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DNA synthesis & Gene Expression

..
Room: 7338, sthanaset@kku.ac.th, http://www.champa.kku.ac.th/thanaset/

:
1. . .
2. :
2540.
2. Lehninger AL, Nelson DL and Cox MM. Principles of Biochemistry.
2nd edition. Irving Place, New York: Worth publishers, 1993.
3. Lewin B. Gene VII. New York; Oxford University Press Inc., 2000.
4. Mathews CK, Van Holde KE, and Ahern KG. Biochemistry. 3rd
edition. Sanfrancisco, California: Benjamin/Cummings, an imprint
of Addison Wesley Longman, 2000.
5. Nelson DL and Cox MM. Lehninger Principles of Biochemistry. 3rd
edition. New York: Worth pub1ishers, 2000.
6. Voet D and Voet JG. Biochemistry. 3rd edition. Wiley International
edition. Hoboken, New Jersey: John Wiley & Sons. Inc. , 2004.



1. DNA (DNA Synthesis) DNA
(DNA replication)
1.1 DNA E. coli (DNA
replication in E.coli)
1.2 DNA (DNA replication
in Eukaryotic cells)
2. (Gene expression)
2.1 RNA (RNA Synthesis)
(Transcription)
2.2 (Protein Synthesis)
(Translation)
3.
3.1
3.2

:
DNA replication, Transcription,
Translation Regulation of Gene Expression

RNA

DNA

double strand

Central dogma

DNA replication

DNA
Reverse
transcription

Transcription

RNA

RNA replication

Translation

Protein

DNA replication
=
= DNA Synthesis

DNA replication
1. (Semiconservative)
- DNA
DNA DNA
(template)

Semiconservative
replication
Parental
duplex

Two daughter
duplexes

2.
(Bidirectional)
- (Origin of
replication)
2
ORI

E.coli

ORI

*** origin of
replication

oriC
ORI

ORI

Eukaryote

ORI

(Replication fork)
Bidirectional
replication

ORI

ORI

ORI

ORI

ORI

ORI

ORI

ORI

ORI

ORI

*** origin of
replication
1

DNA replication in E.coli

: DNA polymerase
DNA polymerase E.coli 3
- DNA polymerase I
- DNA polymerase II
- DNA polymerase III
DNA replication
E.coli DNA polymerase III

 DNA polymerase
E.coli
DNA polymerase I ()

polypeptide 3 activities
1. Polymerase activity
2. 3

5 exonuclease activity

3. 5

3 exonuclease activity

1. Polymerase activity

phosphodiester
5--P dNTP
3-OH DNA

DNA

5
3

2. 3 5 exonuclease activity
NT
3
phosphodiester
NT

proofreading activity

3. 5

3 exonuclease activity

NT
5
phosphodiester

RNA primer
DNA replication

DNA

excision-repair
activity

DNA polymerase I
protease
Large fragment + Small
fragment
(Klenow fragment)
Polymerase activity
3 5 exonuclease
Activity (proofreading)

5 3 exonuclease
activity

 1: DNA Polymerase E.coli

DNA Polymerase
I
II
III
(Structural gene)*
polA
polB
polC (dnaE)
(Subunits)

>4

>10

(Molecular
weight)
3 5 Exonuclease
(proofreading)
5 3 Exonuclease

103,000

88,000

830,000

16-20

40

250-1000

3-200

1,500

>500,000

DNA

RNA primer

DNA
SOS DNA
Repair

DNA

DNA

DNA
Polymerization rate
(nucleotides / )
Processivity (nucleotides
added before dissociation)
(Biological
function)

DNA polymerase III

DNA replication

E.coli

(subunits)
--->

DNA polymerase III holoenzyme

DNA polymerase III complex

 2: (subunits) DNA polymerase III E.coli

holo
enzyme
2

27,000

10,000

holE

71,000

dnaX

;
DNA polymerase III dimer

2
1
1
1
1

52,000
35,000
33,000
15,000
12,000

DnaX*
HolA
HolB
holC
holD

Clamp-loading
compex load -subunit
DNA
lagging strand

37,000

dnaN

(DNA clamp)
processivity

132,000

polC
(dnaE)
dnaQ
(mutD)

Polymerization
Activity
Core
35Proofreading Polymerase
exonuclease

 subunit DNA Polymerase III

processivity

Sliding clamp

 DNA replication E.coli

:
DNA 2
1. leading strand = DNA

2. lagging strand = DNA

= Okazaki fragment
DNA 5

DNA replisome DNA replicase system

 DNA E.coli :
1. Initiation step:
DNA E.coli ori C
Tandem array of
three 13 bp sequences

Consensus sequence
GATCTNTTNTTTT

Binding sites for DnaA protein,

four 9 bp sequences

Consensus sequence
TTATCCACA

8
( 3)


DNA

DnaA

 3:
oriC E.coli

DnaA protein
52,000
1
oriC
oriC
DnaB protein
300,000
6*

(helicase)
DnaC protein
29,000
1
DnaB
oriC
HU
19,000
2
;
;

Primase (DnaG
60,000
1
RNA primers;
protein)
primosome
SSB
75,600
4*

RNA
454,000
5
DnaA
polymerase

4
400,000
DNA

topoisomerase

II (gyrase)
Dam methylase
32,000
1

5GATC oriC

Supercoiled
template

E.Coli DNA

DnaAATP
+ HU + IHF
HU = histonelike protein
IHF = integration host factor

Initial
complex

2
Open
complex
(DnaBDnaC)6
+ DnaA

P1 nuclease = endonuclease

Penicillium citrinum

Prepriming
complex
DnaB

DnaC +
ADP + Pi

DnaB = helicase

4
Priming and replication

2. Elongation step:

DNA

DNA

DNA 2 leading strand

lagging strand

leading strand
primase RNA primer (~10-60 nt)
DNA pol III RNA primer
dNTP DNA
RNA primer DNA template
Watson & Crick

 leading strand
(DnaB)

 lagging strand

Okazaki fragment ~1,000 - 2,000 nt

RNA primer

3 exonuclease activity

DNA Polymerase I

Polymerase activity
DNA Polymerase I
DNA
RNA primer

RNA primer

AMP--- NAD+ (E.coli)

ATP (Eukaryote)

Enzyme-AMP

Okazaki fragment

DNA Ligase

DNA polymerase III dimer

3. Termination step:
DNA


Tus
DNA E.coli
Termination sites (Ter sites)
DnaB (Helicase)
DNA 2

DNA replication in Eukaryotic cells

S phase interphase
cell cycle

Chromatin fiber

Histone and
nonhistone
proteins

Nucleosome
(11 nm diameter)


:
~10
5: E.coli

3.9 x 106

~ 109

30

850

60-90

103-104

0.67

0.33

24

E.coli

(
1 )

(m/)

(//)

: E.coli 37C Hela cells


(..)
lagging strand E.coli
~ 135 nt

DNA
lagging strand

 (..)
6: DNA polymerase

primase

,
kDa
KM dNTPs,
M
Processivity
()
Processivity (

PCNA)
35 exonuclease
2, 3dideoxy-NTPs

arabiosyl-CTP

aphidicolin

Lagging
strand
4
160-185

DNA polymerase

Leading
strand
2
125

1
40

4 (identical)
125

2-5

10a

0.5

2-4

?
210-230 or
125-140

 (..continued)

DNA
: DNA 2
DNA

Telomerase
DNA
gap 5

gap 5
lagging strand

Template = double strand DNA

Primer = single strand DNA

DNA template: 5 GATCGAACCACGGA 3

DNA primer: 5 CCGTG 3

5 GATCGAACCACGGA 3
CTAGCTTG GTGCC 5

2C : 3T : 2G : 1A

Gene expression

DNA replication

DNA ( RNA
)

DNA ( RNA .
)

DNA

Transcription

RNA

Translation

- Transcription
- Translation

Protein

Transcription:
DNA
RNA
RNA
RNA
3
1. mRNA :

: template

2. tRNA :
tRNA
: mRNA

3. rRNA :
rRNA
:

RNA 3 RNA

(Transcription)
1. RNA RNA polymerase
: RNA 5--P NTP 3-OH
RNA phosphodiester
2. RNA primer
3. DNA 3
RNA

5 template

(antisense/noncoding strnad)

(sense strand)
(nontemplate strand)

 E.coli
E.coli RNA polymerase 1

mRNA, tRNA rRNA

7: RNA polymerase E.coli

(Dalton)

36,500

151,000

RNA
RNA

155,000

70,000a

RNA

11,000
1

70 kDa

Holoenzyme
RNA pol core enzyme

subunit Holoenzyme
DNA promoter

 E.coli 3 initiation
step, elongation step termination step
1. Initiation step :
RNA pol DNA promoter
subunit Holoenzyme

(Pribnow box)

DNA -10
region Pribnow box

 DNA template
3 5
RNA pol DNA
template primer
5 RNA

triphosphate; 5-ppp

riboNT

phosphodiester
5 3
rifampicin

subunit RNA pol
subunit core
enzyme RNA
~7-10 nt

2. Elongation step :
DNA
RNA pol

RNA
RNA
5 3
Actinomycin D

G-C DNA

3. Termination step :
2
- () rho factor-dependent
- rho factor-independent
1. rho factor-dependent
RNA

RNA
RNA pol DNA
activity
ATP-dependent
RNA-DNA helicase

DNA-RNA
polymerase

3 RNA

RNA

RNA polymerase

2. rho factor-independent

Stem-loop structure
3 RNA
RNA pol DNA
Stem-loop structure
hairpin structure
symmetrical G-C rich
segments
3 15-20 nt
stem-loop structure 3
U ~ 4-8

RNA
DNA


E.coli

RNA pol 3
8 : RNA polymerase
RNA
polymerase
RNA polymerase I

RNA polymerase II

Nucleus
(nucleolus)
Nucleus

RNA

RNA polymerase III

Nucleus

Mitochondrial
RNA polymerasea
Chloroplast
RNA polymerasea

Mitochondrion

Pre-mRNA, some small nuclear

RNAs
Pre-tRNA, 5S rRNA, other small
RNAs
Mitochondrial RNA

Chloroplast

Chloroplast RNA

Pre-rRNA (except 5S)

RNA polymerase

 (continued)
. promoter
promoter RNA pol I
Pre-rRNA
promoter RNA pol II

Pre-mRNA TATA
box, regulatory seq.(CCAAT box, GC box) Inr seq.

RNA pol II

Initiation & Elongation steps
Transcription factors

Proteins required for transcription at the RNA polymerase II

promoters of eukaryotes ( Nelson DL and Cox MM. Lehninger Principles of
Biochemistry. 3rd edition. 2000; P. 987)
Transcription
factors

Number
of
subunits

Subunit Mr

Functions

12
1

10,000-220,000
38,000

RNA
TATA box

12,000, 19,000, 35,000

TFIIB

35,000

TFIID

12

15,000-250,000

TFIIE

34,000, 57,000

TFIIF

30,000, 74,000

TFIIH

12

35,000-89,000

Stabilizes binding of TFIIB and

TBP to the promoter
Binds to TBP; recruits RNA
polymerase-TFIIF complex
Interacts with positive and negative
regulatory protein
Recruits TFIIH; ATPase and
helicase activities
Binds tightly to RNA polymerase
II; binds to TFIIB and prevents
binding of RNA polymerase to
nonspecific DNA sequences
UnwindsDNA at promoter;
phosphorylates RNA polymerase;
recruits nucleotide-excision repair
complex

Elongation*
ELL
P-TEFb
SII (TFIIS)
Elongin (SIII)

1
2
1
3

80,000
43,000, 124,000
38,000
15,000, 18,000, 110,000

Initiation
RNA polymerase II
TBP (TATA-binding
protein)
TFIIA

* All elongation factors suppress the pausing or arrest of transcription by the RNA polymerase II-TFIIF complex.

The name is derived from the term Eleven-nineteen Lysine-rich Leukemia. The gene for the factor ELL is the site of chromosomal
recombination events frequently associated with the cancerous condition known as acute myeloid leukemia.


(Termination step)

- RNA (posttranscriptional RNA processing)

mRNA -
**
rRNA tRNA -

RNA (mRNA, rRNA, tRNA)

-

- mRNA :
1. RNA Splicing :
intron
exon template

 intron = primary transcript

intervening sequence

exon = primary transcript

 RNA
intron
histone
- primary transcript mRNA
(heterogeneous nuclear RNA;
hnRNA) small nuclear ribonucleoprotein
particle (snRNP snurps)
intron

ribozyme primary transcript
splicing Self Splicing
intron primary transcript tRNA
ATP

 7-methylguanosine
5 nt RNA
5, 5-triphosphate 5-cap

2. 5 cap :

5-cap mRNA
5-cap ribosome
mRNA

3. poly(A) tail :
A
20-250
3 mRNA

5-cap
splicing

mRNA

- rRNA :
primary transcript rRNA pre-rRNA

- pre-rRNA E.coli

 - pre-rRNA Eukaryote
- nucleolus

*** tRNA

- tRNA :
tRNA E.coli : - 30S pre-rRNA
tRNA tRNA
tRNA : tRNA
: primary transcript 1 tRNA
2-7

Clover leaf

DNA replication
DNA
Transcription
RNA
Translation
Protein

Translation :
(protein synthesis)
peptide
mRNA template
mRNA

5 3
mRNA
Polycistronic mRNA

Monocistronic mRNA

 cytoplasm

polypeptide
(N) (C)

 (Genetic Code) mRNA

=
3

(triplet code codon)

1
AUG = initiation codon
: Met (euk.)
: fMet (prok.)
1
1

degeneracy of genetic code

 codon 1 2

frameshift

universal code

 mitochondria:

Codon

Universal code

Mitochondrial
code

UGA

Stop

Trp

AUA

Ile

AGA

Arg

Met and
initiation
Stop

AGG

Arg

Stop

mRNA tRNA
tRNA codon
mRNA hydrogen
3 tRNA codon
mRNA anticodon

 3 2
anticodon 1
2 codon
Watson-Crick

1
anticodon 3
codon
Watson-Crick

1 anticodon wobble
position


cytoplasm
ribosomal
protein rRNA


ER
RER
tRNA
3
1. Peptidyl site (P site)
2. Aminoacyl site (A site)
3. Exit site (E site)

Polysome Polyribosome
:10-100

 tRNA
tRNA
tRNA
tRNA aminoacyl-tRNA
tRNA
aminoacyl-tRNA synthetase
20

2

1. Amino acid + ATP + enzyme
2. tRNA + enzyme(aminoacyl-AMP)

enzyme(aminoacyl-AMP) + PPi
aminoacyl-tRNA + AMP +
enzyme

 3OH A
3 tRNA
tRNA

tRNAMet
Met

Met-tRNAMet
(methionyl-tRNA)

***


3
initiation, elongation, termination steps

1. Initiation step:
E.coli:
- 30S ribosome ( 16S rRNA )
- 50S ribosome ( 23S rRNA )
- mRNA
- fMet-tRNAfMet
- Initiation factors (IF-1, IF-2 IF-3)
- GTP
- Mg2+
30S initiation
complex

70S initiation
complex

 initiation factors 9

13
( 50)

eIF
initiation
Factor

(IF)

IF-1

tRNA A site

IF-2

fMet-tRNAfMet mRNA
P site

IF-3

50S 30S
P site
30S fMet-tRNAfMet

(eIF)

eIF2

Met-tRNAfMet 40S

eIF2B, eIF3

2 40S

eIF4A

RNA helicase
(secondary structure) mRNA mRNA
40S
eIF4F
(eIF4F complex)

eIF4B

mRNA AUG mRNA

eIF4E

5cap mRNA;
eIF4F

eIF4G

eIF4E poly(A) binding protein (PAB);

eIF4F

eIF5

40S
60S 40S 80S
initiation complex

eIF6

80S
40S 60S

2. Elongation step:
E.coli:
- 70S initiation complex
- aminoacyl-tRNA
- Elongation factors (EF-Tu, EF-Ts
EF-G)
- GTP

1
peptide 3

1. Elongation Step
1

70S initiation
complex

2. Elongation Step 2

3. Elongation Step 3
Translocation

Peptidyl
transferase
= 23S rRNA***
(ribozyme)

EF-G =
Translocase

3. Termination step:


E.coli :
-
(UAG,
UAA UGA)
- Termination factors
Release factors
(RF-1, RF-2 RF-3)
- GTP

Release
factor 1 eRF


(posttranslational modification)
polypeptide

:
: insulin ( preproinsulin)
: (glycosylation) glycoprotein (
glycosyltransferase ER)
: (Disulfide bond formation) insulin (
-SH cysteine)
: (attachment of prosthetic group) Hemoglobin
cytochrome ( heme prothetic group)
: (Methylation) Cytochrome c ( Lys
Glutamic acid )
: (Hydroxylation) Collagen (-OH Lys
proline )

Regulation of Gene Expression :

(constitutive gene expression)
Glycolysis Crebs cycle

inducible protein


repressible protein


(transcription initiation)

 transcription
initiation


1. : regulatory protein,
attenuation, antiterminator
(regulatory protein):
regulatory protein regulatory gene :
mRNA
- activator
- repressor
metabolism
Polycistronic gene

DNA
operon

 :
lac operon & trp operon

lac operon
lactose
Negative regulation

Z = -galactosidase
lactose

allolactose
(IPTG)

allolactose IPTG = inducer

Lac repressor

 glucose lactose
lac operon
Activator

Positive regulation

aporepressor

trp operon

trpB

trpA

 attenuation :
trp operon
transcription translation :

Trp

Trp

 antiterminator :

antiterminator RNA pol DNA
template RNA pol mRNA

2. :

- polycistronic mRNA
- translational frameshift
- translational repressor
- antisense RNA



:
- metabolism

-polypeptide mRNA
-transcription translation

- exon
1. :
:
-promoter,transcriptional activator protein (TAP), enhancer

 TAP 4 : Helix-turn-helix, Zinc finger,

Leucine Zipper Helix-loop-helix
DNA
promoter 1

promoter


response element heat shock elements

2.
:



,
mRNA