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Macromolecular Synthesis1 FIND CHAP TOC
1 he
lical
tur
n =
3.4
nm
Hydrogenbond
A
G
T A
T A
CG
C G
T A
TA
C G
G
C
T
A T
Sugar-phosphatebackbone
Base
A T
T
3′ 5′
A
C
5′3′T A
Figure 1.1
Macromolecular Synthesis1 FIND CHAP TOC
PO O
O
CH2PO
O
P
γ β α
–O
Bases
Sugars
Nucleotides
O
O– O– O–
OP–O
O
O–
O
H2N
CH3
NH2
NH2
5 N
N
1
2 4
3
76
5
2
4
6
8
9
N
NH
N
O
O NH
HN
N
N N
N
NH
N
NH
HN
O
NH2
GuanineAdenine
NH2
CytosinePyrimidine Uracil Thymine
Purine
CH2OH5′
3′
1′
2′
4′
1′
O
OH
CH2OHOHOH O
OH OH2-Deoxyribose Ribose
CH2OH
1′O
3′ dCMP
3N
1
O
O
O
NH
HN
O NH
N
N
OH
N
N
O
5′ dGTP
N
NH
P O
O
CH2–O
O–
O
NH2
OH
N
N
O
5′ dGMP
N
NH
9N1
Figure 1.2
Macromolecular Synthesis1 FIND CHAP TOC
O
O–
O
OCH2
A
5′ 5′
P
P
P
T
5′
3′ 3′ 3′P
PhosphodiesterbondDeoxyribose
Base
OH
C
O
O
O–
O
OCH2P5′
4′ 3′2′
1′
4′ 3′2′
1′
O
O–
O
OCH2P5′5′
Base
O
Base
4′ 3′2′
1′O
OH
G
5′ 5′
PP
T
5′
3′ 3′ 3′P
A
5′ 5′
5′ end 3′ endPP
C
3′
5′3′3′5′
3′OH
OH
C
3′ end 5′ end
5′ 5′
PP
5′
3′ 3′ 3′P P
5′ 5′
P3′ 3′
C AT G G
A
B
Figure 1.3
Macromolecular Synthesis1 FIND CHAP TOC
CH3
Deoxyribose
Deoxyribose
H H
H
H
CytosineGuanine
N
N
CC C
H
C
C
O
H
N C
C
C N
N H
N
N
N H
Deoxyribose
Deoxyribose
H H
H
H
ThymineAdenine
N
N
CC C
H
C
C
N
N C C
C
C N
O
H
N
N
O
C
O
Figure 1.4
Macromolecular Synthesis1 FIND CHAP TOC
GDP dGDPRibonucleotide
reductase
dGTPKinase
CDP dCDP dCTP
ADP dADP dATP
dUTPUDP dUDP
dUMPTHF
Phosphatase
dTMPDHF
Dihydrofolatereductase
Thymidylate synthetase
dTTP
Kinase
THF
DHF
Figure 1.5
Macromolecular Synthesis1 FIND CHAP TOC
O–
O– O
OCH2
P
Base
O
O
O–
O
OCH2P5′
4′ 3′2′
1′
4′ 3′2′
1′
O
O–
O
OCH2P5′5′
Base
O
O
P
O
Base
4′ 3′2′
1′O
OH
HHH
α
β γO
P OO
O
O
P
α
β γO
P OO
Figure 1.6
Macromolecular Synthesis1 FIND CHAP TOC
G
5′ 5′
PP
P
P
T
3′ 3′
A
5′ 5′
PP
C
3′
5′3′3′5′
3′OHOH
OH
Template
Primer
PP
5′
3′P P
5′
P3′
C AT G G
5′
3′
5′
3′
5′
3′
A
B
Figure 1.7
Macromolecular Synthesis1 FIND CHAP TOC
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Table 1.1
Macromolecular Synthesis1 FIND CHAP TOC
Generation I:cells grownin 15N
Replication After centrifugation
Heavy15N – 15N
15N – 15N
Generation II:cells grownin 14N
Generation III:cells grownin 14N
and Hybrid density15N – 14N
15N – 14N 15N – 14N
and
15N – 14N 14N – 14N 15N – 14N 14N – 14N
Hybrid15N – 14N
Light14N – 14N
and
Figure 1.8
Macromolecular Synthesis1 FIND CHAP TOC
DNA Pol III
Primase
Primase
Primase
Leading strand
Lagging strand
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′
5′
3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
5′3′
3′A
Pol III
Pol III
B
Pol III
Pol III
Pol I
Okazaki fragment
C
Pol III
D
Pol III
E
Ligase
Pol III
F
Figure 1.9
Macromolecular Synthesis1 FIND CHAP TOC
5′RNA primer
3′3′
5′ 3′5′
Okazaki fragment
DNA Pol III
Figure 1.10
Macromolecular Synthesis1 FIND CHAP TOC
GA
5′ 5′PP
Nick
C
OH
5′ 5′
PP
5′
P
5′ 5′
P
C AT G G
5′
P
5′
P P
A A
5′
PP
U UU
5′ 5′
P
C
OH
5′ 5′
PP
3′ end
3′ end
5′ end
3′ end
5′ end
3′ end
5′ end
5′ end
3′ end
5′ end
3′ end
5′ end
5′→3′ exonucleaseremoves CMP (or dCMP) at nick
A
GA
5′
P
OH
5′ 5′
PP
5′
P
5′ 5′
P
C AT G G
5′
P
5′
P P
A A
5′
PP
U UU
5′ 5′
P
C
OH
5′ 5′
PP
Polymerase activityadds dCMP ontofree 3′ OH
B
A
Nick translation
5′ 5′
PP
C
OH
5′ 5′
PP
5'
P
5′ 5′
P
C AT G G
5′
P
5′
P P
A A
P
U UU
5′ 5′
P
C
OH
5′ 5′
PP
5′→3′ exonucleaseremoves GMP (or dGMP) at nick
C
P
P
P
C
OH
OH
OH
POH
C
POH
G
P
P
P
G
OH
OH
Figure 1.11
Macromolecular Synthesis1 FIND CHAP TOC
5′A T T ACA T
DnaB helicaseA
GTCGTC DNA Pol III
14
4
2
3DnaG primase
3′
B
GTC
GTC
Pol I
SSB
5′
5′
5′ 3′5′
3′
5′
5′A T T ACA T
GTC
2
3
4DnaG primase
3′ GTC
GTC
SSB
5′
5′
5′A T T ACA T
GTC GTCGTC DNA Pol III
2 1
33′
C
3′5′
SSB
5′
5′5′3′
G TC
5′A T T ACA T
GTC GTC
GTC
DNA Pol III
2
3
3′
D
3′5′
3′5′
5′
5′5′3′
5′3′
GTC
GTC
DNA Pol III5′
DnaB helicase
DnaB helicase
DnaB helicase
Figure 1.12
Macromolecular Synthesis1 FIND CHAP TOC
G
G
T
A B
G
A
T
G
A
C
T
A
T
C
C
A
G
A
T
G
C
T
A
C
T
G
A
T
G
A
G
G
T
C
T
A
T
T
C
C
A
D
G
A
T
G
A
G
G
T
Wild type Mutant
C
T
A
C
T
C
C
A
G
A
T
A
A
G
G
T
C
T
A
T
T
C
C
A
G
G
T
C
G
A
T
G
A
C
T
A
C
C
C
A
G
A
T
A
C
T
A
T
T
Figure 1.13
Macromolecular Synthesis1 FIND CHAP TOC
G
P
GA
PP
C
OH
OH PP PP
AT G G
OH
A
C
P
G
P
A
PP
C
OH PP PP
AT G G
B
C
G
OHP
G
P
A
PP
C
OH PP P PP
AT G G
T
OHP
C
C
3′
5′
3′
5′
3′
5′ 3′3′
5′P 5′P 5′
Figure 1.14
Macromolecular Synthesis1 FIND CHAP TOC
CTAm
G
GmA
TC
A BC
TA m
G
G
GmAT
C
C
GA
TC
T
CT
AG
G
CTAm
G
G
CTAm
G
GATC
GATC
3′ 5′ 3′ 5′
5′ 3′5′ 3′
C
CTAm
G
G
CTAm
G
GmA
TC
C
GmA
TC
3′ 5′
5′ 3′
Figure 1.15
Macromolecular Synthesis1 FIND CHAP TOC
AT rich
DnaA box
AT-rich 13-mer
5′
DnaArecognition sites
T T A T C C A C A 3′
5′ G A T C T NT TNT T T T 3′
Figure 1.16
Macromolecular Synthesis1 FIND CHAP TOC
oriCA
B
C
D
DnaA binds toDnaA boxes ( ) andopens helix at 13-mers ( )
E
Primasesynthesizesprimers
DnaG primase
5′
5′
DnaC loadsDnaB helicase
DnaB
SSB
Figure 1.17
Macromolecular Synthesis1 FIND CHAP TOC
A
fL
fL
fR
fR
terA terA
terB terB
BoriC
oriC
oriCoriC
Figure 1.18
Macromolecular Synthesis1 FIND CHAP TOC
3′ 3′
3′
oriC-dependent replication
DNA lesion
Stalledreplicationforks
D-loop formation
andPri proteins assemble the replisome, using 3′ OH as primer
Recombination functions process the DNA structure
Replication continues
Gap created Double-strandbreak created
DNA nick
Box 1.2
Macromolecular Synthesis1 FIND CHAP TOC
oriC
DNA Pol
FtsZ-ring
SupercoilingCondensins
oriC
Par Par
Figure 1.19
Macromolecular Synthesis1 FIND CHAP TOC
A
B
I (70 min)
I
Initiationat oriC
Initiationat oriC
Chromosomesare complete
Chromosomesare complete
Initiationat oriC
Chromosomesare complete
Initiationat oriC
Chromosomesare complete
C (40 min) D (20 min)
C (40 min) D (20 min)
I (30 min)
C (40 min) D (20 min)
C (40 min)
I
D
Figure 1.20
Macromolecular Synthesis1 FIND CHAP TOC
Membrane
C
Initiation
Membrane
A*T G
G A*T C
C A T G
G A*T C
C A*T G
G A T C
SeqA
SeqA
Figure 1.21
Macromolecular Synthesis1 FIND CHAP TOC
Figure 1.22
Macromolecular Synthesis1 FIND CHAP TOC
Figure 1.23
Macromolecular Synthesis1 FIND CHAP TOC
A
Double helix
Relaxed DNA
under- or
overwinding
Linear DNA
Nick
Linear DNA withends constrained
Protein
“Nicked” circular DNA Circular DNA
Supercoil
Supercoiled DNA
B
C
Figure 1.24
Macromolecular Synthesis1 FIND CHAP TOC
Type I:one strandbrokenDNA with
twosupercoils
One strandpassed throughand resealed
Both strandspassed througheach other andresealed
Type II:both strandsbroken
Figure 1.25
Macromolecular Synthesis1 FIND CHAP TOC
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Table 1.2
Macromolecular Synthesis1 FIND CHAP TOC
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Table 1.3
Macromolecular Synthesis1 FIND CHAP TOC
3′5′
3′ 5′5′ 3′
GG A T C C
C C T A GG
GGA T C C
G GA T C C
C C T A GG
Digest with BamHI
3′ 5′5′ 3′
G GA T C C
C C T A GG
GG A T C C
C C T A GG
Anne
al
C CT A G G
3′5′etc.
etc. etc.
G A T C C GG A T C C
CC T A G G
GG
Ligate
A T C C
CC
etc.
T A G GG
G
5′ 3′
A T C C
G
GG A T C C
C C T A GG
GG A T C C
C C T A GG
Figure 1.26
Macromolecular Synthesis1 FIND CHAP TOC
GG A T C CC C T A GG
G A T CC
GC T A G
G A T CC T A G
CG
G A T CC T A
Mix and ligate
G
Sau3A
BamHI
G A T CC T A G
Sau3A
ori
ori
Figure 1.27
Macromolecular Synthesis1 FIND CHAP TOC
1 2 3 4 5kbp bp
23.0
2,2601,860
1,475
1,180
770755
800
280
9.46.6
4.4
2.32.1
1.5
0.6
0.1
6 7 8
Figure 1.28
Macromolecular Synthesis1 FIND CHAP TOC
6.6 kbA
B
4.1
PstI HindIII
2.62.3HindIII PstI
1.4HindIII
1.2
1.4
1.2HindIII
HindIII HindIIIPstI PstI
1.1
1.4
2.3PstI2.3
1.7PstI1.7
1.2
0.6
1.1 0.6 2.3 1.2 1.4
Figure 1.29
Macromolecular Synthesis1 FIND CHAP TOC
Step 1 DNA isolation and digestion
Step 2 DNA electrophoresis
Step 3 DNA transfer
Step 4 DNA hybridrization
A
Figure 1.30
Macromolecular Synthesis1 FIND CHAP TOC
1 Overlay
Circle of membrane filter
Save plate
Agar petri plate with colonies or plaques
2 Remove membrane
3 Denature DNA on membrane
4 Add probe Add probe
5 Wash off unbound probe
6 Develop film
7 Compare with platefrom step 2
Expose X-ray film
Hybridization bag
Film
Signal
DNA on membrane
B
Figure 1.30 cont.
Macromolecular Synthesis1 FIND CHAP TOC
A B C D E F G a b c d e f g
Figure 1.31
Macromolecular Synthesis1 FIND CHAP TOC
Flowchart for Genomic Sequencing
1 Isolate genomic DNA • DNA should be in pieces of >20 kb
2 Shear DNA • DNA fragments will be of random lengths
3 Size fractionate DNA • Collect fragments in size range from 1.5 to 2 kb
4 Construct plasmid library • Inserts are the 1.5- to 2-kb genomic DNA
5 Randomly sequence inserts • Sequencing process is highly automated
• Need ~15,000 sequence runs of ~500 to 600 bases each per megabase of genome DNA
6 Assemble randomly generated sequence information in contiguous segments (“contigs”)
7 Close gaps with directed sequencing • Several hundreds of reactions needed
8 Analyze sequence • Bioinformatics allows “annotation” of ORFs, etc.
Size Range of Genomes
Organism Size (Mbp) Comments
Mycoplasma genitalium 0.58 Smallest cell genome
Treponema pallidum 1.14 Causes syphilis
Helicobacter pylori 1.67 Causes duodenal ulcers
Sulfolobus solfataricus 2.25 Found in Yellowstone hot springs
Bacillus subtilis 4.20 Soil bacterium; “model” for development
Escherichia coli 4.64 Intestinal bacterium; “model” for genetics and physiology
Pseudomonas aeruginosa 6.26 Causes respiratory infections
Streptomyces coelicolor 8.4 Antibiotic-producing soil bacterium
Box 1.4
Macromolecular Synthesis1 FIND CHAP TOC
Cut byrestrictionendonuclease
Not arestriction site
RestrictionsiteAmpr
Cloned gene
Not cut byrestrictionendonuclease
Mutation
Figure 1.32
Macromolecular Synthesis1 FIND CHAP TOC
3′5′
5′
3′ 5′
5′ 3′
3′ 5′
5' 3'
3′ 5′
3′
5′ 3′
5′ 3′5′
Cycle 1
Cycle 3
Step A. Denature templateStep B. Anneal primers
Cycle 2 Step AStep B
Step C. Polymerize
5′ 3′5′
5′ 3′5′
5′
3′ 5′
5′ 3′
Step C
5′3′
5′
5′3′
5′3′
5′3′
5′3′
3′ 5′5′
5′
5′
Step AStep BStep C
Cycle 4–30Step AStep BStep C
3′ 5′5′
5′3′
3′
5′ 3′3′ 5′
5′ 3′
5′ 3′
5′3′5′ 3′
5′ 3′
5′3′
5′ 3′
5′ 3′3′ 5′
5′ 3′3′ 5′
5′ 3′
Figure 1.33
Macromolecular Synthesis1 FIND CHAP TOC
5′3′
3′5′
T A C
A T G
A T C
3′
3′
5′T A C
AAT T G C G A T A G
CCG5′
GGA T C C
A T C
T A G
5′
3'
3′A T G
CAT A T C A T C T A
TCC
5′
A GGT A
T A G
Figure 1.34
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