Lytic and lysogeny

Genes involved in lytic and lysogeny - Dr.SS

1. Microbial Taxonomy and Diversity – Lytic and lysogeny cycle Dr. S. Sivasankara Narayani Assistant Professor Department of Microbiology Ayya Nadar Janaki Ammal College Sivakasi 01/10/20 Dr.SS

2. UNIT – III CLASSIFICATION OF VIRUS Viruses: Physical, chemical and biological properties. Baltimore classification. Types of viruses based on structure and Host. Cultivation of viruses: Cell line - embryonated eggs - cytopathic effect. Structure of viruses: TMV- T4bacteriophage. Replication of virus: Lytic and lysogenic cycle. Phage typing. 01/10/20 Dr.SS

3. Intro l is a model organism for molecular biology. It has been studied from the late 1940s to the present. l is a bacteriophage - a virus that infects bacteria (in this case,  E. coli ) has a proteinaceous head and tail head contains ds linear DNA genome is 48 kb in length- encodes 40 genes 21 structural genes (head and tail) 19 genes for regulation of infection cycle l dependent on host enzymes for growth (uses host RNAP and DNAP). So, l promoters have the usual &endash;10 and &endash;35 conserved regions. This is in contrast to bacteriophage T7, which encodes its own RNAP that recognizes different promoter sequence

4. Intro 1. lytic cycle- the classic mechanism of viral infection infection, production of 100-200 new viral particles, lyse host cell and infect new cells exponential growth of infectious particles 2. lysogenic cycle- l integrates its DNA into  E. coli  chromosome integrated l DNA called a prophage l chromosome replicates with  E. coli  chromosome prophage can remain in this state, or can be induced ( l DNA is excised) in response to certain signals and return to lytic cycle

5. genes l lands on cell surface, injects its DNA lytic- l takes over cell machinery, goes on to infect more cells, orŠ lysogeny as a prophage. Need induction to return to lytic cycle. l lysogens are immune to further infection by l.

6. growth grow a lawn of bacteria in a petri dish small circular regions where bacteria not growing- phage have infected and lysed cells absence of bacterial growth = plaque = l growth wt  l causes turbid plaques (mixed lytic and lysogenic growth) mutant l can undergo strictly lytic growth. Get clear plaque phenotype (no growth in middle of plaque). Mutations in genes cI , cII , cIII --> clear plaques.

7. l regulation- complicated! Why study l? Model for temporal control of gene expression early genes --> late genes (regulated cascade of gene expression / regulation) 2. Model developmental system l - lytic or lysogenic insights into how eukaryotic cells differentiate during development

8. gene expression early- certain genes transcribed early, including enzymes for DNA synthesis and DNA replication. late- structural genes transcribed packaging and lysis- l particles self-assemble (DNA, head, and tail form an infectious particle) timing is critical - don't want new virus made until ready to lyse cell

9. Immediate early genes N - transcribed from pL promoter cro - transcribed from pR promoter no genes initially transcribed from promoter pR ', though pR ' is a strong promoter

10. cro - negative regulator of lysogenic cycle (-) regulator of cI (encodes l repressor) favors lytic growth

11. N - antiterminator protein regulated readthrough of transcription terminators specific for certain transcription units effect of N- readthrough of tR1, tR2, tL1, tL2 antitermination functions via sequences called  nut  sites (N-utilization sites) 2 such sites ( nutL and nutR ) are in DNA and encoded into RNA. Probably the RNA sequence is the recognition element. In the presence of N, transcription takes place through  nut  site to make RNA. N gains access to and interacts with transcription machinery here - termination of transcription no longer takes place, so additional genes transcribed ( cII and Q from pR ; cIII from pL ). This leads to production of delayed early genes

12. Delayed early genes 2 needed for DNA replication 7 recombination genes (lysogeny is a site-specific recombination event) -  int  and  xis  genes 3 regulators1. cII - positive transcriptional activator unstable protein- short 1/2 life- rapidly degraded activates initial expression of cI ( l repressor) 2. cIII - stabilizes cII at protein level protects cII protein from degradation --> longer 1/2 life 3. Q- antiterminator turns on late genes - causes regulated read through of tR ' functions via specific sites called  qut  sites (Q-utilization sites) favors lytic growth - Q gains access to transcription machinery, read through occurs therefore genes for lysis, head, and tail transcribed

13. Other regulators 1. cro - immediate early gene favors lytic cycle 2. cI - l repressor necessary for establishment and maintenance of lysogenic growth only gene required for maintenance of lysogenic growth

14. cI - l repressor- three regulatory activities 1. (-) regulator of transcription from pR and pL 2. (+) regulator of pRM ("repressor maintenance") - cI controls its own expression through a positive autoregulatory loop 3. (-) regulator of pRM - negative autoregulatory loop

15. cro - lytic growth 1. (-) regulator of pRM 2. (-) regulator of pR , pL - only when high [ cro ]

16. cII - (+) regulator leading to higher levels of transcription of genes important for lysogenic growth 1. pRE - "repressor establishment" 2. pINT -  int  codes for integrase (site-specific recombination of l into  E. coli  chromosome) 3. pANTI -Q - antisense of Q gene

17. Lysogenic cycle cII and cIII positively regulate cI (which has (+) or (-) autoregulation) cII and cIII necessary for establishment of lysogeny cI necessary for establishment and maintenance of lysogeny

18. Lytic cycle Q and cro lead to expression of late genes - head, tail, lysis if late genes transcribed, have lytic cycle - self-assembly of viral particles If all the immediate and delayed early proteins are present in  E. coli , which cycle will occur? It is a fine balance.

19. l control region - immunity region OR and OL - operators with similar but not identical sequences OL separated into suboperators OL1, OL2, and OL3 OR separated into suboperators OR1, OR2, and OR3

20. What proteins bind to these operators? cro and cI proteins are similar in 1°, 2°, and 3° structure, but are not identical have different DNA binding affinities cro : OR3 > OR2 = OR1, and OL3 > OL2 = OL1 cI : OR1 > OR2 = OR2, and OL1 > OL2 = OL3 cro and cI bind to DNA as dimers dyad symmetry in operators each monomer recognizes a 1/2 site of the suboperators

21. Lysogenic growth pRE is a weak promoter, i.e. has poor match to consensus sequences at &endash;10 and &endash;35 and thus requires (+) activator- cII protein (delayed early gene) remember that cIII proteins stabilizes the cII protein from pRE , antisense cro is transcribed - can't be transcribed into cro protein. From pR , cro is transcribed, therefore have two complementary RNA stands base pair, inhibiting translation of cro . This is down regulation of the lytic cycle. from pANTI -Q, antisense Q, RNA is transcribed. Get down regulation as above. Also called "antisense" regulation.

22. cI can act as a (+) or (-) regulator of pRM with low to intermediate [ cI ], then cI acts as (+) regulator with high [ cI ], cI acts as (-) regulator these are autoregulatory loop

23. cII protein (+) regulator of pRE promoter weak promoter- non-consensus sequences at &endash;10 and &endash;35 (only 3 of 6 bases match consensus at each position) footprinting experiment - cII binds upstream of RNAP, and overlaps a little. cII probably helps RNAP recognize promoter. cII stimulates closed complex and open complex formation with 100-fold effect.

24. complex operators OL and OR 1. each operator has dyad symmetry OR1 and OL1 are more similar than OR1 and OR3 - makes sense because recognized by l repressor OL3 similar to OR3 2. proteins bind to OR1 and OL1 - overlaps the &endash;10 sequence and block 3. binding l repressor to OR2 stimulates transcription of pRM 4. binding to OR3 blocks transcription of pRM 5. no ribosome-binding site for transcript made from pRM ; the first three bases of the RNA are AUG (initiating Met). Usually a ribosome binding site (Shine- Delgarno sequence) 5' to the AUG of initiating Met - not present here, so this RNA not translated efficiently. By contrast, the transcript initiated at pRE has a good ribosome binding site for cI and is translated efficiently.

25. Cooperative binding occurs at OR1 and OR2, and OL1 and OL2 once one molecule of l repressor is bound to OR1, very likely that second molecule of cI will bind to OR2. The end result it that there is almost simultaneous binding of cI to OR1 and OR2 binding to OR3 not cooperative cro does not exhibit cooperative binding, only cI does

26. l repressor cI binds to OR1 and OR2 - no transcription from pR (blocking &endash;10 and &endash;35 regions). But binding to OR2 stimulates transcription from pRM - (+) regulation in l lysogen, only cI gene expressed   Lytic cycle Q antiterminator - a delayed early gene. Transcription initiates at pR ' (a strong promoter), but get readthrough of tR ' because Q functions at  qut  site. Transcription of lysis, head, and tail genes takes place. cro initiated from pR - cro a (-) regulator of pRM ("repressor maintenance") cro therefore represses lysogen

27. Hard to tell which cycle will win out. cII protein- unstable, short 1/2 life cIII - stabilizes cII proteins that degrade cII are encoded by  E. coli key gene is Hfl gene which encodes Hfl protease lots of Hfl protease --> little cII (lytic) little Hfl protease --> lots of cII (lysogenic) l monitors the level of Hfl Production of Hfl protease dependent on  E. coli  growth conditions 1. rich nutrient media results in highly active Hfl protease favoring lytic growth 2. poor nutrient media results in less Hfl protease, resulting in more cII favoring lysogenic growth To induce lytic cycle from a lysogen, need to get rid of l repressor. Need more than starvation of host - DNA damage needed. Immunity l lysogens "immune" from infection by wt  l cI (made from lysogen) binds to OR1 and OL1 of incoming wt  l - blocks transcription of pR and p

28. Clarifications Questions to think [email protected] 01/10/20 Dr.SS

29. 01/10/20 Dr.SS