B3206 Microbial Genetics

Transcription

1 Prof. Fahd M. Nasr Faculty of Sciences Lebanese University Beirut, Lebanon B3206 Microbial Genetics 1

2 "Genetics of Microorganisms" Prokaryotic M.G. Genetic switches Lecture VII cos Nu1 A B C Nu3 D E FI FII Z U V G T H M L K I J Lyzogeny in details Head/tail genes Recombination Regulation Replication Lysis cos A...I J att int xis red gam ciii N ci cro cii O P Q S R cos P L P R /P RM P RE P R' t L nutl P RM nutr t R1 ciii N ci cro cii O L /P L O R /P R P RE ci ciii N ci ci ci cro cii 2

3 Lyzogeny in details Same intial events Expression of N and cro from P L and P R Expression of cii (right) and ciii (left) expression of l repressor ci cii is a positive regulator binds P RE and assists the RNA Pol. trasncription of mrna ci ci binds to O R and induces its own expression from P RM Establishment of lysogeny 3

4 Promoters in the right and left control regions of phage l N P L ci P RM P R cro O L1 O L2 O L3 O R3 O R2 O R1 Transcription in the l control regions in lytic and lysogenic growth 4

5 Expression of ci repressor gpcii is unstable proteolysis by host proteins (hfla and hflb genes) gpcii is stabilized by gpciii and camp ci repressor Binds to O L and O R and blocks gene expression First expressed from P RE When bound to O R (O R1 and O R2 ) ci expressed from P RM P RE -induced high expression P RM -induced low expression 5

6 Effects of repressor binding 6

7 Repressor and Cro bind in Different Patterns to Control Lytic and Lysogenic Growth 7

8 Lyzogeny revisited Involves integration of l genome host chromosome prophage int gene recombinase site specific recombination att sequences attp = POP and attb = BOB O = 15bp central core integration of l genome two direct repeats attl BOP and attr POB Site-Specific Recombination/ Integration 8

9 Integration of l genome Requires Int (product of int gene) IHF (Integration Host Factor) (a and b encoded by genes hima and himb) DNA gyrase Formation of Holliday structure and resolution integration of l genome into the E. coli genome 9

10 Prophage induction Excision of the phage genome requires Genes int and xis (exicionase) recombination between attl and attr (BOP and POB ) release of l genome as dscccdna prophage induction Lyzogeny expression of int gene Lytic pathway Free phage (dscccdna) no int expression Prophage expression of int and xis genes Expression of int and xis genes How int gene is expressed During lytic pathway N (left) induces P L -initiated transcription ends at b region unstable transcript no int (integrase) or xis (excisionase) During lyzogeny cii (right) induces ci from P RE and int from P I P I initiated transcription ends at T (before b region) stable mrna integrase integration of l genome in E. coli chromosome 10

11 Expression of int gene T P I I J sib int b attp xis t L nutl ciii N ci cro cii O L /P L P RM O R /P R nutr t R1 P RE Transcript 2 Transcript 1 Expression of int gene (integrase) No expression of int and xis genes Transcript 1 Substrate to RNase III Transcript 2 Normal ter. stable 11

12 How to make the right decision? Actors: ci repressor and cro antirepressor same specificity to O L and O R Conditions favor lytic pathway cro binds O R and O L blocks expression of ci and reduces expression from P L and P R Conditions favor lyzogeny camp + ciii cii ci (from P RE ) ci binds O L and O R and inhibits transcription Decision depends on whether cro or ci gains access to operators λcii controls the decision between lytic and lysogenic Growth 12

13 SOS response Regulatory system in bacteria (E. coli) responds to DNA damage or inhibition of DNA replication Upon activation 20 SOS genes Expressed at high rate (survival factors) capacity for DNA repair + inhibition of cell division + restoration of DNA replication Form the SOS regulon SOS pathway Controlled by the interplay of LexA (repressor) RecA (multifunctional protein) induced state uninduced state How does it work LexA (ci) = a protein of two domains joined by a flexible linker region LexA acts as a dimer 13

14 CTCT reca CTCT lexa CTCT SOS genes NTNT NTNT NTNT OFF low expression OFF low expression OFF low expression CT NT DNA repaired DNA damage reca lexa SOS genes ON ON ON reca CT NT CT NT CT NT Figure Regulation of the SOS response in E. coli. 14

15 How does it work? All SOS genes are repressed by LexA DNA damage signal of unknown nature RecA is activated co-protease Cleavage of LexA in the linker region Expression of all SOS genes Damage repair level of inducing signal Dimerization RecA inactive LexA dimerizes and blocks the SOS system Ct Nt DNA bind. The SOS response: uninduced state lexa gene reca gene LexA protein RecA protein (inactive) Represses transcription of genes involved in various DNA repair processes 15

16 The SOS response: induced state lexa gene Active RecA stimulates LexA to cleave itself reca gene LexA protein Cleaved LexA protein RecA protein becomes activated by DNA damage No repression; transcription of Repair genes takes place SOS response: details Transition between induced and repressed states co-protease activity of RecA Treatments SOS response Damage DNA (UV, chemicals, ) Interfere with DNA replication Nalidixic acid DNA gyrase Starvation of a thymine auxotroph Activate RecA 16

17 Functions of SOS genes Direct repair of DNA UvrA + UvrB excision repair RuvA + RuvB + RecA daughter strand gap repair Inhibition of cell division Lon (SOS gene) SulA Ftsz (septation) Mutagenesis UmuC + UmuD RecA UmuD' UmuD'2UmuCRecA mutasome proofreading activity of DNA Pol. DNA Pol. Proceeds past the damaged site Prophage induction SOS response prophage induction Lyzogeny insertion of l genome into host genome prophage released lytic pathway ci repressor LexA (2 domains + flexible linked) Nt Ct DNA binding Connector Dimerization 17

18 How does the phage switch from one state to the other? System is bistable, with two stable steady states: lysogeny: repressor levels are high, cro inactive, levels of Cro low. lysis: levels of Cro are high, ci inactive, levels of repressor low. Cro then triggers production of other phage genes needed to continue lytic growth. SOS activation of RecA cleavage of LexA lytic genes derepressed lysis Nt Ct DNA binding Connector Dimerization 18

19 RecA cleaves repressor rendering it unable to dimerize and hence inactive Once cleaved, ci repressor is unable to dimerize 19

20 Prophage induction Cleavage of ci <<< cleavage of LexA Damage is not severe DNA repair occurs before complete cleavage of ci no prophage induction High levels of damage ci cleavage completion prophage induction Differential sensitivity of LexA and ci SOS induction reversible Prophage induction irreversible Role of genetic switches Motivation key role in development What distinguishes different cell types is the complement of genes which are expressed Cell differentiation a cell "chooses" to express a certain gene profile "Choice" is based on signals from the environment These signals are often the result of gradients of chemical messengers (morphogens) A switch-like response allows a gradient to set up differentiation into a discrete number of cell types 20

21 DNA repair systems Direct repair system Excision repair system Base excision Nucleotide excision Mismatch repair system errors of DNA replication Recombination repair system (daughter strand repair system repair of ds breaks 21

22 Direct repair system The end 22