BACTERIOPHAGE Regulation of Gene Expression in Bacteriophage BMB400, 4_4_04 BACTERIOPHAGE Regulation of Gene Expression in Bacteriophage 1 • Phage – A virus, it is one kind of vector. • Bacteriophage – a phage (virus) that infects bacteria • Simple “organism” – Basic structure consists of DNA or RNA surrounded by a protein coat – Vary greatly in shape and size 2 1

BMB400, 4_4_04 3 4 2

• Phage cannot replicate on their own – Infect a host cell BMB400, 4_4_04 Visible effects on DNA during viral infection T4 phage DNA Pre-infection Post-infection5 • Phage cannot replicate on their own – Infect a host cell • Once inside the host cell, they either remain quiescent (as prophage) or use the cell’s replication machinery to produce many copies of themselves – This replication may or may not lead to destruction of the host cell (lytic) 6 3

Bacteriophage BMB400, 4_4_04 • Why are phage important? – They cause diseases – They are useful tools in molecular biology • Phage are often used as “replacement vectors” - Part of their DNA is removed and replaced with other DNA of interest (e.g., human DNA) - This recombinant DNA is repackaged into phage; phage infect host bacteria - Phage use bacteria to replicate, therefore replicating the DNA of interest 7  bacteriophage as a replacement vector 8 4

toxin insertion Lysis or Lysogeny BMB400, 4_4_04 Phage conversion Dormant prophage – integrated bacteriophage – carries genes that alter the phenotype of the microbe - best examples are pathogens and toxin production toxin prophage Corynebacterium diptheriaea Phage produces diptheria toxin insertion site C.diptheriaea without phage strain produces no toxin Does not cause diptheria This is what makes people sick 9 Lysis or Lysogeny • Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium (bacteriophage T4) • Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively – No progeny – No lysis of the host • Bacteriophage lambda can do either. 10 5

The lytic pathway of bacteriophage infection BMB400, 4_4_04 The lytic pathway of bacteriophage infection 11 Phage life cycle: Lytic vs. Lysogenic pathways 12 6

• The phage genome integrated into the host BMB400, 4_4_04 Elements of lysogeny • The phage genome integrated into the host bacterial genome is a prophage. • Bacterium carrying the prophage is a lysogen. • Lysogens are immune to further infection by similar phage because the phage functions are repressed in trans. • Induction of the lysogen leads to excision of the prophage, replication of the phage DNA, and lysis of the host bacterium. 13 Site-specific recombination in bacteriophage . X-Ray structure of integration host factor (IHF) in complex with a 35-bp target DNA. 14 7

– products are usually enzymes to shut down host BMB400, 4_4_04 The Bacteriophage T4 • Early translation – products are usually enzymes to shut down host and synthesize viral nucleic acids • Late transcription/translation produces structural proteins 15 Lytic virus T4 has linear DNA with specific hydroxymethylcytosine, instead of cytosine, resistent to endonuclease of the host Produce enzyme in DNA replication that similar to those host enzyme in large amount Encode three group protein (early, middle and late proteins) 16 8

A genetic map of bacteriophage . BMB400, 4_4_04 A genetic map of bacteriophage . 17 The Bacteriophage lambdha Lytic and lysogenic virus Genome: double strand DNA, has cohesive ends (cos) Early transcription begin on PL and PR to give the product of N and Cro protein (regulator) N antiterminator (produce O, P (induce replication), Cll and Clll The antiterminator is not effective before termination before the Q gene (little Q protein is produced, Q is antiterminator to transcribe the late genes)) Cro regulator between lytic dand lysogenic pathways by blocking PL and PR (binding OL and OR (site 1, 2, 3) CII (aktivator protein by activate PE to express CI and integrase) and CIII is stabilization of CII. 18 9

Transcription is regulated by • Repression (initiation) BMB400, 4_4_04 lytic pathway lysogeny pathway 19 Lytic and lysogeny pathways Transcription is regulated by • Repression (initiation) • Activation (initiation) • Anti-termination (elongation) 20 10

 repressor (CI) is a dimer; monomer has 236 amino acids. BMB400, 4_4_04 Repressor structure  repressor (CI) is a dimer; monomer has 236 amino acids. C-terminal domain: protein-protein interaction; dimerization and cooperativity Connector N-terminus: DNA binding; Helix-Turn-Helix motif operator repressor can bind cooperatively to operator sub-sites. operator operator oR2 oR1 21 Cro structure Cro is a dimer Monomer has 66 amino acids Has only one protein domain Does NOT display cooperativity DNA binding; Helix-Turn-Helix motif; also dimerization operator oR3 22 11

Bacteriophage : Events leading to lysis BMB400, 4_4_04 Page 1409 Theoretical repression curves for  pR (right) and a simple © 2004 John Wiley & Sons, Inc. Voet Biochemistry 3e repressor–operator system such as that of the lac operon (left). Bacteriophage : Events leading to lysis • lysis or lysogeny (cI or Cro?) ? • Both lysis and lysogeny: – PR, PL, active : synthesize N, Cro – antitermination by N : synthesize cIII, cII, Q • Lysis: – Low [Cro] : binds OR3, shuts off PRM (cI) – High [Cro] : shuts off PR and PL – antitermination by N protein to produce Q Protein which activate late genes 24 12

MOLECULAR GENETIK ANALYSIS Factors favoring lysogeny BMB400, 4_4_04 Factors favoring lysogeny • High multiplicity of infection – More templates produce more of the CII protein, which stimulates PRE. – Phage sense that it is too crowded. • Poor nutrient conditions for host – Low [glucose] leads to increase in [cAMP]. – Increased [cAMP] will repress the host gene hflA. – Less HflA (a protease) leads to less degradation of the CII protein. 25 MOLECULAR GENETIK ANALYSIS 26 13

BMB400, 4_4_04 14 Material Genetik Cetak biru dari semua jenis makhluk hidup sebagian besar berupa DNA 27 ǙDZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ­Ç ÅB TIFFÅiLZWÅj êLí£ÉvÉçÉOÉâÉÄ QuickTimeý Dz Untai tunggal DNA adalah rangkaian nukleotida dimana untai baru selalu dilekatkan pada posisi 3` 28 14

4 dNTP BMB400, 4_4_04 SEKUENSING 15 Prinsip Sekuensing Sintesis DNA in vitro dengan DNA Polymerase dan penggunaan 2`, 3` dideoxynucleotida. Hilangnya molekul 3`-OH menyebabkan berthentinya reaksi sintesis untai DNA pada point dimana molekul dideoxynukleotida disintesis, karena tidak ada reaksi yang bisa dilakukan tanpa adanya molekul tersebut Sintesis untai DNA baru diawali pada posisi spesifik dengan menggunakan primer yang menempel pada ujung 3` pada daerah yang akan disekuensing Reaksi sintesis DNA menggunakan deoxynucleotida dan dideoxynucleotida akan menghasilkan beberapa fragment DNA yang memiliki residu nukcleotida yang serupa Perbedaan ukuran fragment DNA dapat dianalisis dengan polyacrilamide gel elektroforesis Dua Teknik Sekuensing: Metode Enzymatic (Metode Sanger) Metode Degradasi Kimiawi (Metode Maxam - Gilbert) 29 5` DNA Utas tunggal 3` 3` Primer 5` 4 dNTP DNA Polymerase ddTTP ddCTP ddGTP ddATP DNA baru dideoxynukleotida Primer Sekuensing DNA Hasil PCR Kloning fragment PCR pada vektor Sekuensing PCR fragmen dengan Primer Universal yang komplemen dengan vektor UniversalPri mer Vektor 30 15

QuickTimeýDz Primer 2 BMB400, 4_4_04 A G C T ǙDZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ­ÇÅB TIFFÅiLZWÅjêLí£ÉvÉçÉOÉâÉÄ QuickTimeýDz 31 Sekuensing Fragmen DNA berukuran panjang Kloning fragment DNA pada vektor Sekuensing PCR fragmen dengan Primer Universal yang komplemen dengan vektor Sekuensing dengan penyusunan primer baru yang kompleme dengan hasil sekuensing dengan primer universal UniversalPri Vektor mer Primer 1 Primer 2 Vektor Primer 3 32 16

BMB400, 4_4_04 17 Sekuensing Fragmen DNA Secara Random Purifikasi fragment DNA Digesti Fragmen DNA dengan sonikasi atau enzim restriksi Ligasi Fragment DNA pada Vektor Sekuensing DNA dengan universal primer Alignment sekuen fragmen DNA dari DNA target Purifikasi DNA Digesti UniversalPri mer Ligasi dan Sekuensing Alignment 33 17