Week 3 - Transcriptional Regulation in Bacteriophage Flashcards
1
Q
Bacteriophage (phage)
A
bacterial viruses
• protein head with genome
• parasites
2
Q
Three major morphological classes
A
- icosahedral tailless
- ICOSAHEDRAL TAILED
- filamentous
3
Q
T-even phages
A
inject their DNA into bacterial cells
• eg T4 phage
tail sheath extended –> contracted
protein needle of lysozome pierces cell membrane
DNA injected through receptor
• through cell wall into bacterial cytoplasm via cell membrane
4
Q
After the genome is injected
A
there’s a switch to decide if it’s going to be active (lytic - immediately replicate viral genome) or dormant (lysogenic)
5
Q
Lytic cycle of phages
A
- λ phage enters bacterial cell
- transcription, translation, and replication
- assembly, packaging
- lysis, λ phage released
- λ phage attaches to bacterial cell
to start cycle again
6
Q
Lysogenic cycle
A
- λ enters the bacterial cell
- repression
- integration
- -cellular reproduction– - induction (into lytic cycle)
7
Q
Lytic cycle
A
DNA replication and lysis of host cell to release progeny phage
8
Q
Lysogenic cycle
A
DNA insertion into a specific site in the bacterial chromosome, latency as a prophage
9
Q
Prophage can be induced to
A
excise and enter the lytic cycle
10
Q
Lytic development is divided
A
into 2 periods
11
Q
Lytic development is divided into 2 periods
A
a phage infective cycle is divided into the
• early period (before replication)
• late period (after the onset of replication)
12
Q
A phage infection generates
A
a pool of progeny phage genomes that replicate and recombine
13
Q
Usually phage has genes whose function is to
A
ensure preferential replication of phage DNA
14
Q
Lytic development is accomplished by a pathway in which
A
phage genes are expressed in a particular order
15
Q
Complete lytic development
A
Induction
• phage attaches to bacterium
• DNA injected into bacterium
Early development
• enzymes for DNA synthesis are made
• replication begins
Late development
• genomes, heads, and tails are made
• DNA packaged into heads, tails attached
Lysis
• cell is broken to release progeny phages
16
Q
Lysogenic = insertion of DNA
dormant =
A
prophage
17
Q
Lytic = DNA replication
A
immediately
18
Q
If host is in a good environment
A
dormant –>
host and phage multiply
then excise genome and replicate itself
19
Q
Lytic development is controlled by a
A
cascade
20
Q
Cascade
A
a sequence of events, each of which is stimulated by the previous one
21
Q
Transcriptional regulation is divided into stages…
A
at each stage one of the genes that is expressed encodes a regulator needed to express the genes of the next stage
• ordered expression of groups of genes during phage infection
22
Q
Early genes
A
(in lytic cycle)
transcribed by host RNA polymerase following infection
• include or comprise regulators required for expression of the middle set of phage genes
23
Q
Middle genes
A
(in lytic cycle)
includes regulators to transcribe late genes
24
Q
Early phage genes are transcribed by
A
host RNA polymerase type of gene product = regulator genes • RNA polymerase, • sigma factor, or • antitermination factor
25
Middle phage genes
| early product causes transcription of middle genes
regulator genes
• sigma factor, or
• antitermination factor
structural genes
• replication enzymes, etc.
26
Late phage genes
| middle product causes transcription of late genes
structural genes
| • phage components
27
The first genes expressed (early genes)
must be expressible by host RNA polymerase
| • they're phage regulatory proteins that hijack host RNA polymerase
28
Antitermination factors
early genes
29
Early middle late genes general
early genes = antitermination factors
middle genes = regulators to transcribe late genes
late genes = structural
30
2 types of regulatory events control the lytic cascade
• control at initiation
or
• control at termination
31
Control at initiation (lytic)
* replace the sigma factor of the host enzyme with another factor that redirects specificity to phage initiation
* synthesis of a new (phage) RNA polymerase
* new sets of genes are distinguished by different promoters from those originally recognized by the host RNA polymerase
32
New sets of [lytic] genes are distinguished by
different promoters from those originally recognized by the host RNA polymerase
33
Original host RNA polymerase
holoenzyme with σ70 recognizes one set of promoters
| • makes new sigma factor or RNA polymerase
34
Phage sigma factor
causes host enzyme to recognize new promoters
| replace with factor that the phage can control
35
Phage RNA polymerase
recognizes new set of promoters
36
Control at initiation
Regulator proteins in phage cascades may sponsor
• initiation at new (phage) promoters
or
• cause the host polymerase to read through transcription terminators (antitermination)
37
Control at termination
* depends on the arrangement of genes
* early genes lie adjacent to the genes that are to be expressed next, but are separated by terminator sites
* if termination is prevented, the polymerase reads through genes into the other side
* some promoters continue to be recognized
38
Control at termination steps
promoter --> early region --> terminator --> next region
• antitermination factor keeps the RNA polymerase on, read through the terminator, longer transcript with early and late region
• host RNA polymerase runs through termination sequences = antitermination
• phage genes have antitermination factors N and Q
39
Phage antitermination factors
N and Q
40
Lambda can replicate through
a lytic or lysogenic life cycle
41
Lambda genes are
clustered according to function
42
The cos elements of lambda
allow circularization after infection of host
43
Stage: early
Activity:
• host RNA polymerase transcribes N and cro from PL and PR
44
Stage: delayed early
Activity:
pN permits transcription from same promoters to continue past N and cro
45
Stage: Late
Activity:
transcription initiates at Pr' (between Q and S)
| and pQ permits it to continue through all late genes
46
Recombination of genes to
insert into host
47
Infects host then
circularizes genome
| • originally linear
48
Enters cell, begins transcription of
a few essential genes
• then genes to left and right
• then head and tail genes
49
cIII gene function
positive regulator
50
N gene function
antiterminator
51
cI gene function
repressor
52
cro gene function
antirepressor
53
cII gene function
positive regulator
54
The lambda regulatory region
* cIII
* tL
* N
* nutL
* PL/OL
* cI
* PRM .//. PR/OR
* cro
* nutR
* tR1
* PRE
* cII
55
PL and PR promoters lie
on either side of the cI gene
56
Associated with each promoter is
an operator at which repressor proteins bind to prevent RNA polymerase from initiating transcription
(promoter = PR operator = OR)
(promoter = PL operator = OL)
57
The sequence of each operator
OVERLAPS with the promoter it controls
• provides a pressure point at which entry into the lytic cycle can be controlled
(either RNA polymerase can bind promoter
OR
something binds operator that prevents RNA polymerase from binding)
58
The lytic cycle depends on
antitermination by pN
59
The lytic cycle depends on antitermination by
pN
60
Lambda has 2 intermediate early genes
N and cro
| • transcribed by host RNA polymerase from promoters PL and PR
61
cro
trascriptional repressor that prevents expression of the cI gene
62
N gene
encodes an antitermination factor that acts at nut sites
causing RNA polymerase to
• continue transcription past the ends of the 2 immediate early genes (overrides the transcription termination sequences of tL and tR)
and
• transcription of the delayed early genes
(acts on 2nd transcription process, extends protein to the left)
63
pQ
the product of a delayed early gene
• another antiterminator that allows RNA polymerase to transcribe the late genes
(genes required for phage assembly)
64
Immediate early
| • transcription from PR and PL
N and Cro are transcribed and translated
65
Delayed early
| • antitermination by N at tL and tR
transcription of delayed early genes
• cIII
• cII
• Q
66
Delayed early continuation
| • Cro (repressor) binds
binds OR
• shuts off PR and PRM
(cI gene off)
binds OL
• shuts off PL
--> all early genes switched off
67
Late expression
| • antitermination by Q
• activation of PR'
• transcription of late genes
(head and tail genes required for new phage particles)
• Cro represses all of early genes, pQ activates late expression
68
Overview of lytic infectin
1. immediate early
• N and cro are transcribed
2. Delayed early
• N antiterminates
• cII and cIII are transcribed
3. Delayed early continuation
• Cro binds to OL and OR
4. Late expression
• Cro represses cI and all early genes
• pQ activates late expression
69
At the beginning of lytic infection
* injects double stranded linear DNA --> circularizes
* host RNA polymerase doesn't distinguish phage promoters (look like host promoters) --> RNA polymerase binds
* transcribes cro and N (N extends both ways)
70
Lambda immediate early and delayed early genes are needed for
both lytic and lysogenic infection
71
Lambda immediate early and delayed early genes are required for both lytic and lysogenic cycles
• the transcriptional circuit for the lytic cycle is interlocked with the circuit for establishing lysogeny
• when lambda enters a host cell, the lytic and lysogenic pathways start in the same way
(BOTH require the expression of N and cro)
• lysogey requires the delayed early genes cII-cIII
--> the 2 life cycle transcriptional pathways diverge
• the critical gene in maintaining lysogeny is the lamda repressor (cI)
72
The transcriptional circuit for the lytic cycle
is INTERLOCKED with the circuit for establishing lysogeny
73
When lambda enters a host cell
lytic and lysogenic pathways start in the same way
| • BOTH require the expression of N and cro
74
Lytic and lysogenic pathways start in the same way
BOTH require the expression of N and cro
75
Lysogeny requires
the delayed early genes cII and cIII
--> the 2 life cycle transcriptional pathways diverge
• the critical gene in maintaining lysogeny is the lambda repressor cI
76
The critical gene in maintaining lysogeny is
the lambda repressor - cI
77
Lytic and lysogenic both transcribe cro and N
| • early genes expressed, then
decide if its going to make genes to package itself (lytic) or go dormant (with cI gene - lysogenic)
78
PL and PR
promoters, lie on either side of the cI gee
79
PRM
the promoter required for transcription of the cI gene
| also requires PRE
80
The promoter required for transcription of the cI gene
PRM
| also requires PRE
81
PRE
also influcences the transcription of the cI gene
82
Left goes
N --> cIII
83
Right goes
cro --> cII --> head and tail
84
cI (repressor) is in the middle
of PL/OL and PR/OR
also between N and cro
• cI has its own promoter (regulated separately from genes it controls)
85
Lysogeny is maintained by
the lambda repressor protein
86
The lambda repressor
* coded by the cI gene
* required to maintain a lysogenic cycle
* is a transcriptional repressor
* made as a monomer but becomes a dimer, can bind L and R operators = RNA polymerase can't produce anything L or R
87
The lambda repressor acts at
the OL and OR operators
• to block transcription of the immediate early genes
--> thus preventing the lytic cycle from proceeding
88
The binding of the lambda repressor to OR
also stimulates transcription of cI - its own gene - from PRM
89
Lysogeny is stable because
the control circuit ensures that so long as the level of lambda repressor is adequate, there is continued expression of cI
(repressor high = repressor activated)
(cI stimulates its own production)
90
The lambda repressor uses
a helix-turn-helix motif to bind DNA
91
The lambda repressor binds DNA as
a dimer
• monomeric lambda repressors dimerize through their C-terminal domains
• one monomer binds one half site, other monomer binds the other half site
92
Monomeric lambda repressors dimerize through
their C-terminal domains
93
The DNA-binding site of the lambda repressor is
a (partially) palindromic sequence of 17 bp
94
The N-terminal domain of the lambda repressor consists of
5 alpha helices
| the C terminal structure is unknown
95
Lambda repressor dimers bind
cooperatively to the operator
96
Lambda repressor dimers bind cooperatively to
the operator
97
Lambda repressor dimers bind cooperatively to the operator
* repressor binding to one operator increases the affinity for binding a second repressor dimer to the adjacent operator
* the affinity is 10x greater for OL1 and OR1 than other operators, so they are bound first
* promotes RNA polymerase binding
98
The affinity (of lambda repressor binding to an operator) is 10x greater for
OL1 and OR1 than other operators
| --> they are bound first
99
Cooperativity allows repressor to bind the OL2/OR2 sites
at lower concentrations
100
The binding site for RNA polymerase at PRM
overlaps with OR
| mostly OR3
101
Lambda repressor maintains
an autoregulatory circuit
102
Lambda repressor maintains an autoregulatory circuit
* lambda repressor bound at OR2 contacts RNA polymerase and stabilizes/promotes its binding to PRM
* PRM is the promoter for transcription of the lambda repressor
* this is the basis for autoregulatory maintenance
* lambda repressor binding to the OR simultaneously blocks entry into the lytic cycle (block cro) and promotes its own synthesis
103
Lambda repressor bound at OR2
contacts RNA polymerase and stabilizes/promotes its binding to PRM
104
PRM is the promoter for
transcription of the lambda reprssor
105
The lambda repressor binding to the OR simultaneously
blocks entry into the lytic cycle (block cro)
and
promotes its own synthesis
106
Lambda repressor occupies operator
promotes RNA polymerase binding = synthesizes itself
107
Lambda repressor's autoregulatory circuit
N -- PL/OL -- cI -- PRM//PR/OR -- cro
• repressor prevents RNA polymerase from binding PR (and PL)
• RNA polymerase (moves back to binding at PR
108
The lambda repressor binds DNA
with alpha helices 2 and 3
109
The cII and cIII genes are also needed to
establish lysogeny
• when lambda DNA enters a new host cell, there is no repressor --> N and cro are transcribed
• pN allows transcription to extend further (allowing cII and cIII to be transcribed)
• the delayed early gene products cII and cIII are positive transcriptional regulators and are necessary for RNA polymerase to initiate transcription at the promoter PRE
• cII acts directly on the promoter and cIII protects cII from degradation
• transcription from PRE leads to synthesis of the lambda repressor and also blocks the transcription of cro
110
pN
(product of N)
• allows transcription to extend further
--> cII and cIII are transcribed
111
The delayed early gene products
cII and cIII
• are positive transcriptional regulators and are necessary for RNA polymerase to initiate transcription at the promoter PRE
112
The delayed early gene products cII and cIII are positive regulators necessary
for RNA polymerase to initiate transcription at the promoter PRE
113
cII
acts directly on the promtoer (PRE)
114
cIII
protects cII from degradation
115
Transcription from PRE leads to
synthesis of the lambda repressor and also blocks the transcription of cro
116
CII binds
directly to host RNA polymerase
| • binds RNA polymerase to PRE promoter --> produces first lambda repressor
117
Summary of lysogenic infection
* PR and PL active = synthesize N and Cro
* antitermination by N = synthesizes cIII, cII, and Q
* cII and cIII cause repressor synthesis to be established and also trigger inhibition of late gene transcription
* cII stimulates expression from PRM (cI repressor) by binding PRE
* cIII stabilizes cII
* cI repressor shuts of expression from PR, PL, and PR' (no lytic functions), stimulates PRM and therefore its own synthesis
118
Summary of lysogenic genes
* immediate early = N and cro are transcribed
* delayed early = N antiterminates, cII and CIII are transcribed
* lysogenic establishment = cII acts at PRE, cI is transcribed
* lysogenic maintenance = repressor binds at OL and OR, cI is transcribed from PRM
119
First molecules of the lambda repressor made
sit on N and cro, stimulate lambda repressor to be made from PRM
120
Lambda repressor sits and represses
transcription of late lytic genes
| • maintains reperessor by stimulating itself
121
Breaking the lysogenic circuit
* a prophage is induced to enter the lytic cycle when the lysogenic circuit is broken
* this happens when the lambda repressor is inactivated
* absence of a repressor allows RNA polymerase to bind at the PL and PR promoters starting the lytic
122
A prophage is iduced to enter the lytic cycle when
the lysogenic circuit is broken
| • this happens when the lambda repressor is inactivated
123
Absence of a repressor allows
RNA polymerase to bind at the PL and PR promoters
| --> starting the lytic cycle
124
A phage can excise itself when
eg nutrients gone (=bacteria gone)
| and go to lytic cycle
125
RNA polymerase initiates at PR
makes cro mRNA
126
RNA polymerase cannot initiate at PRM
in absence of a repressor
127
RNA polymerase initiates at PL
makes N mRNA
128
The DNA-binding form of the lambda repressor
is a dimer
• the dimeric structure of the lambda repressor is crucial in maintaining lysogeny
• cleavage of the repressor between the 2 domains reduces the affinity for the operator and induces a lytic cycle
• cleavage occurs under certain adverse conditions
129
Cleavage of the repressor between the 2 domains
reduces the affinity for the operator and induces a lytic cycle
130
Monomers (of lambda repressor) are in equilibrium with
dimers, which binds to DNA
131
Cleavage of monomers (of lambda repressor)
disturbs equilibrium
| --> dimers dissociate
132
What determines the balance between lysogeny and the lytic cycle?
the delayed early stage when both Cro and lambda repressor are being expressed is common to lysogeny and the lytic cycle
• the critical event is whether cII causes sufficient synthesis of lambda repressor to overcome the action of Cro
• lysogeny will result if the lambda repressor occupies the operators, otherwise Cro occupies the operators resulting in the lytic cycle
133
Both Cro and repressor are expressed at
the delayed early stage
• repressor acts on OL and OR
• Cro acts on OL and OR
134
Lysogeny requires repressor to
take over OL and OR
135
Lytic cycle requires Cro to
take over OL and OR
136
cII protein binds
the RNA polymerase that will transcribe the lambda repressor
137
Battle between Cro and the lambda repressor
* Cro = transcriptional repressor, sits on L and R promoter
* Cro sits on PR = affects PRM (overlaps)
* if the lambda repressor dominates, RNA polymerase on cI --> lambda repressor made
* cro dominates = repressor off, early genes off, head and tail transcribed
138
The Cro repressor is needed for
lytic infection
• Cro is responsible for preventing the synthesis of the lambda repressor protein
• Cro binds to the same operators as the lambda repressor, but with different affinities
• the affinity of Cro for OR3 is greater than its affinity for OR2 or OR1
• when Cro binds to OR3 it prevents RNA polymerase from binding to PRM and blocks transcription from the maintenance of repressor protein
• when Cro binds to other operators at OR or OL, it prevents RNA polymerase from expressing immediately early genes, which indirectly blocks repressor establishment
139
Cro is responsible for preventing
the synthesis of the lambda repressor protein
140
Cro binds to the same operators as the lambda repressor
but with different affinities
• lambda repressor sits on OR1 then OR2, not OR3
• Cro sits on the same thing but in the opposite direction - OR3 OR2 OR1
141
When Cro binds to OR3
it prevents RNA polymerase from binding to PRM and blocks transcription from the maintenance of repressor protein
142
When Cro binds to operators at OR or OL
it prevents RNA polymerase from expressing immediate early genes, which indirectly blocks repressor establishment
143
The cos elements of lambda DNA allows
allows circularization
144
What allows circularization?
the cos elements of lambda DNA
145
Process of circularization
start with linear molecule of lambda DNA
• base sequences of the 5' overhangs, known as cohesive ends or cos elements, are complementary to each other
• by forming base pairs between the single-stranded ends, the linear DNA molecule can circularize
• cohesive ends are 12bp in length
--> infection of bacterial cell -->
• produces an open circle containing 2 single-stranded breaks
(double-stranded circle)
• host DNA ligase converts the nicked circular DNA molecule into a covalent circle
• DNA topoisomerases then convert the relaxed closed covalent circular DNA into supercoiled DNA
(supercoiled to look like host)
146
Cos elements
single-stranded overhangs that are complementary to each other --> can circularize
147
Circularize when
either integration OR lysis
148
The new phages need
linear - but the DNA is circularized
149
Lambda DNA replication during the lytic cycle
2 types of lambda DNA replication during the lytic cycle
• bidirectional/theta θ replication
• rolling circle replication
150
Bidirectional/theta θ replication
begins at a specific site on the circular DNA
• produces 2 replication forks which move in opposite directions around the lambda DNA circle
• make many copies of itself
151
Rolling circle replication
generation of long concatemers that are required to package DNA into the phage head
• produces multiple copies of the lambda genome into a suitable form for packaging into phage capsids
• makes copies in linear form - because must be linear to put back phage capsids
152
Bidirectional lambda DNA replication
* 2 growing points start at the same site and move in opposite directions until they meet at opposite sides of the circle
* a region called the replication bubble contains newly synthesized DNA, grows as DNA synthesis continues
* the function of theta replication is to increase the number of templates available for transcription and to provide circular DNA molecules for the next stage of replication
* makes 2 new double-stranded circular DNA molecules but are interlinked (un linked by host enzymes)
153
The function of theta (bidirectional) replication is to
* increase the number of templates available for transcription
* to provide circular DNA molecules for the next stage of replication
154
Replication bubble
* in bidirectional/theta replication
* contains newly synthesized DNA
* grows as DNA synthesis continues
155
Rolling circle lambda DNA replication
* begins with a nick (single-stranded break) at the origin of replication
* the 5' end is displaced from the strand
* the 3' end acts as a primer for DNA polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template
* the 5' end continues to be displaced as the circle rolls, and is protected by SSBs until discontinuous DNA synthesis makes it a dsDNA again
* multiple copies of the phage genome are synthesized, all joined together in long concatemers
156
Steps of rolling circle replication
1. nick is made in the + strand of the parental duplex
(O = origin)
2. the 5' end is displaced and covered by SSBs
3. polymerization at the 3' end adds new deoxyribonucleotides
4. attachment of replisome and formation of Okazaki fragments
157
In rolling circle replication, the 5' strand is pulled away
leaving a single strand to act as a template
158
Rolling circle replication's purpose is to
make linear DNA from a circular template so that it may be packaged into the phage capsid/head
159
Packaging of DNA in phage head
• ends of the DNA molecule in the lambda particle always have single-stranded 5' ends
• long concatemers must be cut at their cos sites to generate these termini
• cutting at cos sites is accomplished by a sequence-specific terminase (lambda ter gene)
• this endonucleases cuts at cos sites generating 5' overhangs
• cutting and packaging are somehow coupled
• tail is added to packaged head
--> mature phage particle
160
Ends of the DNA molecule in the lambda particle always have
single-stranded 5'-ends
161
Phage DNA replication circuit
1. absorption
2. DNA injection
3. DNA circularization
4. ligation
5. supercoiling
(6a. θ-mode replication)
6b. rolling circle replication
7. packaging of DNA in phage head
8. addition of tail
162
Site specific recombination (general)
X - A B - Y
(X and Y = bacterial DNA)
(A and B = ends of phage DNA)
integration = insertion of the A and B sequences between the X and Y sequences
• promoted by integrase enzymes
excision = reversal of integration, excision of A and B sequences
• note the integration mechanism and excision mechanism use different reacting sequences
163
Site specific recombination (specifics)
* the bacterial attachment site is called attB, consisting of the sequence components BOB'
* the attachment site on the phage is called attP, consisting of the compounds POP'
* the sequence O is common to attB and attP = the CORE SEQUENCE - recombination occurs within it
• the prophage is bound by 2 new att sites (the products of the recombination) called attL and attR
integration (attB x attP) requires the product of the phage gene - int
• codes for an integrase enzyme Int and a
• bacterial protein called integration host factor (IHF)
(doesn't generate the same sequence that the host/phage already had or would come back out)
excision (attL x attR) requires the product of the gene xis
• codes for an excisionase enzyme - Xis
• in addition to Int and IHF
164
Integration
| attB x attP
requires the product of the phage gene int
| • codes for an integrase enzyme Int and a bacterial protein called integration host factor (IHF)
165
Excision
| attL x attR
requires the product of the gene xis
• codes for an excisionase enzyme Xis
• in addition to Int and IHF
166
Bacteriphages form plaques on a bacterial lawn
* phage numbers can be counted using a plaque assay
* grow bacteria on a nutrient agar plate, colonies grow and appear as a lawn
* phage particles are dded and infect the bacteria
* infected cell lyses ad releases new phage particles, which in turn infect more bacteria
* multiple copies of infection result in destruction of bacteria within a localized area, giving rise to a clear, transparent circular region
* this region is called a plaque
167
Temperate phage generates
```
turbid plaques
(temperate = lysogenic)
```
168
Mutants of phage that have lost the capacity to lysogenize form
clear plaques
| lytic
169
Dormant prophages can be induced to enter the lytic cycle when
the repressor dimer is inactivated
• induction happens when the repressor dimer is cleaved in the connector region
• this happens under certain adverse conditions, when the life of host bacteria is under threat
• conditions eg high temp (.37C) or by UV
• in some mutants of phage lambda (CIts) this occurs at 37C (mutant cI protein is thermolabile)
• another mutant has no functional lambda repressor (CI-)
170
Phages with a mutation in the cI gene
* when lambda phages are placed on a bacterial lawn, they will infect bacteria, forming plaques where large numbers of bacteria have been attacked
* in wild type phages, some bacteria will have been lysed by lytic phages, whereas other bacteria will live on with a copy of the phage in its genome - plaques will be cloudy
* in cI mutants, all bacteria in a plaque will have been killed by phages, since the phages can never enter the lysogenic cycle because they cannot produce a functional lambda repressor = clear plaque
171
Clear plaques
all host cells lysed by lytic phages
172
Turbid plaques
some cells lyesd, some lysogenized
| halo
173
Mutations in E. coli can effect how the phage interacts with the host
* wild type bacteria containing the hflA (high frequency oflysogenization) locus
* MUTANTS int his gene have a high frequency of lysogenization
* this is because in wild type E. coli the hflA gene product degrades the cII gene product which the phage needs for lysogeny
* hence in wild type E. coli some phages will normally enter the lytic cycle
* in hflA-mutants E. coli will rarely be lysed, most infecting phages will be in the lysogenic cycle
174
Phage mutants with defects in rolling circle replication
* E. coli produces a factor called Exo V - which degrades linear phage DNA
* wild type phages produce a factor which stops this degradation
* this factor is a product of the gamma gene
* phage lambda mutants in the gamma gene can't switch to rolling circle replication = can't produce concatemers = cannot package DNA into new phage heads
* a possible plan B for lambda is to produce circular (rather than linear) concatemers
* for this they need a recombination factor - a protein encoded by a gene called red
* mutants deficient in both genes (red- gamma-) cannot ever enter the lytic cycle, unless they are helped by the host cellular machinery
* wild type E. coli produces a recombination factor that can compensate for phage deficiencies in the red gene
* this recombination factor is encoded by E. coli's RecA gene
* if a RecA- mutant of E. coli is infected by a red- gamma- mutant phage, there will be only lysogeny, never lysis = no plaques
175
In phage lambda, genes are organized into functional groups whose expression is controlled by
individual regulatory events
176
N codes an antiterminator that allows expression of
leftward and rightward groups of delayed early genes from the early promtoers (PL and PR)
177
The delayed early gene Q has a similar function, allowing
transcription of all late genes from PR'
178
The lytic cycle is repressed, and the lysogenic state maintained, by expression of
the cI gene
• whose product is a repressor protein, the lambda repressor
• which acts on operators OL and OR to prevent use of the promoters PL and PR
179
Lambda repressor levels are maintained by
expression from PRM
180
Lambda repressor is a dimer and uses
helix-turn-helix motif to bind DNA
181
Cleavage of the lambda repressor dimer results in
a loss of lysogeny due to the inability of lambda repressor to bind DNA
182
Cro binds the
same sites as the lambda repressor but has different affinities
183
Cro binding to OR3
prevents synthesis of the lambda repressor from PRM
184
Establishment of lambda repressor synthesis requires
* the product of the cII gene and
| * transcription from PRE
185
cIII gene product is required to
stabilize the cII product against degradation
186
By turning off cII and cIII expression
Cro acts to prevent lysogeny
187
By turning off all genes except its own (cI), the lambda repressor acts
to prevent the lytic cycle
188
The choice between lysis or lysogeny depends on
whether lambda repressor or Cro occupies the operators in a particular infection
• the stability of cII protein in the infected cell is the primary determinant of this outcome
189
The stability of cII protein in the infected cell is
the primary determinant of lysis v lysogeny
190
upon lambda bacteriophage infection, lambda DNA
circularizes through cohesive ends (cos sites)
191
During the lytic cycle, lambda phage DNA replicates
by 2 mechanisms
• theta (bidirectional) replication
followed by
• rolling circle replication
192
Theta replication provides a means of
rapidly multiplying copies of the phage genome
193
Rolling circle replication produces
linear cocatemers of repeating copies of the phage genome, separated by cos sites
194
Cleavage of the concatemers by phage encoded nuclease allows
packaging of the DNA into phage heads and subsequent assembly of the new phage
195
Lysogeny requires the phage genome to
integrate into the host bacterial genome
| • by site-specific recombination
196
Site specific recombination allows
the phage genome to integrate itself into the bacterial genome
==> lysogeny
197
Site-specific recombination between
a site on the phage genome (attP)
and a site on the bacterial genome (attB)
• a core sequence is shared between both sites
198
Integration (attB x attP) requires
the phage Int gene (integrase)
| and a bacterial protein IHF
199
Excision (attL x attR) requires
the phage Xis gene (excisionase)
| along with Int and IHF
200
Site-specific recombination involves
breakage and reunion of DNA strands
