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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

DNA

RNA

Protein

DNA replication

Reverse transcription

RNA replication

Translation

Transcription

Central dogma

DNA replication

= DNA Synthesis

=

DNA replication

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

Parental duplex

Two daughterduplexes

Semiconservativereplication

2. (Bidirectional)- (Origin of

replication) 2 ORI

ORI

ORI ORI

ORI ORI

ORI

ORI ORI

ORIORI

ORIORI

ORI ORI

(Replication fork)

Bidirectionalreplication

E.coli

Eukaryote

*** origin of replication

oriC

*** 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

DNA 5 3

phosphodiester 5--P dNTP 3-OH DNA

NT 3 phosphodiester

NT

proofreading activity

2. 3 5 exonuclease activity

3. 5 3 exonuclease activity

NT 5 phosphodiester

RNA primer

DNA replication

DNA

excision-repair

activity

DNA polymerase I

protease

Large fragment(Klenow fragment)

Small fragment

+

Polymerase activity 3 5 exonucleaseActivity (proofreading)

5 3 exonucleaseactivity

1: DNA Polymerase E.coli DNA Polymerase

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

(Subunits)

1 > 4 >10

(Molecular weight)

103,000 88,000 830,000

3 5 Exonuclease (proofreading)

5 3 Exonuclease

DNA Polymerization rate (nucleotides / )

16-20 40 250-1000

Processivity (nucleotides added before dissociation)

3-200

1,500

>500,000

(Biological function)

DNA RNA primer

DNA SOS DNA Repair

DNA DNA

DNA replication E.coli

(subunits) --->

DNA polymerase III holoenzyme

DNA polymerase III complex

DNA polymerase III

2: (subunits) DNA polymerase III E.coli

holo

enzyme

2 132,000 polC (dnaE)

Polymerization Activity Core

2 27,000 dnaQ (mutD)

35Proofreading Polymerase exonuclease

2 10,000 holE

2 71,000 dnaX ; DNA polymerase III dimer

2 52,000 DnaX* Clamp-loading 1 35,000 HolA compex load -subunit 1 33,000 HolB DNA 1 15,000 holC lagging strand 1 12,000 holD

4 37,000 dnaN (DNA clamp)

processivity

subunit DNA Polymerase III processivity

Sliding clamp

:

DNA 2

DNA replication E.coli

DNA 5 3

1. leading strand = DNA

2. lagging strand = DNA

= Okazaki fragment

DNA replisome DNA replicase system

DNA E.coli :

1. Initiation step:

8 ( 3)

DNA E.coli ori C

Consensus sequenceGATCTNTTNTTTT

Consensus sequenceTTATCCACA

Tandem array ofthree 13 bp sequences

Binding sites for DnaA protein,four 9 bp sequences

DNA DnaA

3: oriC E.coli

DnaA protein 52,000 1 oriC oriC

DnaB protein (helicase)

300,000 6*

DnaC protein 29,000 1 DnaB oriC

HU 19,000 2 ; ;

Primase (DnaG protein)

60,000 1 RNA primers; primosome

SSB 75,600 4* RNA polymerase

454,000 5 DnaA

DNA topoisomerase II (gyrase)

400,000

4

Dam methylase 32,000 1 5GATC oriC

Supercoiledtemplate

Initialcomplex

Opencomplex

1

2

3

4

Preprimingcomplex

DnaAATP+ HU + IHF

(DnaBDnaC)6+ DnaA

DnaC +ADP + PiDnaB

Priming and replication

E.Coli DNA

P1 nuclease = endonuclease Penicillium citrinum

DnaB = helicase

HU = histonelike proteinIHF = integration host factor

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

(DnaB)

leading strand

lagging strand

Okazaki fragment ~1,000 - 2,000 nt

RNA primer

5 3 exonuclease activity

DNA Polymerase I

Polymerase activity DNA Polymerase I

DNA RNA primer

RNA primer

Okazaki fragment

DNA Ligase

Enzyme-AMP

AMP--- NAD+ (E.coli)ATP (Eukaryote)

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

Nucleosome(11 nm diameter)

Histone and nonhistoneproteins

Chromatin fiber

:

5: E.coli

E.coli

( 1 )

3.9 x 106 ~ 109

(m/)

30 3

(//)

850 60-90

1

1 103-104

0.67 8

0.33 24 : E.coli 37C Hela cells

~10

(..)

lagging strand E.coli ~ 135 nt

DNA lagging strand

6: DNA polymerase DNA polymerase primase

Lagging strand

Leading strand

4 1 4 (identical) 2 ? , kDa

160-185 40 125 125 210-230 or 125-140

KM dNTPs, M

2-5 10a 0.5 2-4 ?

Processivity ()

Processivity ( PCNA)

35 exonuclease b 2, 3- dideoxy-NTPs

arabiosyl-CTP

?

aphidicolin

(..)

(..continued)

DNA : DNA 2 DNA

Telomerase DNA gap 5

gap 5 lagging strand 5

Template = double strand DNAPrimer = single strand DNA

5 GATCGAACCACGGA 3DNA template:

DNA primer: 5 CCGTG 3

5 GATCGAACCACGGA 3GTGCC 5

2C : 3T : 2G : 1A

CTAGCTTG

DNA

RNA

Protein

DNA replication

Translation

Transcription

Gene expression

DNA ( RNA )

DNA ( RNA . ) .

- Translation- Transcription

Transcription: DNA RNA

RNA

RNA 3

1. mRNA :

: template

2. tRNA : tRNA

: mRNA

RNA 3 RNA

1. RNA RNA polymerase: RNA 5--P NTP 3-OH RNA phosphodiester

(Transcription)

2. RNA primer

3. DNA 3 5 template RNA

3. rRNA : rRNA

:

(sense strand)

(nontemplate strand)

(antisense/noncoding strnad)

5

5

3

3

5 3

E.coli RNA polymerase 1 mRNA, tRNA rRNA

E.coli

(Dalton)

1

36,500 2 RNA

151,000 1 RNA RNA

155,000 1

70,000a 1

11,000 1 a 70 kDa

7: RNA polymerase E.coli

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

DNA -10 region Pribnow box

(Pribnow box)

DNA template 3 5

5 RNA triphosphate; 5-ppp

riboNT phosphodiester 5 3

subunit core enzyme RNA ~7-10 nt

rifampicin subunit RNA pol

RNA pol DNA template primer

2. Elongation step :

DNA RNA pol RNA

RNA 5 3

Actinomycin D G-C DNA

2 - () rho factor-dependent- rho factor-independent

1. rho factor-dependent

RNA RNA RNA pol DNA

activity ATP-dependent RNA-DNA helicase

3. Termination step :

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

RNA polymerase

RNA

RNA polymerase I Nucleus (nucleolus)

Pre-rRNA (except 5S)

RNA polymerase II Nucleus Pre-mRNA, some small nuclear RNAs

RNA polymerase III Nucleus Pre-tRNA, 5S rRNA, other small RNAs

Mitochondrial Mitochondrion Mitochondrial RNA RNA polymerasea

Chloroplast Chloroplast Chloroplast RNA RNA polymerasea

a RNA polymerase

8 : RNA polymerase

. promoter promoter RNA pol I Pre-rRNA

(continued)

promoter RNA pol II Pre-mRNA TATA box, regulatory seq.(CCAAT box, GC box) Inr seq.

RNA pol II Initiation & Elongation steps Transcription factors

9 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

Initiation RNA polymerase II TBP (TATA-binding protein) TFIIA TFIIB TFIID TFIIE TFIIF TFIIH Elongation* ELL

P-TEFb SII (TFIIS) Elongin (SIII)

12 1 3 1 12 2 2 12 1 2 1 3

10,000-220,000 38,000 12,000, 19,000, 35,000 35,000 15,000-250,000 34,000, 57,000 30,000, 74,000 35,000-89,000 80,000 43,000, 124,000 38,000 15,000, 18,000, 110,000

RNA TATA box 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

* 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 (post-transcriptional RNA processing)

mRNA -**

rRNA tRNA -

RNA (mRNA, rRNA, tRNA) -

- mRNA :

1. RNA Splicing :

intron exon template

intron = primary transcript

intervening sequence

exon = primary transcript

- primary transcript mRNA (heterogeneous nuclear RNA; hnRNA) small nuclear ribonucleoproteinparticle (snRNP snurps)

RNA intron histone

intron ribozyme primary transcript splicing Self Splicing

intron primary transcript tRNA ATP

7-methylguanosine 5 nt RNA

5, 5-triphosphate 5-cap

5-cap mRNA 5-cap ribosome mRNA

2. 5 cap :

3. poly(A) tail :

A 20-250 3 mRNA

mRNA

5-cap splicing

- rRNA : primary transcript rRNA pre-rRNA

- pre-rRNA E.coli

I

- 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

RNA

DNA replication

Protein

Translation

Transcription

(protein synthesis) peptide

mRNA template mRNA

Translation :

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 Met and

initiation AGA Arg 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 enzyme(aminoacyl-AMP) + PPi

2. tRNA + enzyme(aminoacyl-AMP) aminoacyl-tRNA + AMP + enzyme

3-OH A 3 tRNA

tRNA

tRNAMet

Met-tRNAMet

Met

(methionyl-tRNA)

***

3

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

eIF initiation Factor

(IF)

IF-1 tRNA A site

IF-2 fMet-tRNAfMet mRNA P site

IF-3 50S 30S 30S fMet-tRNAfMet P site

(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; eIF4FeIF4G eIF4E poly(A) binding protein (PAB);

eIF4F

eIF5 40S 60S 40S 80S initiation complex

eIF6 80S 40S 60S

13 ( 50)

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

EF-G)- GTP

70S initiation complex

1 peptide 3

1. Elongation Step 1

2. Elongation Step 2

Peptidyltransferase= 23S rRNA***(ribozyme)

3. Elongation Step 3

Translocation

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

allolactose IPTG = inducer

Z = -galactosidase

lac operon

lactose allolactose Lac repressor

(IPTG)

lactose

Negative regulation

glucose lactose lac operon

Activator

Positive regulation

trp operonaporepressor

trpB trpA

trp operon transcription translation :

Trp

Trp

attenuation :

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