macromolecular synthesis · macromolecular synthesis 2 find chap toc o h o n o o 5′exon 5′ g...

Macromolecular Synthesis2 CHAP TOCFIND

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Table 2.1


4′ 3′2′

1′

4′ 3′2′

1′

O

OH OH

Base

CH25

1423

O–

Phosphates

NTP

O

P

O–

P

O

OO

O–

β αγ

P O–O

Ribose

A

C

B

Purines

NH2

NH

Adenine

NN

N

NH

N

N

Guanine

NNH

NH2

O OPyrimidines

NH2

Cytosine

N

ONH

Uracil

NH

ONH

O–

O– O

OCH2

P

Base

O

O–

O

OCH2P5′

4′ 3′2′

1′

O

O–

O

OCH2P5′5′

Base

O

O

P

O

Base

OH

OHOHOH

α

β γO

P OO

O

O

P

α

β γO

P OO

O O

Figure 2.1


5′ A C U C A UU AC G

(G U)G CC

CG

A Hairpin

AC

GA U

A U

U

C C G G C 3′

5′

3′

A C U C A UU AC G

(G U)G CC

CG

G

B Pseudoknot

AC

GA U

A U

UAU C A C

CG

UU

GCC

Figure 2.2


O

HO

N

O

O

5′ exon

5′

G

Intron

Intron

Lariat

3′G

GOHGroup I

A RNA Introns B Protein inteins

exon

3′GAG

exon

3′exon

5′mRNA ineukaryotes

Final product

exon

5′ exon

5′ exon

N-exteinSer Intein

1 2

2

2

2

1

1

exon

3′

GAOH

exon

Group II

3′GG AOHexon

OC

O

HO

C-exteinSerAsn

N O shift

O N shift

Ser

Ser-Intein

Intein

OH

SerAsn

TransesterificationIntermediate

Branched protein

H2N

H2N

NH2

OC

O

NH

C

O HO

Ser

Ser

Spliced protein

AsnNH

O

O

Ser-Intein+

O

O

Asn

NH

Ser

Box 2.2


5′

3′

Amino acid arm

Anticodonarm

Anticodon5′ 3′

A

A

ρ

ΨGG

T

C

CG

G

Pu

Pu

Py

U

ACCPu

Figure 2.3


β + β′

Active site

Downstream DNA

RNA Pol core Promoter complexwith RNA Pol holoenzyme

Elongation complex

RNA

Secondarychannel

α + ω

σσ

Figure 2.4


B

3′

5′

3′

5′

3′

5′

3′

5′

3′

5′

G

P

P

P

PP

A

5′

3′OHOH

OH PP P PP

C GA T T

P

5′

3′

A

5′ end 3′ end

3′ end 5′ end

Template

RNA

5′ 3′3′ 5′

5′

Figure 2.5


–35region

–35 –10 +1

–10region

Transcribedregion

Startsite

Spacer SpacerPromoter region

Consensussequences

N17 N5–9

A

C

B

5′DNA

– – – T TGACANNNNNNNNNNNNNNNNN NNNNNNN NNNNN(A/G)

NNNN(A/G)

T A T TA A

T TGACA A/GT A T TA A

– – – A AC TGTNNNNNNNNNNNNNNNNN NNNNNNA NNNNN(T/C)A T A AT T3′

5'

RNA

Figure 2.6


5′DNA

A

RNA

A

T

A

T

A

U

T

A

U

A

T

A

C

G

C

G

C

G

A

T

A

C

G

C

C

G

C

T

A

U

A

T

A

C

G

C

G

C

G

C

G

C

A

T

A

T

A

U5′3′

5′B

5′3′

3′5′

3′

3′5′

Coding strand

Template strand

DNA

RNA

Figure 2.7


5′RNA Pol

RNA Pol

–35

SigmaPuNTP

–10 +1

3′

A

5′3′

D

5′

–35 –10Sigma

p-p-p-Pu

+1 +10–50

RNA Pol

+30

3′

B

C

5′3′

+10

Transcription bubble

Terminator

–10

5′ ppp

NTPs

PPi

+30

Sigma

5′ ppp

OH 3′

A—G

Figure 2.8


RNA Pol

DNA

RNA 3′ end

B

A

5′3′

5′DNA

CGCA T T TGCC T CCGGT AGGAG

UUUUCG UOH 3′

GC T T T TGAC T3′ GCG

A

Dissociation ofnascent RNA

G GAC TTG

Hairpinloop

A

C

GU C U

UU

U

A

AA

A

C

T T T T G

T A A ACGGAInverted repeat

GC rich

RNA Pol

GGCCA T CC T CCGA A A AC TGA

5′

3′

Run of A’s

5′

G UG AC GC GU AC GC GG CAUUUC

CCCU

–T

UA

GGC GU AC GC

G AG

G U

Figure 2.9


RNA Pol

RNA Pol

Block to translation

rutRho (ρ)termination site

Dissociation

RNA Pol reaches pause(termination) siteATP

ADP + Pi

Termination site

RNA Pol

Rho

Rhorut

NH2

rut

NH2

5′

5′

5′

3′

Figure 2.10


5′16S tRNA

Spacers

Nucleaseprocessing

23S 5S

5′ 3′ 5′3′ 5′3′ 5′3′16S tRNA 23S 5S

3′

Figure 2.11


H

H

R

O

OH

H2O Peptide bond formation

CN CH

H

H

C

O

OH

R

N CH

H

H

R

O

CN CH

H

H

C

O

OH

R

N C

Figure 2.12


Primary structure

Secondary structures

Amino acids

Amino acids

Hydrogen bonds

α-helix

Tertiary structure

Quaternary structure

N

N

CC

C

N

β-sheet

α-helices

β-sheet

Drawn in “ribbon” form

Antiparallelβ-sheet

Figure 2.13


AG C AU

Tag codons

A

5′

5′

5′

tmRNA enters A site

tmRNA UUA terminates translation

tRNA

3′P A

C

GG

C

G

C

Ala

3′5′

5′

P A

C

GG

C

G

C

AG C AG C AU A

5′

Ala

5′

5′ 3′

5′

P

G

mRNA

Ribosome shiftsto translatingTag condons

Tag

ClPprotease

G G

AG C AG C AU A

AlaAla

A

P

N CAU A AU AA

Ala Ala

aa5′5′

Release factor

Box 2.3


70S

50S

Proteins

23S(2.9 kb)

5S(0.12 kb)

16S(1.54 kb)

Subunits 30S

31 (L1 – L31) 21 (S1 – S21)

rRNA +

Figure 2.14


5′

3′

3′

Amino acid + tRNA + ATP

Amino acid

Adenine

Aminoacyl-tRNA+ AMP + PPi

Aminoacyl-tRNA

synthetase

CH2+NH3

O

O

P

O

H

OO–

C CO

O

R

Figure 2.15


5′3′

5′ 3′

U

CA

A

U

G

Anticodon

Codon

Figure 2.16


5′ 3′P A

A

AU

A

U

U

A

AU

A

U

U

A

AU

A

U

U

A

AU

A

U

U

MetEF-Tu

CA U

AC C AC C

GTP

GTP

GDP

GTP

+ GDP + Pi

A A UEF-Tu

GTP

GTP

GDP

EF-Tu

UA G

Ile

GG

U

Peptide bond formationB

A

Ts

Ts

Tu

CG

G

EF-Tu

5′ 3′

5′

P A

U

GA

A

U

C

E

G

AC

G

C

U

Met

AU U AU UCA U

tRNA

EF-Ts

Leu

5′ 3′P

U

GA

A

U

C

E

G

AC

G

C

U

Met

CA UA

A

AU

A

U

U

Leu

5′ 3′P

U

GA

A

U

C

E

G

AC

G

C

U

CA UA

A

AU

A

U

U

LeuMet

Leu

E

U

GA

A

U

C

Figure 2.17


Acceptorstem

5′

3′

B

A

30S subunit 50S subunit

P E E P A

tRNAA

anticodon

Figure 2.18


5′3′ 5′

mRNA

A

3′A

U

G

tRNA anticodon loop

C

G

CorU

5′3′ 5′

mRNA

B

3′G

C

U

tRNA anticodon loop

A

U

AorC

5′3′ 5′

mRNA

C

3′A

U

G

tRNA anticodon loop

C

I

AorCorU

Figure 2.19


5′ NNNNNAGGAGG AUGU – – –– 3′––N5–10

CCUCCAUU

A3′ 5′

3′ end of16S rRNA

mRNAShine-Dalgarnosequence

Initiationcodon

Figure 2.20


P

IF2 + GDP+Pi

30S subunit+

IF3

tRNA fMet

+IF2-GTP

50S subunit

70S initiationcomplex

5′ 3′

5′

P A

U

GA

A

U

C

fMet

5′ 3′

U

GA

A

U

C

fMet

IF2

IF3

GTP

P5′ 3′GA U

IF3

S-D

5′ 3′ mRNAGA US-D

Figure 2.21


(CH2)2

C

CH3

NH2

S

COOHH

Methionine

N

(CH2)2

C

CH3

S

COOH

Formylgroup

H

H

N-Formylmethionine

HC

O

Figure 2.22


Peptidyltransferase

C

C

O

O

O C

NH

O

HR

C

C

NH

H

fMet

Leu

R

H

P site A site

C

C

O

O

NH2

HRLeu

P site A site

O C

O

C

C

NH

O

H

fMet

R

H

Figure 2.23


(CH2)

CH3

Formylgroup

S

N

A

A

Formylgroup

C C

O

O CH

H

R3

CHNH

C

O

CH

R2

C

(CH2)2

CH3

S

H2N C

O

CH NH

C

R2

NH

O

O

B

N-FormylmethionineB

CH NH

H2N C

R3

C

R2

Figure 2.24


Releasefactor

5′ 3′P A

5′ 3′

X

XX

X

X GU A

X

Dissociation

Polypeptide

30S

50S

Figure 2.25


5′

Initiationcodon

TIR TIR

Leader Intercistronicspacer, –1 to40 bases

A

5′ 3′

Stopcodon S-DS-D

Initiationcodon

B

Stopcodon

3′

Figure 2.26


Ribosome

Initiationcodon

Initiationcodon

A

B

A

U C G U G A C U A C G A U G

U G U G

U A

G U

A C

C A U C C

U U G C A U C CA U

A U

C G

A U

U A

C G

G C

Figure 2.27


PheG

lnRibosom

e

rutA A A G T C

ρ terminationsiteGene Y Gene Z

5′RNA U U U C A G

pYZ

rutrut

ρ terminationsite

TIRGene Y Gene Z

TIR

TIR TIR

RNA Pol

NH2NH2

NH2

A

B

5′

rutpYZRNA Pol

RNA Pol dissociated by Rho (ρ)

Rho

rut

Ph

e

U U U U A G

U U U U A G

Polypeptide fragment of gene Y

mRNA fragment

Ribosome dissociates

ρ siteTIRGene Y Gene Z

Not transcribed

TIR

NH2

C

5′

rut

rut

A A A A T CpYZ

Ph e

A A A A T C

Figure 2.28


ATP

ADP+

GroES

GroES+

ATP

GroELmultimers

Unfoldedprotein

Foldedprotein

Figure 2.29


N

D

Innermembrane

Outermembraneif a gram-negativebacterium

Periplasm

Cytoplasm

C NC N C NC

Translocase

Lep cleavessignal peptide

SecYEGcomplex

Presecretory protein

Signal sequence

F

A

N

Innermembrane

Outermembrane

Periplasm C NC C

Cotranslationaltranslocation

N

N C

NC

B

SecB

SecB

Sec A

Cytoplasm

ATP Sec A ADP

Pi

N

Innermembrane

SRP

Outermembrane

Periplasm C NC NC

C

SRP

FtsY

N

D

Innermembrane

Outermembraneif a gram-negativebacterium

Periplasm

Cytoplasm

C NC N C NC

Translocase

Lep cleavessignal peptide

SecYEGcomplex

Presecretory protein

Signal sequence

F

A

N

Innermembrane

Outermembrane

Periplasm C NC C

Cotranslationaltranslocation

N

N C

NC

B

SecB

SecB

Sec A

Cytoplasm

ATP Sec A ADP

Pi

N

Innermembrane

SRP

Outermembrane

Periplasm C NC NC

C

SRP

FtsY

Figure 2.30


YscD,FYscl-LYscO-ULcrD

VirGPP

TolC

HlyB

HlyD

ADP

C

ATP

IM

CP

OM PulD

Type IIType I Type III

YscC

LspA

SecA

SecB

S

Syc

SecD-F,Y

ADPATP YscN

ADPATP

PulCPulE-O

NN

Box 2.6


Outermembrane

Exchange reaction

Innermembrane

Periplasm

Exported protein

Periplasm

N Cys Cys CGly Leu

S S

Cys

SH

Cys

SHC

SH

DsbA

Exportedprotein

N Cys

SH

Cys CGly Leu DsbA

Outermembrane

Innermembrane

N

CysS S

CysC

N

Figure 2.31


Promoter

Activatorsite

PolycistronicmRNA

Operator

A B C

Negativeregulation

A

B

mRNA

OFF

Positive regulation

ON

ActivatorRNA Pol

Activator sites

80

60

40

% o

f pro

mot

ers

touc

hed

20

0–80 –60 –40 –20

Base pairs coordinate0 20 40

Operators

Repressor

Figure 2.32


TerminatorPromoter

3′5′Coding strand

Transcribed strand

Reading frames

mRNA 5′

1

5′3′

3′

Stop

X X X

5′

NH2 COOH

A U G UAAS-D

23

4

56

Stop

Stop

StopX A A U X G A U X X X G A U X A A U X X X X X X

X U AStop Stop

3′ Sequencereadssame incodingstrand

A X X X X X X X X U A

ORF

G X X X X X X U G A X X X

Stop

Figure 2.33


5′ 3′ mRNA

+1+1

+1

pORFA lacZS-D

N N CLacZ protein

orfA

Fusion jointTranscriptionalfusion:

A

Promoter andtranscription site: orfA geneTIR : lacZ gene

Protein : LacZ

(c)OrfA

ATG

5′ 3′ mRNA

+1

pORFA ′lacZ

N COrfA′ - ′LacZ fusion protein

orfA′

Fusion jointTranslationalfusion:

B

Promoter andtranscription site: orfA geneTIR : orfA gene

Protein : OrfA′ - ′LacZ

S-D

ATG

Figure 2.34


pET-15b5,708 bp

CCCGCGAAATAGATCTCGAT TAATACGACTCACTATAGGG CGGATAACAAGAAT TGTGAG T TCCCCTCTAGAAATAAT T T TGT T TAACT TTAAGAAGGAG

RBSXbaIBglII lac operator T7 promoter

His Tag

A

GCAGCAGCCATATACCATGG TCATCATCATCATCACAGCA GCCGCGCGGCGCGGCCTGGT AGCCATATGCTCGAGGATCCGGCTGCTAACAAAGCCCGAl y S e r S e r H iMe t G s H i s H i s H i s H i s H i s S e r S l P r oA r gG l ye r G l y L e uV a S e r H i sMe t L e uG l uA s pP r oA l a A l a A s n L y s A l a A r g

AGT TGGCTGCAAGGAAGCTG TGCCACCGCTGAGCAATAAC CCT TGGGGCTAGCATAACC CTCTAAACGGGTCT TGAGGGGT T T T T TGl uA l aG l u L eL y s G uA l a A l a A l a Th r A l aG l uG l n E nd

NcoI NdeI XhoI BamHI

Bpu1102I Thrombin T7 terminator

EcoRI 5706Bpu1102 I 267pET-15b sequence landmarks

T7 promoterT7 transcription startHis Tag coding sequenceMultiple cloning sites (NdeI - BamHI)T7 terminatorpBR322 originbla coding sequence

453–469452362–380

319–335213–25938824643–5500

NcoI 389XbaI 428 BglII 494

SphI 691SgrAI 535

EcoNI 751

DrdII 939

MluI 1216BclI 1230

BstEII 1397BmgI 1425ApaI 1427

BssHII 1627

HpaI 1722

BsaXI 1875

PshAI 2061

EagI 2284NruI 2319BspMI 2399

BsmI 2704MscI 2791

Bpu10I 2926

Tth111I 3565BsaAI 3572

AccI 3590Bst1107I 3591

SapI 3704

BspLU11I 3820

AlwNI 4236

ori 3882

Eam1105I 4713BsaI 4774

PstI 4958

PvuI 5083ScaI 5193

SspI 5517AatII 5635

BamHI 319XhoI 324NdeI 331

ClaI 24HindIII 29

AP 4643–5500

lacI 866–1945

Figure 2.35


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Table 2.2


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Table 2.3

macromolecular synthesis · macromolecular synthesis 2 find chap toc o h o n o o 5′exon 5′ g...

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