1. 9/21/2010 1 Control of Gene Expression Dr. Ir. Giyanto, MSi Laboratorium Bakteriologi Tumbuhan Departemen Proteksi Tanaman Fakultas Pertanian Institut Pertanian Bogor

2. 9/21/2010 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, Biological Science, Bogor Agricultural University, Indonesia PhD. April 1, 1999 – March 24, 2004. Department of Bioinformatics and Genomics, Graduate School of Information Science, 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 of Biological Science, Nara Institute of Science and Technology, JAPAN June 1, 2001 – March 30, 2002. Teaching Assistant at Laboratory of Microbial Cell Biology, Graduate School of Biological Science, Nara Institute of Science and Technology, JAPAN November 1, 2002 – February 28, 2003. Research Assistant at Graduate School of Biological Science, Nara Institute of Science and Technology, JAPAN April 1, 2004 – March 30, 2005. Postdoctoral fellow at Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, JAPAN April 1, 2005 – now. Staff member at Laboratory of Plant Bacteriology, Department of Plant Protection, faculty of Agriculture, Bogor Agricultural University, INDONESIA. Genomes: Sizes and Numbers of Genes GenomeGroupSize (kb)Number of genes Eukaryotic nucleus Saccharomyces cerevisiae Caenorhabditis elegans Arabidopsis thaliana Homo sapiens Yeast Nematode Plant Human 13,500 (L) 100,000 (L) 120,000 (L) 3,000,000 (L) 6,000 13,500 25,000 100,000 Prokaryote Escherichia coli Hemophilus influenzae Methanococcus jannaschii Bacterium 4,700 (C) 1,830 (C) 1,660 (C) 4,000 1,703 1,738 Viruses T4 HCMV (herpes group) Bacterial virus Human virus 172 (L/C) 229 (L) 300 200 Eukaryotic organelles S. cerevisiae mitochondria H. sapiens mitochondria Yeast Human 78 (C) 17 (C) 34 37 Marchantia polymorpha ChloroplastLiverwort121 (C)136 Plasmids F plasmid pSymA pSymB pMLa pMLb E. coli Sinorhizobium Mesorhizobium 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 2

3. 9/21/2010 3 Gene expression is controlled at: Transcription—initiation,termination, not elongation Processing—in EuK RNA splicing, modification, transporting translation -- in ProK coupled transcription and 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 to particular site on DNA *constitutive expression!!

4. 4 9/21/2010 Gene regulatory mechanism: Negative control: Repressor binds to operator, gene turn off Positive control: Activator binds to operator, gene turn on *inducible repressible Gene regulatory mechanism: Negative control: lac operon gal operon trp operon Positive control: ara operon mal operon tol operon Global Regulatory Mechanism: Catabolic repression ntr operon, general stress response, etc [1] [2] [3]

5. 9/21/2010 5 Negative control—Bacteria Repressor,Operator **polycistronic mRNA Positive control—ProK, EuK Cis-acting sites, trans-acting factors **monocistronic mRNA

6. 9/21/2010 Gene Expression Regulator bind to DNA ? Positive regulationNegative regulation YES No OFF ON OFF Gene Regulation Negative control Positive control 6

7. Coding region Control region 7 Regulatory gene 9/21/2010 Metabolic regulation The lac operon The lac operon controls utilization of Lactose operon

8. Log Cell No 9/21/2010 E.coli growing on glucose and lactose After a "lag period", the cells start to Lag period grow on lactose Cells stop growing when all glucose is used up Time Genes of the lac operon control this utilisation of lactose as a carbon source During the lag period, new enzymes are synthesised ß-galactosidase (lacZ) - splits lactose into glucose and galactose lactose permease (lacY) - transports lactose across the cell membrane lactose transacetylase (lacA) -inactivates toxic galactosides 8

9. 9/21/2010 9 How lac I gene control lac ZYA gene transcription ? Negative regulation-- lac genes are transcribed unless it was turned off by a repressor protein Repressor–-lac I product Inducer—lactose (allolactose), IPTG *isopropyl thioglactoside (Gratuitous inducer)

10. 9/21/2010 Induction of gene expression in lac operon Inducers Structure of the lac operon — 1 All 3 genes (Z, Y, A) are expressed from the same promoter (p) as a POLYCISTRONIC mRNA ZYA p o DNA I regulatory gene Polycistronic mRNA LacI repressor Normally, transcription from the single promoter is blocked by LacI repressor protein. It binds to a special DNA sequence, the OPERATOR, next to the promoter 10

11. Polycistronic mRNA produced Lactose 9/21/2010 Structure of the lac operon — 2 However, in the presence of lactose, the operon is switched on by inactivation of the lacI repressor protein (an example of Negative Control) LacI repressor I ZYA DNA p o L L L L RNA polymerase ( ) can now bind to the promoter and begin transcription Promoter and operator of the lac operon lacI Repressor coding region 11

12. 9/21/2010 12 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’

13. 9/21/2010 13 Two models for repressor action Regulation of gene expression The Trp operon

14. 9/21/2010 14 The trp operon Repressor: trpR gene coded （ inactive different locus ） Corepressor : trytophane With two different separate mechanism Attenuation control Structure of trp operon

15. 9/21/2010 15 Regulation of trp operon by TrpR repressor inactive Active repressor

16. 9/21/2010 16 Attenuation : a mechanism that controls the ability of RNA polymerase to read through an attenuator, which is an intrinsic terminator located at the beginning of a transcription unit. The changes in secondary structure that control attenuation are determined by the position of the ribosome on mRNA. The external circumstances is influenced ribosome movement in the leading sequence of mRNA.

17. 9/21/2010 17 The trp operon—leader sequence Chorismic acid to tryptophane

18. 18 9/21/2010 The structure on the leader region 1 23 4 12 34 123 4 21 34