Transcript Document

Chapter 8
Major Shifts in Prokaryotic
Transcription
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
gp28+host core
10 - end
Late genes
gp33+gp34+host
core
Figure 8.1 Temporal control of
transcription In phage SPO1infected B. subtilis.
(a) Early transcription is directed by
the host RNA polymerase holoenzyme,
including the host σ factor (blue); one
of the early phage proteins is gp28
(green), a new σ 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 σ factor.
(c) Late transcription depends on the
host core polymerase plus gp33 and 34.
Evidence for σ switching model

Genetic studies
mutations in gene 28 prevent early-tomiddle switch;
mutations in gene 33 or 34 prevent
middle-to-late
switch

Biochemical studies
purification of RNA polymerase
Figure 8.2 Subunit compositions of RNA polymerases in SP01 phageinfected B. subtilis cells.
Polymerases were separated by chromatography and subjected to SDSPAGE to display their subunits. Enzyme B (first lane) contains the core
subunits (β', β, and α), 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 δ and σ subunits, respectively.
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 [3H]UTP 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 phageinfected cells at 10 minutes post-infection; and late RNA (red) collected from phageinfected 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 δ is clearly competed best by middle RNA, and the
product made by C plus δ 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.
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 σ factors that associate with the host core
RNA polymerase and change its specificity from early to
middle to late. The host σ 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.
8.2 The RNA Polymerase
Encoded in Phage T7

T7 (E. coli phage, small genome)
Temporal control of transcription in T7
genes
Class I
Expressi
on stage
early
product
Class II
middle
Class II proteins
Class
III
late
Class III proteins
Phage RNA
polymerase, ect.
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 σ 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.
SUMMARY
Phage T7, instead of coding for a new σ 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.
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.
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 EcoRIHincII fragment (blue); the latter is on a 770 bp fragment (red).
Figure 8.7 Specificities of σA and 6E.
Losick and colleagues transcribed plasmid p213 in vitro with RNA
polymerase containing σA (lane 1) or σE (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 σA enzyme
transcribed only the vegetative gene, while the σE enzyme transcribed both
Figure 8.8 Specificity of σE 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 σE (middle lane) or σB plus σc (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 σE
made this transcript.
SUMMARY
When the bacterium B. subtilis sporulates, a whole new
set of sporulation-specific 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 σ factors that displace the vegetative σ factor
from the core RNA polymerase and direct transcription of
sporulation genes instead of vegetative genes. Each σ factor
has its own preferred promoter sequence.
8.4 Genes with Multiple
Promoter



The B. subtilis spoVG Gene
The Anabaena Glutamine
Synthetase Gene
The E. coli glnA Gene
Figure 8.10 Resolution of
RNA polymerases that
transcribe the spoVG gene
from two different promoters.
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).
Figure 8.11 Specificities of
σ B and σ E.
LOSiCk and
colleagues purified sigma
factors σ B and σ 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 σ E, caused
initiation selectively at the
downstream promoter (P2).
Lane 5, containing σ B,
caused initiation selectively
at the upstream promoter
(P1). Lane 6, containing both
σ factors caused initiation at
both promoters. The other
lanes were the results of
experiments with other
fractions containing neither σ
factor.
Figure 8.11 Overlapping promoters in B.subtills spoVG.
P1 denotes the upstream promoter, recognized by σ 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
σ E; the start of transcription and -10 and -35 boxes for this promoter are
indicated in blue below the sequence.
Summary
Some prokaryotic genes must be transcribed
under conditions where two different σ factors
are active. These genes are equipped with two
different promoters, each recognized by one of
the two σ factors. This ensures their expression
no matter which factor is present and allows for
differential control under different conditions.
8.5 The E. coli Heat Shock
Genes


htpR gene, σ32 (σH)
Comparison of σ32 and σ70 gene:
-35 sequence
sequence
σ70
σ32
TTGACA
CNTTGAA
space
16-18
13-15
-10
TATAA
CCCCATNT
SUMMARY
The heat shock response in E. coli is
governed by an alternative σ factor, σ 32 (σ H)
which displaces σ 70 (σ A) and directs the
RNA polymerase to the heat shock gene
promoters.
The accumulation of σ 32 in response
to high temperature is due to stabilization
of σ 32 and enhanced translation of the
mRNA encoding σ 32 .
8.6 Infection of E. coli by
Phage λ
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.
Figure 8.12 Lytic versus lysogenic infection by phage λ.
Blue cells are in the lytic phase; yellow cells are in the lysogenic phase;
green cells are uncommitted.
Summary
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.
Lytic Reproduction of λ 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.
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.
Figure 8.15 Effect of N on leftward
transcription.
(a) Map of N region of λ 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.
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).
Figure 8.17
Figure 8.18
Figure 8.19
Figure 8.20 Map of the PR' region of the λ, 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.
5 proteins (N, NusA, NusB,
NusG and S10) collaborate in
antitermination at theλ
immediate early terminators.





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
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
λrepressor.

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.
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).
Figure 8.22 Binding of CII at the -35 box of both PRE and PI promoters of λ,
phage.
Ptashne and colleagues performed a DNase footprint analysis of the interaction
between CII and two early λ. 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.
Figure 8.23
Summary
Phage λ 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.
Autoregulation of cI Gene During
Lysogeny




Repressor turns off
interrupting lytic circle
PRM activating
repressor
synthesis
OR controls leftward transcription
of cI
OR1+OR2
repressor
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.
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.
Figure 8.26 Analysis of the effect of λ 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.
Figure 8.27
Figure 8.28 Principle of intergenic
suppression to detect interaction
between λ 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.
Summary
Figure 8.29 Selection for intergenic suppressor of λ 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 λ PRM promoter with adjacent λ OR; (2)
a λ prophage containing the λ cI gene (light green) driven by a weak lac promoter.
Into these bacteria, Susskind and colleagues placed plasmids bearing mutagenized
rpoD (σ 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 σ factor (blue) and the mutant repressor (green), which permits transcription
of the kanamycin resistance gene from PRM.
Figure 8.30 Activation by contacting σ.
The activator (e.g., λ 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 σ, 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.
SUMMARY
Intergenic suppressor mutation studies show
that the crucial interaction between repressor
and RNA polymerase involves region 4 of the σ
subunit of the polymerase. This polypeptide
binds near the weak -35 box of PRM, which
places the σ region 4 close to the repressor
bound to OR2. Thus, the repressor can interact
with the σ factor, recruiting RNA polymerase to
the weak promoter. In this way, OR2 serves as
an activator site, and λ repressor is an activator
of transcription from PRM. It stimulates
conversion of the closed promoter complex to
the open promoter complex.
Determining the Fate of a Infection:
Lysis or Lysogeny
Whether a given cell is lytically or lysogenically infected by phage
λ 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.
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.
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
λ operators and inducing the lytic cycle. In this way, progeny
λ phages can escape the potentially lethal damage that is
occurring in their host.
Figure 8.32 Inducing the λ
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.