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
01-10-2020
Dr.SS

2. UNIT–III
CLASSIFICATION
OFVIRUS
 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.
01-10-2020
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. lregulation-
complicated!
Whystudyl?
 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. geneexpression
 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. Immediateearly
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
regulatorof
lysogeniccycle
 (-) 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. Delayedearly
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. Otherregulators
 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-lrepressor-
threeregulatory
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-lyticgrowth
 1. (-) regulator of pRM
 2. (-) regulator of pR, pL - only when high [cro]

16. cII-(+)regulatorleading
tohigherlevelsof
transcriptionofgenes
importantforlysogenic
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. Lysogeniccycle
 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. Lyticcycle
 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. lcontrolregion-
immunityregion
 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. Whatproteins
bindtothese
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. cIcanactasa
(+)or(-)
regulatorof
pRM
 with low to intermediate [cI], then cI acts as (+)
regulator
 with high [cI], cI acts as (-) regulator
 these are autoregulatory loop

23. cIIprotein
 (+) 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
operatorsOL
andOR
 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. Hardtotell
whichcyclewill
winout.
 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
Questionsto
think
sivasan91@gmail.com
01-10-2020
Dr.SS

29. 01-10-2020
Dr.SS