (promoters). - . - . - RNA.
RNA
RNA .
-
140
1. RNA 5- ATP, GTP, CTP UTP. - , 2 3 - .
2. 3- 5- . - ( 4.1). DNA ( -).
3. RNA DNA. RNA , DNA - (template). C, T, G A DNA, RNA G, A, C U.
4. DNA , .
5. RNA 5 3. 3 -. DNA.
6. RNA , DNA , .
7. 5- RNA RNA - . 3- . 5 RNA .
RNA : (1) RNA DNA -, , (2) , (3) (4) DNA.
-
141
4.1 RNA RNA . 3- - 5- .
- E. coli - DNA- . RNA E. coli RNA-.
RNA . mRNA, rRNA tRNA , RNA - .. RNA, , .. miRNA.
-
142
RNA E. coli RNA E. coli
400.000 daltons. . , . (core enzyme) RNA (core RNA polymerase) - RNA. . - , , , - . , 2 (holoenzyme). RNA .
in vitro RNA , . in vitro RNA in vivo E. coli. , (initiation factor).
RNA . . 329 -. 85 - (-carboxyterminal domain, -CTD) DNA. 1320 . -CTD (. ).
RNA - -
-
143
- RNA. RNA - . . RNA (. . 5). RNA E. coli . - E. coli. - - -. RNA . - - , E.coli - . - . - DNA, RNA ( 4.3)
RNA DNA . (DNase protection method footprinting) DNA RNA 70 bp. "" (dimethylsulfate protection method).
RNA DNA. . :
-
144
RNA DNA, - ' , - DNA RNA. DNA, - .
100 10 35 bp . 35 10 - , DNA RNA +1 - . 10 Pribnow box TATAAT. , . - 35 TTGACA . - . 4.2, - (consensus sequences). RNA "" .
E. coli. (rrn) E. coli RNA - . rrn AT, 20 bp 40 60 bp -, . 35. upstream element UP element. RNA CTD, . .
-
145
4.2 70 RNA E. coli. () . () 35 10, ( - ). () ( ) . -.
"" "" (strong and weak promoters) , RNA . RNA - - 35 10 -.
, -, RNA . , RNA . - 35 10. - 70 - - - RNA -
-
146
, 35. Bacillus subtilis - .
E. coli , 70 : 28, 32, 38 54 ( 4.1). , .. 32 , - .
4.1 E. coli.
. 32 28 70. 70 DNA 10 35 . 38 70 70. - 54 12 24 . - - RNA .
-
-
147
RNA -, DNA , .
RNA E. coli . - "" (closed complex). -35 - . "" (open complex) RNA . 10 AT CG - .
DNA RNA ( 4.4). 11 +3 . - ( 4.3). (NTP uptake channel), RNA (RNA exit channel) RNA DNA . DNA , . DNA , - - . - (nontemplate strand NT channel) -. , (template strand channel T). 11 DNA, .
-
148
4.3 RNA . DNA, , 10 35 - . - DNA RNA.
, RNA
. , . - . ATP GTP ATP . DNA (). - DNA. , , DNA.
-
149
, - . , , RNA DNA . DNA . - RNA , . (abortive initiation), .
in vivo in vitro , -. RNA . .
-. ( 3 ) Pribnow box, .
RNA 10 bp -
, DNA RNA - . - RNA. , . DNA, . (DNA) , . - DNA . , RNA ' -
-
150
. DNA. , DNA, - DNA .
4.4 . () RNA 7580 bp ( 50 +20). () . () RNA .
-
151
RNA DNA . ( 810) ( 4.4). DNA . .
. 3-5 - . RNA DNA , , . 89 RNA DNA. DNA DNA-RNA.
RNA . - , . (pyrophosphorolytic editing) - - PPi. , . . - (hydrolytic editing) - RNA, . Gre , .
5 RNA - , - - . in vitro . -
-
152
- (-32P-NTP) , - -32P-NTP -.
DNA. : RNA RNA , RNA DNA RNA DNA.
: (intrinsic terminators) (termination factors) . - :
1. DNA . - ( 4.5). (hairpin) RNA ( ) - DNA (cruciform structures).
2. G+C ,
3. A+T , 6-8 U RNA ( 4.5). G+C.
. - . , RNA , RNA -
-
153
(hairpin). , (U) (A) , - RNA DNA ( 4.6).
4.5 () DNA - . coli . - . () 3 mRNA -. mRNA (stem and loop structure). U .
, , (-dependent termi-nation).
-
154
4.6 RNA. () (U). (b) RNA-RNA DNA-RNA U DNA. (c) poly(U)-poly(dA) .
-
155
, - . A RNA . 50 kDa - . RNA, P, ATP RNA ATP RNA DNA. RNA, 40 - (rut sites, Rho utilization), C. RNA DNA - . . . -. - .
E. coli . . (antitermination factors).
RNA - DNA . . - .
. RNA . : , , mRNA, . . . 3 DNA , .
-
156
RNA
, - RNA. (primary transcript) RNA. (processing) - . - :
)
) mRNA 3 , CCA 3 tRNA - (capping) mRNA 5 ,
) .
RNA mRNA, rRNA tRNA - . RNA.
) mRNA
mRNA . mRNA , mRNA . - -, mRNA . mRNA 5 3 . mRNA -.
-
157
, mRNA, mRNA. 3 RNA , . , . poly(A) E. coli - mRNA - poly(). - RNA mRNA. - 3 - RNA 3 RNA.
mRNA - (polycistronic) . - . (cistron) DNA - . mRNA -. mRNA 30008000 . mRNA - (untranslated sequences). - (leader seque-nce) . mRNA .
mRNA , - mRNA. 3 7, -, mRNA RN . RN III .
-
158
) tRNA tRNA
(precursor molecules). . 20% RNA , . tRNA .. tRNATyr, .. tRNAGly- tRNAThr E. coli , . tRNA - . 5 3 , . : RNA .
, tRNATyr E. coli, , 4.7. 85 , 41 5 225 3 . tRNA . 1. 3'- .
: , RN F, 3 , RN D, - CCA - 3 tRNA. -tRNA (pre-tRNA) E. coli.
2. 5' . RN P 5 tRNA E. coli. RN P -. RN P RNA . RNA 375 (130.000 daltons) , 20.000 daltons. RNA . RNA , .
-
159
4.7 tRNATyr E. coli
-
160
3. . tRNA (modification 4.7). .
tRNA 3 CCA. tRNA 4.7 3 . tRNA CCA tRNA - .
) rRNA
RNA - RNA (spacer regions). tRNA. RNA tRNA - ( ) 5.000 . 4.8 -, rRNA tRNA. 16S- -23S-5S- , tRNA , , .
. - RN - pre-16S pre-23S RNA. - tRNA pre-5S rRNA. - (secondary processing) 16, 23, 5 16S, 23S 5S . 3- tRNA - RN D. - in vitro.
-
161
rRNA rRNA .
4.8 - rRNA tRNA E. coli. .
rRNA , - , - , .
:
1. (stable) RNA . mRNA , .
2. .
-
162
3. - .
4. A, U, G C - .
- . - .
- , , - . mRNA . mRNA , mRNA (5 3) . - .
- RNA - . . . rRNA - (spacer regions) . .
-
163
-, (operons). - mRNA. . . , , mRNA . - . . - : - . - , -.
. , . - (structural genes). RNA . - . "" (constitutive).
. "" . DNA. - (regulatory genes).
: .
-
164
DNA. - RNA - mRNA. (repressors) DNA (operator). - .
(effector molecules), . (inducers) DNA. - - (lac operon), - .
, DNA RNA - . (activa-tors).
RNA , , . RNA . DNA - . NtrC - 150 bp . DNA , .
-
165
-, . . . , - mRNA DNA.
- , - DNA- -.
(lac operon) E. coli -
Jacob Monod '60. , DNA- - .
Z, Y . Y . lacZ - (-galactosidase) . lac (lactose permease) - . lacA - , .
E. coli --
-
166
. - , 20 . - . - - . . . (coordinately expressed) , . mRNA . -, . .
, , lac repressor , CAP CRP. i, (negative control) - , . . i - ( 4.9).
. (operator) (Plac) . RNA . . . (inducer). - - :
-
167
) , . , . ( ) .
) , RNA - - - .
4.9 , . 6000 bp DNA. i . lac i (Plac) . (Olac) 26 bp lac Z.
. ( 4.10).
-
168
4.10 (a) () (b) ().
, : . , . ,
-
169
, , . .
(positive control) -. E. coli, - . . . E. coli , . -, , . (catabolite) mRNA (catabolite repres-sion).
- . cAMP . , cAMP - . , cAMP -- . - , cAMP . cAMP. catabolite activator protein (CAP) cAMP receptor protein (CRP). cAMP-CAP
-
170
, CAP (CAP site). () RNA ( 4.11). in vitro cAMP-CAP in vivo - . cAMP-CAP -. cAMP-CAP ( ), ( ).
CRP CAP - - (combinatorial control) - .
4.11
-
171
. - , . , -. , . (attenuation). , Trp tRNAs.
, mRNA, - , - .
cAMP-CAP. (a) lac mRNA . (b) , () - . cAMP cAMP-CAP CAP, RNA - lac mRNA. (c) . , cAMP , cAMP-CAP CAP RNA -.
-
172
-
5
, , ,
.
-
174
1. DNA, - - - , DNA , - DNA . .
2. . .
3. RNA - RNA.
4. .
5. (intervening sequences) (introns) - .
6. mRNA : (cap) 5 mRNA (200 ) poly(A) - 3 - mRNA.
7. mRNA .
8. mRNA , . .
-
175
RNA -
RNA - RNA.
RNA - rRNA. (proc-essing) 18S, 28S 5.8S RNA.
RNA mRNA (precursor mRNA, pre-mRNA). - - RNA RNA.
RNA 5S RNA, tRNA RNA (small nuclear RNA, snRNA).
RNA , - . RNA , , , DEAE Sephadex.
RNA - -, , Amanita phalloides. RNA -, RNA RNA -.
RNA . 500.000 daltons - . RNA - 12 15 , . RNA S. cerevisiae . RNA - -.
-
176
RNA - (2) E. coli. - - E. coli. E. coli. RNA , .
RNA , E. coli, - , . , .
RNA - . - RNA - . . (carboxyterminal domain, CTD). RNA 26 - 52. 10 .
- CTD . , - , .
CTD . , .
-
177
RNA - RNA E. coli.
- . - -. - - (5 ) -. upstream sequences ( ) . , - ( ) (downstream sequences) , , , (.., 5S RNA tRNA) , , - , transcription factors- DNA. - RNA , , RNA .
-- -: (1) (general transcrip-tion factors) , - (2) - (regulatory transcription factors) - .
DNA in vitro, RNA . In vivo DNA ,
-
178
, - (mediator complex), .
- - - - in vitro , - , , . - , in vitro, DNA .
, - in vitro -, .
RNA -. in vivo in vitro ( 5.1).
1. , 25 30 bp - TATAAA(T). TATA box Pribnow box - .
2. TATA box "" (initiator) PyPyANT/APyPy , RNA.
3. TFIIB-BRE (TFIIB Response Element).
4. (DPE Downstream Promoter Element).
5. , ,
-
179
75 bp, GG(T/C)CAATCT. CAAT box.
6. GC box, GGGCGG..
- RNA . .
5.1 - RNA . TATA box . .
, , . - (regulatory sequences) . : (promoter proximal elements) (upstream activator sequences) (enhancers) (insulators) (silencers) -
, - (boundary elements).
, (activa-tors) (repressors), -
-
180
. , , .. -.
Pol II (preinitiation complex) . RNA , DNA, . TFIID (transcription factor IID) TATA. [ - ]. O TFD . box (TATA-box binding protein, TBP).
5.2 - TBP DNA, - .
TBP , 30 kD, DNA, (, bending). TBP DNA , , ( 5.2). TBP
-
181
( 180 ) DNA -. TBP - TAFs . TATA binding protein associated factors. TBP - , , . .
in vitro - TFIIA (2 - , 3 ). TFIIA , - . TFIIB - ( 5.3). - RNA - TFIIF -. - , TFIIE TFIIH. O F 2 , TFIIH . TFIIH 9 2 ATP, . - ATP ATP TFIIH - ATP DNA . TFIIH - ATP - (CTD) RNA , . TFIIH - . - DNA. (40) 2 daltons, . 70% .
-
182
5.3 RNA in vitro (- ).
-
183
in vitro RNA - DNA . In vivo DNA . - - , - (mediator complex), - ( 5.4).
5.4 in vivo. -, , - .
- . - DNA - . (CTD) , - DNA .
-
184
. 20 .
(chromatin remodellers) - - DNA.
CCAAT GGGCG 10-15% - . GC box Sp1 (stimu-latory protein 1) CCAAT box , - -, .
rDNA RNA - . - . - (18S, 28S 5.8S) RNA , - DNA , . (species specific).
T (18S, 28S 5.8S) RNA - . 5S RNA .
- . -. , (core promoter, core element) (45 +20)
-
185
5.5 RNA .
. 50-70 bp 180 107 bp - upstream control element, UCE. GC, . RNA - . UBF-upstream binding factor, UCE ( 5.5) - . -
-
186
1 (selectivity factor 1, SL 1) . , RNA .
UBF GC -. SL1 - . - TBP - RNA , SL1. TAFI . TBP (TBP-associated factors) . TFIID-TAFII - RNA .
RNA - ( 5S, tRNA snRNA) . - . - (downstream) DNA . ( 5S RNA tRNA). -- - - . O - RNA (small nuclear RNA, snRNA) - RNA , .. box .
tRNA (internal control regions). tRNA, D TC, A box B box. tRNA , TFIIIC TFIIIB . TFIIIC A box B box ( 5.6). TFIIIB , 50 A box. TFIIIB
-
187
, DNA TFIIIC. O TFIIIB . TBP . - TFIIIB RNA . - ATP. TFIIIB, o TFIIIC - , . - (assembly factor) TFIIIB (initiation factor). - +10 +20 +50 +60 tRNA.
5.6 tRNA RNA .
-
188
5S Xenopus laevis. 45 96. TFIIIB TFIIC TFIIIA. TFIIIA - ( 5.7). O TFIIIA (81 99 bp) C box TFIIIC TFIIIB, TFIIIC, tRNA. TFIII TBP tRNA. . TFIIIA .
RNA (snRNA) , PSE (proximal sequence element) oct (octamer) .
- RNA : RNA . - . - DNA- RNA -. - .
TBP, box , - , - . TBP box. TBP TBP-DNA -
-
189
. TBP, TAF - . TBP RNA -.
5.7 - 5S RNA RNA .
-
190
- . -. DNA - . - , .
, RNA .
RNA : - . , - elongation factors, . , P-TEFb. - hSPT5 TAT-SF1. - TFIIS, . TFIIS , , - . TFIIS - (proofreading) . -, , -
-
191
. TFIIS RN - RNA. RNA -.
. RNA .
rDNA, - , - 1000 bp 3 , 18 - .
RNA . - (U) 4 5 U GC. - .
RNA . , >1000 bp 3 mRNA. 3 mRNA - .
-
. . mRNA . -
-
192
DNA RNA . - .
, - DNA RNA mRNA DNA . - - mRNA . - - , . mRNA DNA . DNA - mRNA. DNA mRNA (intervening sequences) (introns) mRNA (exons). - (split or interrupted genes).
- .. -7700 bp- - ( 5.8). , , - (coding region) 5 3 mRNA . (R-loop) DNA. -. - tRNA rRNA. -
-
193
. - .
5.8 . () - DNA mRNA R-loop. () (). (L, 17) RNA (AG) - DNA-RNA. () (Leader, 17) (AG).
-
194
RNA DNA . . RNA (RNA splicing) - . mRNA - RNA 15S DNA - . . 15S RNA - DNA, DNA RNA -.
mRNA .
RNA
mRNA
mRNA RNA. , mRNA - . RNA (heterogeneous nuclear RNA) . mRNA .
RNA mRNA. : (1) (5 3) (2) RNA (RNA splicing) . , RNA .
-
195
1.
5 (capping) - . - , (G) 7 5-5 -, , mRNA - .
- 5 RNA RNA . -- GTP - RNA - . -, 5 -. "" RNA. 5' - 5' 5' ( 5.9).
(CTD) RNA 5 -.
5 , mRNA - mRNA.
3 mRNA -, . mRNA, mRNA 3 -poly(A)- 20 200 mRNA. , .
-
196
5.9 5 mRNA .
cDNA mRNA AAUAAA 10 35 poly(A). -
-
197
3 . - - GU, U, 50 . - . - , 350 kDa (Cleavage and Polyade-nylation Specificity Factor, CPSF) 3 4 , - - AAUAAA. , - 200 kDa, (Cleavage Stimu-latory Factor, CStF), (Cleavage Factors) CFI CFII, CPSF-RNA ( 5.10). CStF GU U - - . , -- (PAP), -. CPSF CStF, - , CTD - RNA .
, poly(A) : 12 200 250 . - poly(A) poly A (Poly-A Binding Protein II, PAB II), PABI poly(A) . PABII PAP . , , PABII poly(A) 200 250 -.
mRNA. mRNA poly(A) AAUAAA. 3 RNP U7snRNP.
-
198
poly(A) mRNA. - , mRNA poly(A).
5.10 3 mRNA .
-
199
2. . RNA (RNA splicing)
RNA RNA 50% 90% -. mRNA - RNA (RNA splicing). 5 ( ) - RNA, 3 . .
- (excision and splicing) . -. , mRNA, - . . GU 5 AG 3 . GU-AG - : " GU AG". 5 3 (5 and 3splice sites).
(splicing) . -. . lariat branched circle (G) 5 (A) 20 40 - 3 . - (branch point). 2-5 (G) - 3-5 (- 5.11). - ,
-
200
trans- 5.11. (G) 2- . () 3- () 3 (lariat). (transesterification) 3- - 3 - lariat. - , , . - ATP - .
- , . . . , pre-mRNA , mRNA . - .
RNA RNA (small nuclear ribonucleoproteins,snRNPs). RNA (small nuclear RNA, snRNA), U1, U2, U4, U5 U6 100 300 , (U), - RNA. RNA . snRNP, snRNP. (systemic lupus erythema-tosus, SLE) - anti-Sm antibody- U1, U2, U4 U5 snRNP. , - snRNP. -
-
201
. -, in vitro U1 snRNP, , U1snRNP.
5.11 pre-mRNA. trans- - . - .
snRNA, - - : () snRNP, () snRNA pre-mRNA
snRNP . - 5 , - - RNA. - - spli-
-
202
ceosome. 5 snRNA 150 . snRNP. ATP .
- . U1snRNP 5 , pre-mRNA. U2AF - , 3 . BBP-Branch point Binding Protein. , early complex (E). U2snRNP BBP. complex. () 5 ( 5.12).
- U4snRNP, U6snRNP U5snRNP complex. U4 U6snRNP RNA , U5snRNP . snRNP - . U1snRNP 5 U6snRNP pre-mRNA. . U4snRNP U6snRNP U2snRNP RNA-RNA ( 5.13). - C RNA. trans-, 5 3 U5snRNP. mRNA snRNP. snRNP - ,
-
203
. snRNP . 5-2- ( 5.12).
5.12 - (spliceosome) RNA.
-
204
RNA . ( kb) , -. , . -, 5 U1snRNP U6snRNP ( 5.13).
5.13 snRNA pre-mRNA . (a) 5 - U1snRNA U6 snRNA. (b) U2snRNA . (c) U6snRNA U2snRNA. (d) BBP, U2snRNP.
, - ,
-
205
. . RNA . (CTD) , 5 CTD 3 , -, . , . - .
- . SR proteins ( ) (exonic splicing enhancers) - . SR U2AF 3 U1snRNP 5 . SR - , - (alternative splicing).
(pre-mRNA) RNA . RNA , -. RNA - (heterogeneous nuclear ribonucleoproteins, hnRNPs) mRNA, pre-mRNA . pre-mRNA hnRNP - pre-mRNA - . mRNA -.
-
206
, .. , ( ), , ..
mRNA . mRNA .
mRNA - - 6 . . - .
(alternative splicing)
- RNA - mRNA (pre-mRNA) - mRNA . mRNA : . mRNA () (exon skipping), - , - poly(A), mRNA. - . - - , 60%. - - . .
- . SR -
-
207
- .
RNA (RNA Self-splicing)
pre-mRNA. - (group I and II self-splicing introns). - RNA - in vitro RNA .
(group II introns) pre-mRNA, RNA , 5.14b. .
5.14 RNA . pre-mRNA (spliceosome).
-
208
. 3- - (G) RNA 5 . - trans- G 5 . , . 3- 3 . ( 5.14c). , G - (internal guide sequence) 5 . .
400 1000 - , , - . In vivo .
RNA - . - pre-mRNA. RNA - snRNA . . - RNA . - RNA.
-
209
tRNA
tRNA - - tRNA. 5 , RN P , - RN P E. coli.
, , tRNA - 10 30 . - (processing) (splicing) . pre-mRNA. . 1. -
(splicing endonuclease) .
2. ATP. -
.
rRNA
- RNA: 5S, 5.8S, 18S 28S. - . : () - 5S RNA rRNA, () RNA , RNA ( 5.15).
5.15 rRNA Xenopus. (untranscribed spacer).
-
210
rRNA -. 50 . 18S, 5.8S RNA, 28S RNA - (untranscribed spacer). 12000 bp. RNA ( , primary transcript). .
5S RNA - . 600 bp . Xenopus 24000 5S . H .
rRNA (processing) . . . - pre-rRNA , o , pre-RNP. rRNA - RNA (small nucleolar RNA, snoRNA). RNA RNA 87 275 , U3 RNA RNA . snRNA, snoRNA - - (snoRNP). snoRNP - pre-rRNA pre-mRNA . - 5.16.
-
211
5.16 rRNA - (45S) .
-
212
- .
. - . mRNA - .
- - . . . - , . DNA , .
:
1. (transcriptional control) 2. RNA (processing control) 3. mRNA (trans
port control) 4. (translational control) 5. (posttranslational control).
- . . - .
-
213
- . . .. .
(transcription factors) . - DNA - .
- (processing control). - mRNA , - . - , , ( -).
-
214
-
6 tRNA &
DNA
. - , (repli-cation) DNA, , (expression) , : , (trans-cription) DNA mRNA, , (translation) mRNA . ,
-
216
(post-translational modifications), - , . , in vivo , , . mRNA . - RNA (, retroviruses) - ' mRNA: - DNA ( , reverse transcription) , mRNA . , RNA, ' mRNA. , , DNA DNA, DNA RNA, RNA RNA, RNA DNA. - . , mRNA .
tRNA - DNA mRNA -
() , mRNA "" ( ) 3 1, ( 6.1, , 7.1). , 20 , (genetic code). , mRNA , . , RNA , tRNA (transfer-RNA, Crick - '60). tRNA ( 73 95 -
-
217
) : (1) , (2) , (antico-don) (codon) mRNA. - (: o , tRNA -). 6.1 tRNA - . - tRNA .
tRNA -tRNA (aminoacyl-tRNA synthetase) - - 2' 3' tRNA (, 3' ), . tRNA (charged) - tRNA -tRNA aa-tRNA (aminoacyl-tRNA).
mRNA
SH | CH2 | CH O / \ / NH2 C | O
/
ACA |||
UGU
CH3 | CH O / \ / NH2 C | O
/
- ACA
||| UGU
-
218
20 tRNA ( - , . ) . , tRNA tRNA1Tyr tRNA2Tyr. (tRNA) , Tyr-tRNA1 Tyr-tRNA2 .
tRNA
tRNA. - ( , 90% 95% ) . , ( ) , - (R = G A), (Y = C ) ( 6.2). tRNA 73 95 76 .
tRNA ( ) , (clover-leaf structure). ( Watson-Crick) G:C A:T, , G:U, G: A: ( tRNA -, .., = , D = I = , ). , - tRNA. tRNA - (arms stems) (extra arm) (loops). / . - :
(acceptor amino acid arm stem) - 7 8 . 7 tRNA,
-
219
3' , - 5'-CC-3'. CCA ( tRNA) CCA . 3' CC 2'- 3'-, , tRNA.
D (D arm) D (D loop) , (dihydrouridine). D ( 10 13 22 25) - . tRNA tRNA, - 11 24. D 7 11 (in-sertion) (deletion) 17 ( 0, 1 2 ) 20 ( 1, 2 3 ). , tRNAPhe - 16 18 ( 17 17) 17 . 20. D 14, 18, 19 21, A, G, G A, .
(anticodon arm) - (anticodon loop) . 5 - 7 , 3 - . , ( 33). - ( 32 37, ).
TC (TC arm) TC TC , (, pseudouridine). 5 7 , , .
-
220
6.2 tRNA. - , 4 - U, C, A, G, - . - - .
TC (extra arm) - -. , tRNA , 1 (class 1) 3 5 2 (class 2) 13 21 , ( 5) . tRNA 1 2/3 - tRNA .
tRNA (tertiary structure) tRNA
( ). L,
-
221
. tRNA - "L-shaped", "L-".
6.3 tRNA. 1 5 76 3 - CCA.
tRNA
( ) tRNA, . - ' tRNA. , - , tRNA .
() tRNA - . TC , , D . ,
-
222
( , secondary H bonds) (- , tertiary H bonds). ( -) tRNA ' tRNA.
D TC "" (, ) . , - "", -, "". : - mRNA - ( ), - ( ) ).
-
mRNA ( 6.1). DNA - (coding sense strand) DNA U RNA. - (reverse complement) DNA mRNA - , mRNA (. 7.1).
(codons) mRNA - . , .
U ( ), C, A G, 43 = 64 .
-
223
, 20 ( , 20 , 6.1) 20 , , . - (degeneracy) .
, () mRNA 20 , - tRNA. , tRNA , , , ( tRNA) tRNA. , {-} .
6.1 . - .
UUU Phe (F) UCU Ser (S) UAU Tyr (Y) UGU Cys (C)
UUC UCC UAC UGC
UUA Leu (L) UCA UAA Term UGA Term
UUG UCG UAG Term UGG Trp (W)
CUU Leu (L) CCU Pro (P) CAU His (H) CGU Arg (R)
CUC CCC CAC CGC
CUA CCA CAA Gln (Q) CGA
CUG CCG CAG CGG
AUU Ile (I) ACU Thr (T) AAU Asn (N) AGU Ser (S)
AUC ACC AAC AGC
AUA ACA AAA Lys (K) AGA Arg (R)
AUG Met (M) ACG AAG AGG
GUU Val (V) GCU Ala (A) GAU Asp (D) GGU Gly (G)
GUC GCC GAC GGC
GUA GCA GAA Glu (E) GGA
GUG GCG GAG GGG