Transcript Chapter 14

Chapter 14
Phage Strategies
14.1 Introduction
 Bacteriophages are viruses that infect bacteria.
Figure 14.CO: TEM of a T4 phage.
© Dr. Harold Fisher/Visuals Unlimited
14.2 Lytic Development Is Divided into Two
Periods
• A phage infective cycle is divided into the:
– early period (before replication)
– late period (after the onset of replication)
• A phage infection generates a pool of progeny
phage genomes that replicate and recombine.
14.2 Lytic Development Is Divided into Two
Periods
Figure 14.1: A temperate phage may
follow the lytic or lysogenic pathway.
14.2 Lytic Development Is Divided
into Two Periods
Figure 14.2: Phages reproduce in lytic development.
14.3 Lytic Development Is Controlled by a
Cascade
• The early genes transcribed by host RNA
polymerase following infection include, or
comprise, regulators required for expression of
the middle set of phage genes.
• The middle group of genes includes regulators
to transcribe the late genes.
• This results in the ordered expression of groups
of genes during phage infection.
14.3 Lytic Development Is Controlled by a
Cascade
Figure 14.3: Lytic development is a regulatory cascade.
14.4 Two Types of Regulatory Event Control
the Lytic Cascade
 Regulator proteins used in phage cascades may:
 sponsor initiation at new (phage) promoters
 cause the host polymerase to read through
transcription terminators
14.4 Two Types of Regulatory Event Control the
Lytic Cascade
Figure 14.4: A phage may control transcription
at initiation either by synthesizing a new sigma
factor that replaces the host sigma factor or by
synthesizing a new RNA polymerase.
14.4 Two Types of Regulatory Event Control
the Lytic Cascade
Figure 14.5: Control at initiation generates independent mRNAs.
14.4 Two Types of Regulatory Event Control the
Lytic Cascade
Figure 14.6: Control at termination generates a single mRNA.
14.5 Lambda Immediate Early and Delayed
Early Genes Are Needed for Both Lysogeny
and the Lytic Cycle
• Lambda has two immediate early genes, N and
cro, which are transcribed by host RNA
polymerase.
• N is required to express the delayed early genes.
14.5 Lambda Immediate Early and Delayed
Early Genes Are Needed for Both Lysogeny
and the Lytic Cycle
• Three of the delayed early genes are regulators.
• Lysogeny requires the delayed early genes cIIcIII.
• The lytic cycle requires the immediate early
gene cro and the delayed early gene Q.
14.5 Lambda Immediate Early and Delayed
Early Genes Are Needed for Both Lysogeny
and the Lytic Cycle
Figure 14.7: Lambda has two lifestyles.
14.6 The Lytic Cycle Depends on
Antitermination by pN
• pN is an antitermination factor.
– It allows RNA polymerase to continue
transcription past the ends of the two
immediate early genes.
• pQ is the product of a delayed early gene.
– It is an antiterminator that allows RNA
polymerase to transcribe the late genes.
14.6 The Lytic Cycle Depends on Antitermination
by pN
Figure 14.8: Lambda genes are functionally clustered.
14.6 The Lytic Cycle Depends on Antitermination
by pN
Figure 14.9: Similar controls apply
to left and right transcription.
14.6 The Lytic Cycle Depends on
Antitermination by pN
• Lambda DNA circularizes after
infection.
– The late genes form a single
transcription unit.
Figure 14.10: Lambda has three stages of development.
14.7 Lysogeny Is Maintained by the Lambda
Repressor Protein
• The lambda repressor, encoded by the cI gene,
is required to maintain lysogeny.
• The lambda repressor acts at the OL and OR
operators to block transcription of the immediate
early genes.
• The immediate early genes trigger a regulatory
cascade.
– Their repression prevents the lytic cycle from
proceeding.
14.7 Lysogeny Is Maintained by the Lambda
Repressor Protein
Figure 14.11: Lambda has a compact regulatory region.
14.7 Lysogeny Is Maintained by the Lambda
Repressor Protein
Figure 14.12: Repressor maintains
lysogeny
14.8 The Lambda Repressor and Its Operators
Define the Immunity Region
• Several lambdoid phages have different
immunity regions.
• A lysogenic phage confers immunity to further
infection by any other phage with the same
immunity region.
14.8 The Lambda Repressor and Its Operators
Define the Immunity Region
Figure 14.13: RNA polymerase initiates at PL and PR but not at PRM during
the lytic cycle.
14.9 The DNA-Binding Form of the Lambda
Repressor Is a Dimer
• A repressor monomer has two distinct domains.
• The N-terminal domain contains the DNAbinding site.
• The C-terminal domain dimerizes.
14.9 The DNA-Binding Form of the Lambda
Repressor Is a Dimer
Figure 14.14: Repressor has two domains.
14.9 The DNA-Binding Form of the Lambda
Repressor Is a Dimer
• Binding to the operator requires the dimeric form
so that two DNA-binding domains can contact
the operator simultaneously.
• Cleavage of the repressor between the two
domains:
– reduces the affinity for the operator
– induces a lytic cycle
14.9 The DNA-Binding Form of the Lambda
Repressor Is a Dimer
Figure 14.15: Repressor cleavage induces lytic cycle.
14.10 Repressor Uses a Helix-Turn-Helix Motif
to Bind DNA
• Each DNA-binding region in the repressor
contacts a half-site in the DNA.
• The DNA-binding site of the repressor includes
two short α-helical regions that fit into the
successive turns of the major groove of DNA.
14.10 Repressor Uses a Helix-Turn-Helix Motif to
Bind DNA
Figure 14.16: The operator is a 17-bp sequence with an axis of symmetry
through the central base pair. Each half-site is marked in light blue. Base
pairs that are identical in each operator half are in dark blue.
14.10 Repressor Uses a Helix-Turn-Helix Motif
to Bind DNA
Figure 14.18: Repressor binds DNA via two α-helices.
14.10 Repressor Uses a Helix-Turn-Helix Motif
to Bind DNA
• A DNA-binding site is a (partially) palindromic
sequence of 17 bp.
• The amino acid sequence of the recognition
helix makes contacts with particular bases in the
operator sequence that it recognizes.
14.10 Repressor Uses a Helix-Turn-Helix Motif
to Bind DNA
Figure 14.19: Helix-3 determines DNA-binding specificity.
14.11 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.
• Cooperativity allows repressor to bind the
OL1/OR2 sites at lower concentrations.
14.11 Repressor Dimers Bind Cooperatively to the
Operator
Figure 14.21: Each operator contains three repressor-binding sites and
overlaps with the promoter at which RNA polymerase binds..
14.11 Repressor Dimers Bind Cooperatively to
the Operator
Figure 14.22: Lambda repressors bind DNA cooperatively.
14.12 Lambda Repressor Maintains an
Autoregulatory Circuit
• The DNA-binding region of repressor at OR2
contacts RNA polymerase and stabilizes its
binding to PRM.
• This is the basis for the autoregulatory control of
repressor maintenance.
• Repressor binding at OL blocks transcription of
gene N from PL.
14.12 Lambda Repressor Maintains an
Autoregulatory Circuit
Figure 14.23: Helix-2 interacts with RNA polymerase.
14.12 Lambda Repressor Maintains an
Autoregulatory Circuit
• Repressor binding at OR blocks transcription of
Cro, but also is required for transcription of cI.
• Repressor binding to the operators therefore
simultaneously blocks entry to the lytic cycle and
promotes its own synthesis.
14.13 Cooperative Interactions Increase the
Sensitivity of Regulation
• Repressor dimers bound at OL1 and OL2 interact
with dimers bound at OR1 and OR2 to form
octamers.
• These cooperative interactions increase the
sensitivity of regulation.
14.13 Cooperative Interactions Increase the
Sensitivity of Regulation
Figure 14.24: In the lysogenic state, the repressors bound at OL1 and OL2
interact with those bound at OR1 and OR2. RNA polymerase in bound at
PRM (which overlaps with OR3) and interacts with the repressor bound at
OR2.
14.13 Cooperative Interactions Increase the
Sensitivity of Regulation
Figure 14.25: OL3 AND OR3 are brought into proximity by formation of the
repressor octamer, and an increase in repressor concentration allows
dimers to bind at these sites and interact.
14.14 The cII and cIII Genes Are Needed to
Establish Lysogeny
• The delayed early gene products cII and cIII are
necessary for RNA polymerase to initiate transcription at
the promoter PRE.
• cII acts directly at the promoter and cIII protects cII from
degradation.
• Transcription from PRE leads to synthesis of repressor
and also blocks the transcription of cro.
14.14 The cII and cIII Genes Are Needed to
Establish Lysogeny
Figure 14.26: Repressor establishment uses a special
promoter.
14.14 The cII and cIII Genes Are Needed to
Establish Lysogeny
• PRE has atypical sequences at –10 and –35.
• RNA polymerase binds the promoter only in the
presence of cII.
• cII binds to sequences close to the –35 region.
14.15 Lysogeny Requires Several Events
• cII and cIII cause repressor synthesis to be established.
– They trigger inhibition of late gene transcription.
• Establishment of repressor turns off immediate and
delayed early gene expression.
• Repressor turns on the maintenance circuit for its own
synthesis.
• Lambda DNA is integrated into the bacterial genome at
the final stage in establishing lysogeny.
14.15 Lysogeny Requires
Several Events
Figure 14.27: The lysogenic pathway leads
to repressor synthesis.
14.16 The Cro Repressor Is Needed for Lytic
Infection
• Cro binds to the same operators as the lambda
repressor, but with different affinities.
• When Cro binds to OR3, it:
– prevents RNA polymerase from binding to PRM
– blocks the maintenance of repressor promoter
• When Cro binds to other operators at OR or OL, it
prevents RNA polymerase from expressing immediate
early genes.
– This (indirectly) blocks repressor establishment.
14.16 The Cro Repressor
Is Needed for Lytic
Infection
Figure 14.28: The lytic pathway leads to
expression of cro and late genes.
14.17 What Determines the Balance Between
Lysogeny and the Lytic Cycle?
• The delayed early stage when both Cro and
repressor are being expressed is common to
lysogeny and the lytic cycle.
• The critical event is whether cII causes sufficient
synthesis of repressor to overcome the action of
Cro.
14.17 What Determines
the Balance Between
Lysogeny and the Lytic
Cycle?
Figure 14.29: Repressor determines
lysogeny, and Cro determines the lytic
cycle.