T A Macromolecular SynthesisMacromolecular Synthesis 1 FIND CHAP TOC O OP O O P CH2 O P γ β α –O Bases Sugars Nucleotides O O –O O –O P O O O– O H2N CH3 NH2 NH2 5 N N 1

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


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


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


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


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


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


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


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Table 1.1


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


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


5′RNA primer

3′3′

5′ 3′5′

Okazaki fragment

DNA Pol III

Figure 1.10


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


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


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


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


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


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


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


A

fL

fL

fR

fR

terA terA

terB terB

BoriC

oriC

oriCoriC

Figure 1.18


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


oriC

DNA Pol

FtsZ-ring

SupercoilingCondensins

oriC

Par Par

Figure 1.19


A

B

I (70 min)

I

Initiationat oriC

Initiationat oriC

Chromosomesare complete


Initiationat oriC


Initiationat oriC


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


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


Figure 1.22


Figure 1.23


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


Type I:one strandbrokenDNA with

twosupercoils

One strandpassed throughand resealed

Both strandspassed througheach other andresealed

Type II:both strandsbroken

Figure 1.25


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Table 1.2


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Table 1.3


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


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


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


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


Step 1 DNA isolation and digestion

Step 2 DNA electrophoresis

Step 3 DNA transfer

Step 4 DNA hybridrization

A

Figure 1.30


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.


A B C D E F G a b c d e f g

Figure 1.31


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


Cut byrestrictionendonuclease

Not arestriction site

RestrictionsiteAmpr

Cloned gene

Not cut byrestrictionendonuclease

Mutation

Figure 1.32


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


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

Documents

T A Macromolecular SynthesisMacromolecular Synthesis 1 FIND CHAP TOC O OP O O P CH2 O P γ β α –O Bases Sugars Nucleotides O O –O O –O P O O O– O H2N CH3 NH2 NH2 5 N N 1