Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Genes and Bioinformatics > Week 3 - Transcriptional Regulation in Bacteriophage > Flashcards

Week 3 - Transcriptional Regulation in Bacteriophage Flashcards

1
Q

Bacteriophage (phage)

A

bacterial viruses
• protein head with genome
• parasites

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Three major morphological classes

A
  • icosahedral tailless
  • ICOSAHEDRAL TAILED
  • filamentous
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Lytic cycle of phages

A
  1. λ phage enters bacterial cell
  2. transcription, translation, and replication
  3. assembly, packaging
  4. lysis, λ phage released
  5. λ phage attaches to bacterial cell
    to start cycle again
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Lysogenic cycle

A
  1. λ enters the bacterial cell
  2. repression
  3. integration
    - -cellular reproduction–
  4. induction (into lytic cycle)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Lytic cycle

A

DNA replication and lysis of host cell to release progeny phage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Lysogenic cycle

A

DNA insertion into a specific site in the bacterial chromosome, latency as a prophage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Prophage can be induced to

A

excise and enter the lytic cycle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Lytic development is divided

A

into 2 periods

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

A phage infection generates

A

a pool of progeny phage genomes that replicate and recombine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Usually phage has genes whose function is to

A

ensure preferential replication of phage DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Lytic development is accomplished by a pathway in which

A

phage genes are expressed in a particular order

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Lysogenic = insertion of DNA

dormant =

A

prophage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Lytic = DNA replication

A

immediately

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

If host is in a good environment

A

dormant –>
host and phage multiply
then excise genome and replicate itself

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Lytic development is controlled by a

A

cascade

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Cascade

A

a sequence of events, each of which is stimulated by the previous one

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Middle genes

A

(in lytic cycle)

includes regulators to transcribe late genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Middle phage genes

early product causes transcription of middle genes

A

regulator genes
• sigma factor, or
• antitermination factor

structural genes
• replication enzymes, etc.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Late phage genes

middle product causes transcription of late genes

A

structural genes

• phage components

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

The first genes expressed (early genes)

A

must be expressible by host RNA polymerase

• they’re phage regulatory proteins that hijack host RNA polymerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Antitermination factors

A

early genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Early middle late genes general

A

early genes = antitermination factors
middle genes = regulators to transcribe late genes
late genes = structural

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

2 types of regulatory events control the lytic cascade

A

• control at initiation
or
• control at termination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Control at initiation (lytic)

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

New sets of [lytic] genes are distinguished by

A

different promoters from those originally recognized by the host RNA polymerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Original host RNA polymerase

A

holoenzyme with σ70 recognizes one set of promoters

• makes new sigma factor or RNA polymerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Phage sigma factor

A

causes host enzyme to recognize new promoters

replace with factor that the phage can control

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

Phage RNA polymerase

A

recognizes new set of promoters

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

Control at initiation

A

Regulator proteins in phage cascades may sponsor
• initiation at new (phage) promoters
or
• cause the host polymerase to read through transcription terminators (antitermination)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

Control at termination

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

Control at termination steps

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

Phage antitermination factors

A

N and Q

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

Lambda can replicate through

A

a lytic or lysogenic life cycle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

Lambda genes are

A

clustered according to function

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

The cos elements of lambda

A

allow circularization after infection of host

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

Stage: early
Activity:

A

• host RNA polymerase transcribes N and cro from PL and PR

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

Stage: delayed early
Activity:

A

pN permits transcription from same promoters to continue past N and cro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

Stage: Late
Activity:

A

transcription initiates at Pr’ (between Q and S)

and pQ permits it to continue through all late genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

Recombination of genes to

A

insert into host

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

Infects host then

A

circularizes genome

• originally linear

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

Enters cell, begins transcription of

A

a few essential genes
• then genes to left and right
• then head and tail genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

cIII gene function

A

positive regulator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

N gene function

A

antiterminator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

cI gene function

A

repressor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
52
Q

cro gene function

A

antirepressor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
53
Q

cII gene function

A

positive regulator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
54
Q

The lambda regulatory region

A
  • cIII
  • tL
  • N
  • nutL
  • PL/OL
  • cI
  • PRM .//. PR/OR
  • cro
  • nutR
  • tR1
  • PRE
  • cII
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
55
Q

PL and PR promoters lie

A

on either side of the cI gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
56
Q

Associated with each promoter is

A

an operator at which repressor proteins bind to prevent RNA polymerase from initiating transcription
(promoter = PR operator = OR)
(promoter = PL operator = OL)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
57
Q

The sequence of each operator

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
58
Q

The lytic cycle depends on

A

antitermination by pN

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
59
Q

The lytic cycle depends on antitermination by

A

pN

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
60
Q

Lambda has 2 intermediate early genes

A

N and cro

• transcribed by host RNA polymerase from promoters PL and PR

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
61
Q

cro

A

trascriptional repressor that prevents expression of the cI gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
62
Q

N gene

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
63
Q

pQ

A

the product of a delayed early gene
• another antiterminator that allows RNA polymerase to transcribe the late genes
(genes required for phage assembly)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
64
Q

Immediate early

• transcription from PR and PL

A

N and Cro are transcribed and translated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
65
Q

Delayed early

• antitermination by N at tL and tR

A

transcription of delayed early genes
• cIII
• cII
• Q

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
66
Q

Delayed early continuation

• Cro (repressor) binds

A

binds OR
• shuts off PR and PRM
(cI gene off)

binds OL
• shuts off PL

–> all early genes switched off

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
67
Q

Late expression

• antitermination by Q

A

• 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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
68
Q

Overview of lytic infectin

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
69
Q

At the beginning of lytic infection

A
  • 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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
70
Q

Lambda immediate early and delayed early genes are needed for

A

both lytic and lysogenic infection

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
71
Q

Lambda immediate early and delayed early genes are required for both lytic and lysogenic cycles

A

• 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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
72
Q

The transcriptional circuit for the lytic cycle

A

is INTERLOCKED with the circuit for establishing lysogeny

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
73
Q

When lambda enters a host cell

A

lytic and lysogenic pathways start in the same way

• BOTH require the expression of N and cro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
74
Q

Lytic and lysogenic pathways start in the same way

A

BOTH require the expression of N and cro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
75
Q

Lysogeny requires

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
76
Q

The critical gene in maintaining lysogeny is

A

the lambda repressor - cI

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
77
Q

Lytic and lysogenic both transcribe cro and N

• early genes expressed, then

A

decide if its going to make genes to package itself (lytic) or go dormant (with cI gene - lysogenic)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
78
Q

PL and PR

A

promoters, lie on either side of the cI gee

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
79
Q

PRM

A

the promoter required for transcription of the cI gene

also requires PRE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
80
Q

The promoter required for transcription of the cI gene

A

PRM

also requires PRE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
81
Q

PRE

A

also influcences the transcription of the cI gene

82
Q

Left goes

A

N –> cIII

83
Q

Right goes

A

cro –> cII –> head and tail

84
Q

cI (repressor) is in the middle

A

of PL/OL and PR/OR
also between N and cro
• cI has its own promoter (regulated separately from genes it controls)

85
Q

Lysogeny is maintained by

A

the lambda repressor protein

86
Q

The lambda repressor

A
  • 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
Q

The lambda repressor acts at

A

the OL and OR operators
• to block transcription of the immediate early genes
–> thus preventing the lytic cycle from proceeding

88
Q

The binding of the lambda repressor to OR

A

also stimulates transcription of cI - its own gene - from PRM

89
Q

Lysogeny is stable because

A

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
Q

The lambda repressor uses

A

a helix-turn-helix motif to bind DNA

91
Q

The lambda repressor binds DNA as

A

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
Q

Monomeric lambda repressors dimerize through

A

their C-terminal domains

93
Q

The DNA-binding site of the lambda repressor is

A

a (partially) palindromic sequence of 17 bp

94
Q

The N-terminal domain of the lambda repressor consists of

A

5 alpha helices

the C terminal structure is unknown

95
Q

Lambda repressor dimers bind

A

cooperatively to the operator

96
Q

Lambda repressor dimers bind cooperatively to

A

the operator

97
Q

Lambda repressor dimers bind cooperatively to the operator

A
  • 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
Q

The affinity (of lambda repressor binding to an operator) is 10x greater for

A

OL1 and OR1 than other operators

–> they are bound first

99
Q

Cooperativity allows repressor to bind the OL2/OR2 sites

A

at lower concentrations

100
Q

The binding site for RNA polymerase at PRM

A

overlaps with OR

mostly OR3

101
Q

Lambda repressor maintains

A

an autoregulatory circuit

102
Q

Lambda repressor maintains an autoregulatory circuit

A
  • 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
Q

Lambda repressor bound at OR2

A

contacts RNA polymerase and stabilizes/promotes its binding to PRM

104
Q

PRM is the promoter for

A

transcription of the lambda reprssor

105
Q

The lambda repressor binding to the OR simultaneously

A

blocks entry into the lytic cycle (block cro)
and
promotes its own synthesis

106
Q

Lambda repressor occupies operator

A

promotes RNA polymerase binding = synthesizes itself

107
Q

Lambda repressor’s autoregulatory circuit

A

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
Q

The lambda repressor binds DNA

A

with alpha helices 2 and 3

109
Q

The cII and cIII genes are also needed to

A

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
Q

pN

A

(product of N)
• allows transcription to extend further
–> cII and cIII are transcribed

111
Q

The delayed early gene products

A

cII and cIII
• are positive transcriptional regulators and are necessary for RNA polymerase to initiate transcription at the promoter PRE

112
Q

The delayed early gene products cII and cIII are positive regulators necessary

A

for RNA polymerase to initiate transcription at the promoter PRE

113
Q

cII

A

acts directly on the promtoer (PRE)

114
Q

cIII

A

protects cII from degradation

115
Q

Transcription from PRE leads to

A

synthesis of the lambda repressor and also blocks the transcription of cro

116
Q

CII binds

A

directly to host RNA polymerase

• binds RNA polymerase to PRE promoter –> produces first lambda repressor

117
Q

Summary of lysogenic infection

A
  • 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
Q

Summary of lysogenic genes

A
  • 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
Q

First molecules of the lambda repressor made

A

sit on N and cro, stimulate lambda repressor to be made from PRM

120
Q

Lambda repressor sits and represses

A

transcription of late lytic genes

• maintains reperessor by stimulating itself

121
Q

Breaking the lysogenic circuit

A
  • 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
Q

A prophage is iduced to enter the lytic cycle when

A

the lysogenic circuit is broken

• this happens when the lambda repressor is inactivated

123
Q

Absence of a repressor allows

A

RNA polymerase to bind at the PL and PR promoters

–> starting the lytic cycle

124
Q

A phage can excise itself when

A

eg nutrients gone (=bacteria gone)

and go to lytic cycle

125
Q

RNA polymerase initiates at PR

A

makes cro mRNA

126
Q

RNA polymerase cannot initiate at PRM

A

in absence of a repressor

127
Q

RNA polymerase initiates at PL

A

makes N mRNA

128
Q

The DNA-binding form of the lambda repressor

A

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
Q

Cleavage of the repressor between the 2 domains

A

reduces the affinity for the operator and induces a lytic cycle

130
Q

Monomers (of lambda repressor) are in equilibrium with

A

dimers, which binds to DNA

131
Q

Cleavage of monomers (of lambda repressor)

A

disturbs equilibrium

–> dimers dissociate

132
Q

What determines the balance between lysogeny and the lytic cycle?

A

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
Q

Both Cro and repressor are expressed at

A

the delayed early stage
• repressor acts on OL and OR
• Cro acts on OL and OR

134
Q

Lysogeny requires repressor to

A

take over OL and OR

135
Q

Lytic cycle requires Cro to

A

take over OL and OR

136
Q

cII protein binds

A

the RNA polymerase that will transcribe the lambda repressor

137
Q

Battle between Cro and the lambda repressor

A
  • 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
Q

The Cro repressor is needed for

A

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
Q

Cro is responsible for preventing

A

the synthesis of the lambda repressor protein

140
Q

Cro binds to the same operators as the lambda repressor

A

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
Q

When Cro binds to OR3

A

it prevents RNA polymerase from binding to PRM and blocks transcription from the maintenance of repressor protein

142
Q

When Cro binds to operators at OR or OL

A

it prevents RNA polymerase from expressing immediate early genes, which indirectly blocks repressor establishment

143
Q

The cos elements of lambda DNA allows

A

allows circularization

144
Q

What allows circularization?

A

the cos elements of lambda DNA

145
Q

Process of circularization

A

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
Q

Cos elements

A

single-stranded overhangs that are complementary to each other –> can circularize

147
Q

Circularize when

A

either integration OR lysis

148
Q

The new phages need

A

linear - but the DNA is circularized

149
Q

Lambda DNA replication during the lytic cycle

A

2 types of lambda DNA replication during the lytic cycle
• bidirectional/theta θ replication
• rolling circle replication

150
Q

Bidirectional/theta θ replication

A

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
Q

Rolling circle replication

A

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
Q

Bidirectional lambda DNA replication

A
  • 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
Q

The function of theta (bidirectional) replication is to

A
  • increase the number of templates available for transcription
  • to provide circular DNA molecules for the next stage of replication
154
Q

Replication bubble

A
  • in bidirectional/theta replication
  • contains newly synthesized DNA
  • grows as DNA synthesis continues
155
Q

Rolling circle lambda DNA replication

A
  • 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
Q

Steps of rolling circle replication

A
  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
Q

In rolling circle replication, the 5’ strand is pulled away

A

leaving a single strand to act as a template

158
Q

Rolling circle replication’s purpose is to

A

make linear DNA from a circular template so that it may be packaged into the phage capsid/head

159
Q

Packaging of DNA in phage head

A

• 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
Q

Ends of the DNA molecule in the lambda particle always have

A

single-stranded 5’-ends

161
Q

Phage DNA replication circuit

A
  1. absorption
  2. DNA injection
  3. DNA circularization
  4. ligation
  5. supercoiling
    (6a. θ-mode replication)
    6b. rolling circle replication
  6. packaging of DNA in phage head
  7. addition of tail
162
Q

Site specific recombination (general)

A

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
Q

Site specific recombination (specifics)

A
  • 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
Q

Integration

attB x attP

A

requires the product of the phage gene int

• codes for an integrase enzyme Int and a bacterial protein called integration host factor (IHF)

165
Q

Excision

attL x attR

A

requires the product of the gene xis
• codes for an excisionase enzyme Xis
• in addition to Int and IHF

166
Q

Bacteriphages form plaques on a bacterial lawn

A
  • 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
Q

Temperate phage generates

A 
turbid plaques
(temperate = lysogenic)
168
Q

Mutants of phage that have lost the capacity to lysogenize form

A

clear plaques

lytic

169
Q

Dormant prophages can be induced to enter the lytic cycle when

A

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
Q

Phages with a mutation in the cI gene

A
  • 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
Q

Clear plaques

A

all host cells lysed by lytic phages

172
Q

Turbid plaques

A

some cells lyesd, some lysogenized

halo

173
Q

Mutations in E. coli can effect how the phage interacts with the host

A
  • 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
Q

Phage mutants with defects in rolling circle replication

A
  • 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
Q

In phage lambda, genes are organized into functional groups whose expression is controlled by

A

individual regulatory events

176
Q

N codes an antiterminator that allows expression of

A

leftward and rightward groups of delayed early genes from the early promtoers (PL and PR)

177
Q

The delayed early gene Q has a similar function, allowing

A

transcription of all late genes from PR’

178
Q

The lytic cycle is repressed, and the lysogenic state maintained, by expression of

A

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
Q

Lambda repressor levels are maintained by

A

expression from PRM

180
Q

Lambda repressor is a dimer and uses

A

helix-turn-helix motif to bind DNA

181
Q

Cleavage of the lambda repressor dimer results in

A

a loss of lysogeny due to the inability of lambda repressor to bind DNA

182
Q

Cro binds the

A

same sites as the lambda repressor but has different affinities

183
Q

Cro binding to OR3

A

prevents synthesis of the lambda repressor from PRM

184
Q

Establishment of lambda repressor synthesis requires

A
  • the product of the cII gene and

* transcription from PRE

185
Q

cIII gene product is required to

A

stabilize the cII product against degradation

186
Q

By turning off cII and cIII expression

A

Cro acts to prevent lysogeny

187
Q

By turning off all genes except its own (cI), the lambda repressor acts

A

to prevent the lytic cycle

188
Q

The choice between lysis or lysogeny depends on

A

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
Q

The stability of cII protein in the infected cell is

A

the primary determinant of lysis v lysogeny

190
Q

upon lambda bacteriophage infection, lambda DNA

A

circularizes through cohesive ends (cos sites)

191
Q

During the lytic cycle, lambda phage DNA replicates

A

by 2 mechanisms
• theta (bidirectional) replication
followed by
• rolling circle replication

192
Q

Theta replication provides a means of

A

rapidly multiplying copies of the phage genome

193
Q

Rolling circle replication produces

A

linear cocatemers of repeating copies of the phage genome, separated by cos sites

194
Q

Cleavage of the concatemers by phage encoded nuclease allows

A

packaging of the DNA into phage heads and subsequent assembly of the new phage

195
Q

Lysogeny requires the phage genome to

A

integrate into the host bacterial genome

• by site-specific recombination

196
Q

Site specific recombination allows

A

the phage genome to integrate itself into the bacterial genome
==> lysogeny

197
Q

Site-specific recombination between

A

a site on the phage genome (attP)
and a site on the bacterial genome (attB)
• a core sequence is shared between both sites

198
Q

Integration (attB x attP) requires

A

the phage Int gene (integrase)

and a bacterial protein IHF

199
Q

Excision (attL x attR) requires

A

the phage Xis gene (excisionase)

along with Int and IHF

200
Q

Site-specific recombination involves

A

breakage and reunion of DNA strands

Genes and Bioinformatics (10 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions