Major Shifts in Prokaryotic Transcription

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Chapter 8 Major Shifts in Prokaryotic
Transcription
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8.1 Modification of The Host RNA Polymerase
During Phage Infection

• SPO1(B. subtilis phage, large DNA genome)
• Temporal program of transcription

Time of infection Genes expressed RNA polymerase
0 - 5' Early genes Host holoenzyme
5 - 10 ' Middle genes gp28host core
10 - end Late genes gp33gp34host core
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Figure 8.1 Temporal control of transcription In
phage SPO1- infected B. subtilis. (a) Early
transcription is directed by the host RNA
polymerase holoenzyme, including the host s
factor (blue) one of the early phage proteins is
gp28 (green), a new s factor. (b) Middle
transcription is directed by gp28, in conjunction
with the host core polymerase (red) two middle
phage proteins are gp33 and gp34 (purple and
yellow, respectively) together, these constitute
yet another s factor. (c) Late transcription
depends on the host core polymerase plus gp33 and
34.
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Evidence for s switching model

• Genetic studies
• mutations in gene 28 prevent
  early-to-middle switch
• mutations in gene 33 or 34 prevent
  middle-to-late
• switch
• Biochemical studies
• purification of RNA polymerase

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Figure 8.2 Subunit compositions of RNA
polymerases in SP01 phage-infected B. subtilis
cells. Polymerases were separated by
chromatography and subjected to SDS-PAGE to
display their subunits. Enzyme B (first lane)
contains the core subunits (ß', ß, and a), as
well as subunit IV (gp28). Enzyme C (second lane)
contains the core subunits plus subunits V (gp33)
and Vl (gp34). The last two lanes contain
separated d and s subunits, respectively.
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Figure 8.3 Specificities of polymerases B and C.
Pero et al. measured polymerase specificity
by transcribing SP01 DNA in vitro with core
polymerase (a), enzyme B (b), or enzyme C (c), in
the presence of 3HUTP to label the RNA product.
Next they hybridized the labeled RNA to SP01 DNA
in the presence of each of the following
competitors early SP01 RNA (green) made in vivo
in the presence of chloramphenicol (CAM) middle
RNA (blue) collected from phage-infected cells at
10 minutes post-infection and late RNA (red)
collected from phage-infected cells 30 minutes
post- infection, The product of the core
polymerase is competed roughly equally by all
three classes of RNA. On the other hand,
competition for the product made by B plus d is
clearly competed best by middle RNA, and the
product made by C plus d is competed best by late
RNA. These differences are not as dramatic as one
might prefer, but they are easiest to see at low
competitor concentration.
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SUMMARY Transcription of phage SPO1 genes in
infected B. subtilis cells proceeds according to
a temporal program in which early genes are
transcribed first, then middle genes, and finally
late genes. This switching is directed by a set
of phage-encoded s factors that associate with
the host core RNA polymerase and change its
specificity from early to middle to late. The
host s is specific for the phage early genes the
phage gp28 protein switches the specificity to
the middle genes and the phage gp33 and gp34
proteins switch to late specificity.
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8.2 The RNA Polymerase Encoded in Phage T7

• T7 (E. coli phage, small genome)
• Temporal control of transcription in T7

genes Expression stage product
Class I early Phage RNA polymerase, ect.
Class II middle Class II proteins
Class III late Class III proteins
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Figure 8.4 Temporal control of transcription in
phage T7-infected E. coil. (a) Early (class I)
transcription depends on the host RNA polymerase
holoenzyme, including the host s factor (blue)
one of the early phage proteins is the T7 RNA
polymerase (green). (b) Late (class II and III)
transcription depends on the T7 RNA polymerase.
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SUMMARY Phage T7, instead of coding for a new
s factor to change the host polymerase's
specificity from early to late, encodes a new RNA
polymerase with absolute specificity for the
later phage genes. This polymerase, composed of a
single polypeptide, is a product of one of the
earliest phage genes, gene 1. The temporal
program in the infection by this phage is simple.
The host polymerase transcribes the earliest
(class I) genes, one of whose products is the
phage polymerase, which then transcribes the
later (class II and class III) genes.
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8.3 Control of transcription During Sporulation
Figure 8.5 Two types of B.subtilis cells.
(a) B.subtilis vegatative cells and (b) a
sporulating cell. With an endospore developing at
the left end.
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Figure 8.6 Map of part of plasmid p213. This
DNA region contains two promoters a vegetative
promoter (Veg) and a sporulation promoter (0.4
kb). The former is located on a 3050 bp
EcoRI-HincII fragment (blue) the latter is on a
770 bp fragment (red).
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Figure 8.7 Specificities of sA and 6E.
Losick and colleagues transcribed plasmid p213 in
vitro with RNA polymerase containing sA (lane 1)
or sE (lane 2). Next they hybridized the labeled
transcripts to Southern blots containing
EcoRI-Hincll fragments of the plasmid. As shown
in Figure 8.6, this plasmid has a vegetative
promoter in a 3050 bp EcoRI-Hincll fragment, and
a sporulation promoter in a 770 bp fragment.
Thus, transcripts of the vegetative gene
hybridized to the 3050 bp fragment, while
transcripts of the sporulation gene hybridized to
the 770 bp fragment. The autoradiogram in the
figure shows that the sA enzyme transcribed only
the vegetative gene, while the sE enzyme
transcribed both the vegetative and sporulation
genes.
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Figure 8.8 Specificity of sE determined by
run-off transcription from the spollD promoter.
Rong et al. prepared a restriction fragment
containing the spollD promoter and transcribed it
in vitro with B. subtilis core RNA polymerase
plus sE (middle lane) or sB plus sc (right lane)
Lane M contained marker DNA fragments whose sizes
are indicated at left The arrow at the right
indicates the position of the expected run-off
transcript from the spollD promoter (about 700
nt). Only the enzyme containing sE made this
transcript.
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SUMMARY When the bacterium B. subtilis
sporulates, a whole new set of sporulation-specifi
c genes is turned on, and many, but not all,
vegetative genes are turned off. This switch
takes place largely at the transcription level.
It is accomplished by several new s factors that
displace the vegetative s factor from the core
RNA polymerase and direct transcription of
sporulation genes instead of vegetative genes.
Each s factor has its own preferred promoter
sequence.
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8.4 Genes with Multiple Promoter

• The B. subtilis spoVG Gene
• The Anabaena Glutamine Synthetase Gene
• The E. coli glnA Gene

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Figure 8.10 Resolution of RNA polymerases that
transcribe the spoVG gene from two different
promoters.
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Figure 8.10 Resolution of RNA polymerases that
transcribe the spoVG gene from two different
promoters. Losick and his colleagues
purified polymerase from B. subtilis ceils that
were running out of nutrients. The last
purification step was DNA-cellutose column
chromatography. The polymerase activity in each
fraction from the column is given by the red line
and the scale on the left-hand y axis. The salt
concentration used to remove the enzyme from the
column is given by the green line and the scale
on the right-hand y-axis. The inset shows the
results of a run-off transcription assay using a
DNA fragment with two spoVG promoters spaced 10
bp apart, The fraction numbers at the top of the
inset correspond to the fraction numbers from the
column at bottom. The last lane (M) contained
marker DNA fragments. The two arrowheads at the
left of the inset indicate the two run-off
transcripts, approximately 110 and 120 nt in
length. The column separated a polymerase that
transcribed selectively from the downstream
promoter and produced the shorter run-off
transcript (fractions 19 and 20) from a
polymerase that transcribed selectively from the
upstream promoter and produced the longer run-off
transcript (fractions 22 and 23).
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Figure 8.11 Specificities of s B and s E.
LOSiCk and colleagues purified sigma factors
s B and s E by gel electrophoresis and
tested them with core polymerase by the same
run-off transcription assay used in Figure 8.10.
Lane 2, containing s E, caused initiation
selectively at the downstream promoter (P2). Lane
5, containing s B, caused initiation selectively
at the upstream promoter (P1). Lane 6, containing
both s factors caused initiation at both
promoters. The other lanes were the results of
experiments with other fractions containing
neither s factor.
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Figure 8.11 Overlapping promoters in B.subtills
spoVG. P1 denotes the upstream promoter,
recognized by s B the start of transcription and
-10 and -35 boxes for this promoter are indicated
in red above the sequence. P2 denotes the
downstream promoter, recognized by s E the
start of transcription and -10 and -35 boxes for
this promoter are indicated in blue below the
sequence.
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Summary Some prokaryotic genes must be
transcribed under conditions where two different
s factors are active. These genes are equipped
with two different promoters, each recognized by
one of the two s factors. This ensures their
expression no matter which factor is present and
allows for differential control under different
conditions.
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8.5 The E. coli Heat Shock Genes

• htpR gene, s32 (sH)
• Comparison of s32 and s70 gene
• -35 sequence space
  -10 sequence
• s70 TTGACA 16-18 TATAA
• s32 CNTTGAA 13-15 CCCCATNT

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SUMMARY The heat shock response in E.
coli is governed by an alternative s factor, s 32
(s H) which displaces s 70 (s A) and directs the
RNA polymerase to the heat shock gene promoters.
The accumulation of s 32 in response to
high temperature is due to stabilization of s 32
and enhanced translation of the mRNA encoding s
32 .
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8.6 Infection of E. coli by Phage ?
Phage lambda can replicate in either of two
ways lytic or lysogenic. In the lytic mode,
almost all of the phage genes are transcribed and
translated, and the phage DNA is replicated,
leading to production of progeny phages and lysis
of the host cells. In the lysogenic mode, the
lambda DNA is incorporated into the host genome
after that occurs, only one gene is expressed.
The product of this gene, the lambda repressor,
prevents transcription of all the rest of the
phage genes. However, the incorporated phage DNA
(the prophage) still replicates, since it has
become part of the host DNA.
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Figure 8.12 Lytic versus lysogenic infection by
phage ?. Blue cells are in the lytic phase
yellow cells are in the lysogenic phase green
cells are uncommitted.
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Summary
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Figure 8.13 Genetic map of phage lambda. (a)
The map is shown in linear form, as the DNA
exists in the phage particles the cohesive ends
(cos) are at the ends of the map. The genes are
grouped primarily according to function. (b) The
map is shown in circular form, as it exists in
the host cell during a lyric infection after
annealing of the cohesive ends.
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Lytic Reproduction of ? Phage
The immediate early/delayed
early/late transcriptional switching in the lytic
cycle of phage lambda is controlled by
antiterminators. One of the two immediate early
genes is cro, which codes for a repressor of the
cI gene that allows the lytic cycle to continue.
The other, N, codes for an antiterminator, N,
that overrides the terminators after the N and
cro genes. Transcription then continues into the
delayed early genes. One of the delayed early
genes, Q, codes for another antiterminator (Q)
that permits transcription of the late genes from
the late promoter, PR', to continue without
premature termination.
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Figure 8.14 Temporal control of transcription
during lytic infection by phage lambda. (a)
Immediate early transcription (red) starts at the
rightward and leftward promoters (PR and PL,
respectively) that flank the repressor gene (cI)
transcription stops at the rho-dependent
terminators (t) after the N and cro genes. (b)
Delayed early transcription (blue) begins at the
same promoters, but bypasses the terminators by
virtue of the N gene product. N. which is an
antiterminator. (c) Late transcription (gray)
begins at a new promoter (PR') it would step
short at the terminator (t) without the Q gene
product, Q, another antiterminator. Note that O
and P are protein- encoding delayed early genes,
not operator and promoter.
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Figure 8.15 Effect of N on leftward
transcription. (a) Map of N region of ? genome.
The genes surrounding N are depicted, along with
the leftward promoter (PL) and operator (OL), the
terminator (red), and the nut site (green). (b)
Transcription in the absence of N. RNA polymerase
(pink) begins transcribing leftward at PL and
stops at the terminator at the end of N. The N
mRNA is the only product of this transcription
(c) Transcription in the presence of N. N
(purple) binds to the nut region of the
transcript, and also to NusA (yellow), which,
along with other proteins not shown, has bound to
RNA polymerase. This complex of proteins alters
the polymerase so it can read through
the terminator and continue into the delayed
early genes.
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Figure 8.16 Protein complexes involved in
N-directed antitermination. (a) Weak,
non-processive complex. NusA binds to polymerase,
and N binds to both NusA and box B of the nut
site region of the transcript, creating a loop in
the growing RNA. This complex is relatively weak
and can cause antitermination only at terminators
near the nut site (dashed arrow). These
conditions exist only in vitro. (b) Strong,
processive complex. NusA tethers N and box B to
the polymerase, as in (a) in addition, S10 binds
to polymerase, arid NusB binds to box A of the
nut site region of the transcript. This provides
an additional rink between the polymerase and the
transcript, strengthening the complex. NusG also
contributes to the strength of the complex. This
complex is processive and can cause
antitermination thousands of base pairs
downstream in vivo (open arrow).
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Figure 8.17
33
Figure 8.18
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Figure 8.19
35
Figure 8.20 Map of the PR' region of the ?,
genome. The PR promoter comprises the -10 and
-35 boxes. The qut site overlaps the promoter and
includes the Q binding site upstream of the -10
box, the pause signal downstream of the
transcription start site, and the pause site at
positions 16 and 17.
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• 5 proteins (N, NusA, NusB,
• NusG and S10) collaborate in antitermination at
  the? immediate early terminators.

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• NusA and S10 bind to RNA polymerase
• N and NusB bind to the boxB and boxA regions
• N and NusB bind to NusA and S10
• NusA stimulates termination by interfering with
  the binding between upstream part of the RNA
  hairpin and the core polymerase
• N helps NusA bind RNA, preventing hairpin
  formation

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Establishing Lysogeny

• The delayed early genes help establish lysogeny
  in two ways
• Some of the delayed early gene products are
  needed for integration of the phage DNA into the
  host genome
• The products of the cII and cIII genes allow
  transcription of the cI gene and therefore
  production of the ?repressor.

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• The promoter used for establishment of losogeny
  is PRE, which lies to the right of PR and cro.
  Transcription from this promoter goes leftward
  through the cI gene. The delayed early genes cII
  and cIII also participate in this process CII,
  by directly stimulating polymerase binding to PRE
  and PI CIII, by slowing degradation of CII.

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Figure 8.21 Establishing lysogeny. Delayed
early transcription from PR gives cII mRNA that
is translated to CII (purple). CII allows RNA
polymerase (blue and red) to bind to PRE and
transcribe the CI gene, yielding repressor
(green).
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Figure 8.22 Binding of CII at the -35 box of
both PRE and PI promoters of ?, phage.
Ptashne and colleagues performed a DNase
footprint analysis of the interaction between CII
and two early ?. promoters, PRE (a) and PI (b),
In (a), lanes 1-4 contained the following amounts
of CII lane 1, none lane 2, 10 pmol lane 3, 18
pmol and lane 4, 90 pmol. In (b), lanes 1-4
contained the following amounts of CII lane 1,
none lane 2, 18 pmol lane 3, 45 pmol lane
4,100 pmol. The CII footprint in both promoters
includes the -35 box.
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Figure 8.23
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Summary

• Phage ? establishes lysogeny by causing
  production of enough repressor to bind to the
  early operators and prevent further early RNA
  synthesis. The promoter used for establishment of
  lysogeny is PRE, which lies to the right of PR
  and cro. Transcription from this promoter goes
  leftward through the cI gene. The products of the
  delayed early genes cII and cIII also participate
  in this process CII, by directly stimulating
  polymerase binding to PRE CIII, by slowing
  degradation of CII.

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Autoregulation of cI Gene During Lysogeny

• Repressor turns off interrupting
  lytic circle
• PRM activating repressor
  synthesis
• OR controls leftward transcription of cI
• OR1OR2 repressor

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Figure 8.24 Maintaining lysogeny.
(bottom) Repressor (green, made originally via
transcription from PRE) forms dimers and binds
cooperatively to OR1 and 2. The protein-protein
contact between repressor on OR2 and RNA
polymerase (red and blue) allows polymerase to
bind to PRM and transcribe cI. (top)
Transcription (from PRM) and translation of the
cI mRNA yields a continuous supply of repressor,
which binds to OR and OL and prevents
transcription of any genes aside from cI.
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Figure 8.25 Map of the DNA fragment used to assay
transcription from cI and cro promoters. The
numbers denote the distances (in bp) between
restriction sites. The red arrows denote the in
vitro cI and cro transcripts.
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Figure 8.26 Analysis of the effect of ? repressor
on cl and cro transcription in vitro.
Ptashne and colleagues performed run-off
transcription (which actually produced "stutter"
transcripts) using the DNA template depicted in,
Figure 8.25. They included increasing
concentrations of repressor as shown at bottom.
Electrophoresis separated the cl and cro stutter
transcripts, which are identified at right. The
repressor clearly inhibited cro transcription,
but it greatly stimulated cl transcription at low
concentration, then inhibited cl transcription at
high concentration.
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Figure 8.27
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Figure 8.28 Principle of intergenic suppression
to detect interaction between ? repressor and RNA
polymerase. (a) With wild-type repressor and
polymerase, the two proteins interact closely,
which stimulates polymerase binding and
transcription from PRM. (b) The repressor gene
has been mutated, yielding repressor with an
altered amino acid (red). This prevents binding
to polymerase. (c) The gene for one polymerase
subunit has been mutated, yielding polymerase
with an altered amino acid (represented by the
square cavity) that restores binding to the
mutant repressor. Since polymerase and repressor
can now interact, transcription from PRM is
restored.
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Summary
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Figure 8.29 Selection for intergenic suppressor
of ? cI pc mutation. Susskind and colleagues
used bacteria with the chromosome illustrated (in
smalr part) at bottom. The chromosome included
two prophages (1) a P22 prophage with a
kanamycin resistance gene (yellow) driven by a ?
PRM promoter with adjacent ? OR (2) a ? prophage
containing the ? cI gene (light green) driven by
a weak lac promoter. Into these bacteria,
Susskind and colleagues placed plasmids bearing
mutagenized rpoD (s factor) genes (light blue)
driven by the lac UV5 promoter. Then they
challenged the transformed cells with medium
containing kanamycin. Cells transformed with a
wild type rpoD gene, or with rpoD genes bearing
irrelevant mutations. could not grow in
kanamycin. However, cells transformed with rpoD
genes having a mutation (red X) that compensated
for the mutation (black X) in the cI gene could
grow This mutation suppression is illustrated by
the interaction between the mutant s factor
(blue) and the mutant repressor (green), which
permits transcription of the kanamycin resistance
gene from PRM.
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Figure 8.30 Activation by contacting s. The
activator (e.g., ? repressor) binds to an
activator site that overlaps the weak -35 box of
the promoter. This allows interaction between the
activator and region 4 of s, which would
otherwise bind weakly, if at all, to the -35 box.
This allows the polymerase to bind tightly to a
very weak promoter and therefore to transcribe
the adjacent gene successfully.
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SUMMARY Intergenic suppressor mutation
studies show that the crucial interaction between
repressor and RNA polymerase involves region 4 of
the s subunit of the polymerase. This polypeptide
binds near the weak -35 box of PRM, which places
the s region 4 close to the repressor bound to
OR2. Thus, the repressor can interact with the s
factor, recruiting RNA polymerase to the weak
promoter. In this way, OR2 serves as an activator
site, and ? repressor is an activator of
transcription from PRM. It stimulates conversion
of the closed promoter complex to the open
promoter complex.
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Determining the Fate of a Infection Lysis or
Lysogeny
Whether a given cell is lytically or
lysogenically infected by phage ? depends on the
outcome of a race between the products of the cI
and cro genes. The cI gene codes for repressor,
which blocks OR1, OR2, OL1, and OL2, turning off
all early transcription, including transcription
of the cro gene. This leads to lysogeny. On the
other hand, the cro gene codes for Cro, which
blocks OR3 (and OL3), turning off cI
transcription. This leads to lytic infection.
Whichever gene product appears first in high
enough concentration to block its competitor's
synthesis wins the race and determines the cell's
fate. The winner of this race is determined by
the CII concentration, which is determined by the
cellular protease concentration, which is in turn
determined by environmental factors such as the
richness of the medium.
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Figure 8.31 The battle between cI and cro.
(a) cI wins. Enough repressor (green) is made by
transcription of the cl gene from PRM that it
blocks poLymerase (red and blue) from binding to
PR and therefore blocks cro transcription.
Lysogeny results. (b) cro wins. Enough Cro
(purple) is made by transcription from PR that it
blocks polymerase from binding to PRM and
therefore blocks cl transcription. The lytic
cycle results.
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Lysogeny Induction
When a lysogen suffers DNA damage, it
induces the SOS response. The initial event in
this response is the appearance of a co-protease
activity in the RecA protein. This causes the
repressors to cut themselves in half, removing
them from the ? operators and inducing the lytic
cycle. In this way, progeny ? phages can escape
the potentially lethal damage that is occurring
in their host.
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Figure 8.32 Inducing the ? prophage. (a)
Lysogeny. Repressor (green) is bound to OR (and
OL) and cI is being actively transcribed from the
PRM promoter. (b) The RecA co-protease
(activated by ultraviolet light or other
mutagenic influence) unmasks a protease activity
in the repressor, so it can cleave itself. (c)
The severed repressor falls off the operator,
allowing polymerase (red and blue) to bind to PR
and transcribe cro. Lysogeny is broken.