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Control of Gene Expression

Dr. Ir. Giyanto, MSi

Laboratorium Bakteriologi Tumbuhan

Departemen Proteksi Tanaman

Fakultas Pertanian

Institut Pertanian Bogor

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CURRICULUM VITAE

EDUCATION

Bachelor. (September 1,1987- January 25, 1992). Plant Protection Science , Bogor AgriculturalUniversity, Indonesia

Master of Science. ( September 1, 1995 – October 31,1998). Department of Molecular Microbiology, BiologicalScience, Bogor Agricultural University, Indonesia

PhD. April 1, 1999 – March 24, 2004 . Department of Bioinformatics and Genomics, Graduate School of InformationScience, Nara Institute of Sciences and Technology (NAIST), Japan.

JOB EXPERIENCES

February 1, 1992 – April 30,1995. Technical Staff at Faculty of Agriculture, Bogor Agricultural University, INDONESIA

May 1,1995 – October 31, 1998. Lecturer and Research Staff at Faculty of Agriculture, Bogor Agricultural University,INDONESIA.

September 1, 2000 – March 30, 2001. Teaching Assistant at Laboratory of Microbial Cell Biology, Graduate School ofBiological Science, Nara Institute of Science and Technology, JAPAN

June 1, 2001 – March 30, 2002. Teaching Assistant at Laboratory of Microbial Cell Biology, Graduate School ofBiological Science, Nara Institute of Science and Technology, JAPAN

November 1, 2002 – February 28, 2003. Research Assistant at Graduate School of Biological Science, Nara Instituteof Science and Technology, JAPAN

April 1, 2004 – March 30, 2005. Postdoctoral fellow at Department of Bacteriology, Graduate School of MedicalScience, Kanazawa University, JAPAN

April 1, 2005 – now. Staff member at Laboratory of Plant Bacteriology, Department of Plant Protection, faculty ofAgriculture, Bogor Agricultural University, INDONESIA.

Genomes: Sizes and Numbers of GenesGenome Group Size (kb) Number of genesEukaryotic nucleusSaccharomyces cerevisiaeCaenorhabditis elegansArabidopsis thalianaHomo sapiens

YeastNematodePlantHuman

13,500 (L)100,000 (L)120,000 (L)3,000,000 (L)

6,00013,50025,000100,000

ProkaryoteEscherichia coliHemophilus influenzaeMethanococcus jannaschii

BacteriumBacteriumBacterium

4,700 (C)1,830 (C)1,660 (C)

4,0001,7031,738

VirusesT4HCMV (herpes group)

Bacterial virusHuman virus

172 (L/C)229 (L)

300200

Eukaryotic organellesS. cerevisiae mitochondriaH. sapiens mitochondria

YeastHuman

78 (C)17 (C)

3437

Marchantia polymorphaChloroplast Liverwort 121 (C) 136PlasmidsF plasmidpSymApSymBpMLapMLb

E. coliSinorhizobiumSinorhizobiumMesorhizobiumMesorhizobium

100 (C)1.35 MB (C)1.68 MB (C)351.341 kb (C)206.315 kb (C)

29????

----------------------------------------------------------------------------------------------------------------------NOTE: C = circular; L = linear; L/C = linear in free virus, circular in cell

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Gene expression is controlled at:

Transcription—initiation,termination,not elongation

Processing—in EuK RNA splicing, modification,transporting translation

-- in ProK coupled transcriptionand translation

Translation--like transcription

Genes products of:

Structural genes*:functions as: structure proteins, enzymes

regulatory proteins

Regulatory genes:functions: a specific protein in regulation

of gene expression, by binding toparticular site on DNA

*constitutive expression!!

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Gene regulatory mechanism:

Negative control: Repressor binds to operator,gene turn off

Positive control: Activator binds to operator,gene turn on

*induciblerepressible

Gene regulatory mechanism:

Negative control: lac operongal operontrp operon

Positive control: ara operonmal operontol operon

Global Regulatory Mechanism:Catabolic repressionntr operon, general

stress response, etc

[1]

[2]

[3]

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Negative control—BacteriaRepressor,Operator

**polycistronic mRNA

Positive control—ProK, EuKCis-acting sites, trans-acting factors

**monocistronic mRNA

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Gene ExpressionRegulator bind to DNA ?

Positive regulationNegative regulation

YES

No

OFF

ON

ON

OFF

Gene Regulation

Negative control

Positive control

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Coding region

Control region

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Regulatory gene

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Metabolic regulation

The lac operon

The lac operon controls utilizationof Lactose

operon

Log

Cel

l No

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E.coli growing on glucose and lactose

After a "lag period",the cells start to

Lagperiod

grow on lactose

Cells stop growingwhen all glucose isused up

Time

Genes of the lac operon control this utilisation oflactose as a carbon source

During the lag period, new enzymes are synthesised

• ß-galactosidase (lacZ) - splits lactose intoglucose and galactose

• lactose permease (lacY) - transports lactoseacross the cell membrane

• lactose transacetylase (lacA) -inactivates toxicgalactosides

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How lac I gene control lac ZYAgene transcription ?

Negative regulation--

lac genes are transcribed unlessit was turned off by a repressorprotein

Repressor–-lac I product

Inducer—lactose(allolactose), IPTG

*isopropyl thioglactoside(Gratuitous inducer)

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Induction of gene expression in lac operon

Inducers

Structure of the lac operon — 1

All 3 genes (Z, Y, A) are expressed from the samepromoter (p) as a POLYCISTRONIC mRNA

Z Y Ap oDNA

I

regulatorygene

Polycistronic mRNALacI

repressor

Normally, transcription from the single promoter is blockedby LacI repressor protein. It binds to a special DNAsequence, the OPERATOR, next to the promoter

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Polycistronic mRNAproduced

Lactose

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Structure of the lac operon — 2

However, in the presence of lactose, the operon isswitched on by inactivation of the lacI repressorprotein (an example of Negative Control)

LacIrepressor

I Z Y ADNA

p o

LL

L

L RNA polymerase ( ) cannow bind to the promoter and

begin transcription

Promoter and operator of the lac operon

lacI

Repressor coding region

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Induction of lac genes expression------- by inducer

Induceable enzyme—From ~ 5 to 5,000 molecular

Short life of mRNA—Half-life ~3 min

Stable of enzyme

Gratuitous inducer—IPTG

The operator is palindromic—with inverted repeats

5’-TGTGTG------------------------------------------GTGTGT-5’

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Two models for repressor action

Regulation of gene expression

The Trp operon

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The trp operon

Repressor: trpR gene coded（ inactive different locus）

Corepressor: trytophane

With two differentseparate mechanism

Attenuation control

Structure of trp operon

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Regulation of trp operon by TrpR repressor

inactive

Active repressor

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Attenuation: a mechanism that controls the ability ofRNA polymerase to read through an attenuator, whichis an intrinsic terminator located at the beginning of atranscription unit.

The changes in secondary structure that controlattenuation are determined by the position of theribosome on mRNA.

The external circumstances is influenced ribosomemovement in the leading sequence of mRNA.

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The trp operon—leader sequence

Chorismic acid to tryptophane

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The structure on the leader region

12 3 4

1 2

3 4

1 2 3

4

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3 4