From the Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The chromosomes of Escherichia coli and Bacillus subtilis, and some plasmids like R6K, initiate replication from specific origins and the replication forks moving either bi- or unidirectionally, are arrested at sequence-specific replication termini (1-3). Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4, 5), plants (6), Xenopus (7), and human (8).
The replication termini are sequence-specific and bind to cognate
terminator proteins, and the protein-DNA complex arrests replication
forks in an orientation-dependent manner (9, 10). The
terminator protein of E. coli, called Tus, is a 36-kDa
monomer that binds to the
~22-bp1 Ter
() sequence and arrests replication fork by antagonizing helicase-catalyzed DNA unwinding in a polar mode (11-13). The
replication terminator protein (RTP) of B. subtilis, in
contrast, is a homodimer of 14.5-kDa monomers that binds cooperatively
as two interacting dimers to the cognate Ter site of ~30 bp that
comprises an overlapping core and an auxiliary sequence (14-17). The
most frequently used terminus of B. subtilis is Ter I (also
called BS3; Ref. 18), which binds to two interacting dimers of RTP and
arrests replication forks in a polar mode (14-16).
Since a usable in vitro replication system for B. subtilis is yet to be worked out, we have developed an E. coli-based in vitro system to analyze the mechanism of
action of RTP (19). The RTP-BS3 complex of B. subtilis has
been independently shown to block replication fork of E. coli in
vivo in an orientation-dependent manner (20). Both Tus
and RTP block the replication fork of E. coli by impeding
DNA unwinding by the replicative helicase DnaB (11, 12, 19). Detailed
biochemical and biophysical analyses have been made possible in both
the systems by the determination of the crystal structures of RTP at
2.6 Å (21) and of Tus-Ter () complex at 2.7 Å (22). The
DNA binding (23), dimer-dimer interaction (24), and DnaB interaction
domains (25) of RTP have been determined by genetic and biochemical
analyses that used the crystal structure as a guide. Similarly, the DNA
binding domain of Tus has been determined with the help of crystal
structure and genetic analysis (22).
The impact of transcription on the initiation (26, 27) and elongation stages of DNA replication have been reported (28, 29). We have reported previously that Tus and RTP can block RNA chain elongation catalyzed by several prokaryotic RNA polymerases in a polar mode and that the passage of an RNA transcript that invades the terminus from the permissive direction causes functional inactivation of the replication terminus (30). We have suggested that the polar arrest of transcription protects the terminus from functional inactivation by transcriptional invasion (30, 31). The interaction between transcription and termination of DNA replication is of further interest because the replication check points of B. subtilis that are conditionally active under stringent conditions (32) appear to be controlled by transcription.2
In the present work, we endeavored to address four important questions
regarding the interplay between transcription and replication termination. (i) What is the eventual fate of RNA polymerase (RNAP) when it reaches the Tus-Ter () complex that is positioned
in blocking orientation? Is the RNAP arrested or does it dissociate from the DNA leading to authentic termination of transcription? (ii)
Does the passage of transcription through the replication terminus
cause the dissociation of the Tus protein from the DNA-protein complex
thus abrogating replication fork arrest or is it caused by a
transcription-mediated conformational change of the Tus-Ter (
) complex that does not involve removal of Tus? (iii) Are the DNA
and RNA chains blocked at the same site of the terminus? (iv) Are
eukaryotic RNAPs blocked by prokaryotic replication termination proteins, and, conversely, can eukaryotic transcription termination proteins block replicative helicases and a prokaryotic RNAP? In this
report, we present a series of in vivo and in
vitro experiments that address these questions. The results show
that replication terminator proteins arrested RNAPs but did not cause
transcription termination. Transcriptional invasion caused the
terminator proteins to dissociate from the terminus, thus explaining
the mechanism of abrogation of replication fork arrest. The
transcription and replication arrest sites are different. Although the
replication terminator proteins blocked eukaryotic RNAPs, a eukaryotic
transcription terminator protein failed to block T7 RNAP and E. coli DnaB helicase.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA--
For experiments on arrest of T7 RNAP by Tus, the
plasmids pET22b- and pET22b-
rev. were constructed by cloning a
177-bp HindIII fragment of DNA containing the Ter
(
) site in either orientation at the HindIII site of
pET22b(+) plasmid (Novagen). pET22b-BS3 and pET22b-BS3 rev. plasmids
(used in experiments to investigate T7 RNAP blockage by RTP) and
T7A1-BS3 and T7A1-BS3 rev. DNA substrates (used in experiments to
investigate E. coli RNAP blockage by RTP) have been
described previously (30). For experiments on blockage of E. coli RNAP by Tus, the Ter (
) site was cloned in
either orientation downstream of a T7A1 promoter to generate the DNA
substrates T7A1-
and T7A1-
rev. DNA fragment containing the
T7A1-
was obtained by polymerase chain reaction (PCR) with a primer
upstream of the T7A1 promoter (A1 primer, 5
-AGGAGAGACTTAAAGAG-3
) and
the M13 universal primer or the A1 primer and a primer
5
-TATTAACCACTTTAGTTACAACATACTTATTTTA-3
that overlaps the
Ter (
) site. Similarly, a DNA fragment containing the
T7A1-
rev. was obtained by PCR with the A1 primer and M13 universal
primer or the A1 primer and a primer 5
-TAAAATAAGTATGTTGTAACTAAAGTGGTTA ATA-3
that overlaps the Ter (
) site. PCR was carried out
for 30 cycles in each case with each cycle consisting of three steps: 94 °C for 30 s, 55 °C for 60 s, and 72 °C for
120 s. For experiments with Reb1p, the Reb1 binding site (33) from
the plasmid p6.50 (from Dr. Walter Lang, University of Washington,
Seattle, WA) was cloned as a HindIII-SacI or
HindIII-NotI fragment into pET22b plasmid to
generate templates with the blocking and the nonblocking orientations
of Reb1p binding site, respectively.
Proteins--
Purification of Tus, RTP, and T7 RNAP has been
described previously (30, 31). E. coli RNA polymerase
containing 6-histidine-tagged -subunit was purified from the strain
AG2005/
-6His (kindly provided by Dr. Alex Goldfarb, Public Health
Research Institute, New York, NY) according to Kashlev et
al. (34), with some modifications. The fractions from
Ni+-agarose column that contained the RNA polymerase were
pooled and fractionated further on a 1-ml MonoQ (Pharmacia Biotech
Inc.) column (35). The RNA polymerase was eluted with a 0- 0.6 M NaCl gradient in buffer containing 40 mM
Tris·HCl, pH 7.9, 5% glycerol, 0.1 mM EDTA, 1 mM
-mercaptoethanol). Peak fractions were pooled and
used for transcription. For some experiments, we have also used
E. coli RNA polymerase that was purified as described (30). Reb1 protein was purified according to published procedures (33) with
some modifications. Yeast RNA polymerase I and II were kind gifts from
Dr. Walter Lang. S1 nuclease was purchased from U. S. Biochemicals.
EcoRI E111Q mutant protein was a kind gift from Dr. Paul
Modrich (Duke University, Durham, NC).
Transcription Impedance-- Multiple-round transcription reactions were carried out as described previously (30). Single-round transcription reactions in the presence of rifampin were carried out as described below in the appropriate sections.
Fate of E. coli RNAP Blocked by E111Q Mutant Form of EcoRI
Restriction Endonuclease, Tus, and RTP--
To study the fate of
E. coli RNAP when it is blocked by E111Q mutant form of
EcoRI (that binds to DNA site with a very long half-life but
does not cleave DNA), Tus or RTP single-round transcription reaction
was carried out essentially as described below. 50 fmol of the DNA
fragment were incubated with 5-10-fold molar excess of E111Q protein
or 10-20-fold RTP or Tus at 37 °C for 5 min in transcription buffer
containing 30 mM Tris·HCl, pH 7.6, 40 mM KCl,
0.1 mM EDTA, 1 mM DTT, 50 µg/ml bovine serum
albumin, 100 µM each of the four NTPs, and 10 µCi of
[-32P]GTP. 100 fmol of E. coli RNA
polymerase (2-fold molar excess over DNA) was added and incubated at
37 °C for 5 min. Transcription was initiated by adding
MgCl2 to 2 mM, followed by rifampin to 100 µg/ml and heparin to 100 µg/ml. The reaction was carried out at
37 °C for 15 min. The transcription mix was then divided into two
equal halves. To one half, KCl was added (in 1 × transcription buffer containing 2 mM MgCl2) to a final
concentration of 0.5 or 0.7 M KCl. To the other half,
1 × buffer with 2 mM MgCl2 was added. The
reaction was continued for another 30 min at 37 °C. 2 units of
RNase-free DNase I (Pharmacia) was added, and incubation was continued
at 37 °C for 15 min. The RNA was precipitated and run in a 6%
polyacrylamide, 7 M urea gel.
Transcription and Gel Shift Assay to Determine the Fate of RNAP-- To determine the fate of E. coli RNAP when it is blocked by Tus, a gel mobility shift assay was carried out. A single-round transcription reaction was set up in the presence of Tus as described above. After the addition of RNAP, MgCl2, rifampin, and heparin, incubation was carried out at 37 °C for 30 min, followed by addition of 5 µl of the loading dye containing 25% glycerol with bromphenol blue and xylene cyanol dyes (to give a final concentration of 5% glycerol) and was resolved in a 5% polyacrylamide gel.
Analysis of Tus-Ter () Complex by Gel Shift--
The binding
of Tus to 32P-labeled transcription template DNA and the
effect of salt on the binding were analyzed by nondenaturing polyacrylamide gel electrophoresis. The steps of a standard
single-round transcription reaction were followed except that RNAP
dilution buffer was added instead of RNAP. After incubation of the DNA with Tus followed by incubation with RNAP dilution buffer,
MgCl2, rifampin, and heparin were added and further
incubation was carried out for 15 min at 37 °C. Then, KCl in 1 × transcription buffer or 1 × buffer alone was added to the
appropriate tube, and incubation was continued for another 30 min at
37 °C. Sample dye was added to give a final concentration of 5%
glycerol, and the samples were run in a 5% polyacrylamide gel.
Analysis of Fate of the Terminator Protein in the Nonblocking
Orientation--
100 fmol of the pET22b- or pET22b-
rev. plasmid
linearized at the BlpI site was incubated with 65 fmol of
Tus protein at room temperature for 15 min. 0.5-1.0 pmol of T7 RNAP
was added and incubated for 1 min, following which 12 fmol of an
end-labeled (by [
-32P]ATP) 177-bp Ter (
)
fragment was added and incubation was continued for 30 min at 37 °C.
The reaction mix was run in a 5% polyacrylamide gel to monitor
trapping of the Tus protein displaced from transcription template by
the 32P-labeled Ter fragment.
Mapping of the in Vivo Leading Strand Block Site--
E.
coli Tus+ strain TH423 (a kind gift of Dr. T. M. Hill, University of North Dakota School of Medicine and Health
Sciences, Grand Forks, ND) containing the plasmid pUC18- with the
Ter (
) site in replication arresting orientation was
grown and replication intermediates were isolated from it as described
previously (30). The plasmid was run in a 0.8% agarose gel without
ethidium bromide. The replication intermediate, which ran slower than
the fully replicated plasmid was eluted from the gel,
ethanol-precipitated, and dissolved in TE (10 mM
Tris·HCl, 1 mM EDTA, pH 8.0). The DNA was digested with
HindIII, dephosphorylated with shrimp alkaline phosphatase
(U.S. Biochemical Corp.), phenol-extracted, precipitated with ethanol,
and dissolved in TE. The dephosphorylated DNA was end-labeled with
[
-32P]ATP by T4 polynucleotide kinase, passed through
Sephadex G25 spin column, precipitated with ethanol, and dissolved in
TE. The labeled DNA was run in a 6% polyacrylamide, 7 M
urea sequencing gel alongside the pUC18 sequencing ladder.
Mapping of Lagging Strand Block Site-- A unidirectional multiple-round primer extension by PCR was performed with the replication intermediate to determine the lagging strand blockage in vivo. The M13/pUC reverse sequencing primer was annealed to the replication intermediates in which it would anneal to the lagging strand and one parental strand. Extension was carried out by PCR using Vent DNA polymerase (New England Biolabs) for 30 cycles with each cycle having three steps: 94 °C for 30 s, 55 °C for 60 s, and 72 °C for 120 s. The primer extension products were run in a 6% polyacrylamide, 7 M urea gel alongside pUC18 sequencing ladder.
Mapping of Transcription Arrest/Block Sites--
For mapping the
3-ends of the transcripts blocked at the terminator protein binding
sites, scaled-up reactions were carried out in 100-µl volumes. After
assembly, to 10 µl of the reaction mix [
-32P]GTP was
added to monitor transcription impedance by the terminator proteins.
The remainder of the sample (90 µl) was used for mapping. Samples
with and without [
-32P]GTP were incubated at 37 °C
for 1 h, extracted with phenol:chloroform:isoamyl alcohol and
chloroform:isoamyl alcohol, and precipitated with ammonium acetate and
ethanol as described previously (30). The 32P-labeled
sample was run in a 6% polyacrylamide, 7 M urea gel to
confirm transcription blockage. For mapping the 3
-end of RNA generated
in the experiments with RTP, the DNA probe was generated as below; an
NdeI-SacI fragment containing the BS3 site
obtained from the plasmid pET22b-BS3 was end-filled by T4 DNA
polymerase in the presence of TTP and [
-32P]dATP so
that only the NdeI end was labeled. For mapping experiments with Tus, the DNA probe was generated as described below; pET22b-
plasmid was digested with NcoI, end-filling was done by T4
DNA polymerase in the presence of dCTP and [
-32P]
dATP, the labeled DNA was digested with XhoI, and finally
the labeled NcoI-XhoI fragment containing the
Ter (
) site was recovered from a 6% polyacrylamide
gel.
Yeast and T7 RNAP Transcription Blockage by Reb1
Protein--
Templates used for run-off transcription were
BglII-BlpI fragments from pET22b plasmids
containing RebIp binding site in the blocking (at
HindIII-SacI sites) or the nonblocking (at
HindIII-NotI site) orientation. The ~400-bp
BglII-BlpI fragments were modified by ligating a
14-nt oligonucleotide (5-GATCAAAAAACCA-3
) to the BglII end
by T4 DNA ligase to create a 3
overhang to serve as RNAP entry site.
Typical reactions were carried out in a 40-µl volume containing 40 fmol of template DNA, 50 ng of Reb1 protein (500 fmol) in reaction
buffer containing 10 mM HEPES-KOH pH 7.9, 10 mM
MgCl2, 25 mM KCl, 2.5 mM EDTA, 20 mM DTT, 10 units of RNAguard (Pharmacia), 1 µl of 20 mM dinucleotide UpG (Sigma), 0.5 mM each of
ATP, CTP, and UTP, 0.1 mM GTP, and 20 µCi of
[
-32P]GTP. Yeast RNA polymerase I (12 units) or RNAP
II (6 units) were added after dilution in the buffer containing 20 mM HEPES-KOH pH 7.9, 50 mM KCl, 5 mM EGTA, 50 mM EDTA, 2.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mg/ml leuopeptin.
After binding Reb1p or RTP to the corresponding template in the
reaction buffer for 15 min at room temperature, yeast RNAP was added
and incubation was carried out at 30 °C for 30 min, followed by
Dnase I treatment at 37 °C for 30 min. Transcription products were
extracted with phenol:chloroform:isoamyl alcohol and precipitated with
ethanol in the presence of 10 µg of yeast tRNA. RNA was dissolved in
loading buffer (30) and resolved in a 6% polyacrylamide, 7 M urea gel. For transcription by T7 RNA polymerase, all the
conditions were same as above except that the transcription buffer was
as described earlier for T7 RNA polymerase (30) and nucleotides used
were 0.5 mM each of ATP, CTP, and UTP, 12 µM
GTP, 20 µCi of [
-32P]GTP, and 100 ng of purified T7
RNAP.
Yeast RNAP I and II Blockage by RTP-- Templates used were ~400-bp BglII-BlpI fragments from the plasmids pET22b-BS3 and pET22b-BS3 rev. to which the 14-nt oligonucleotide described in the previous section was ligated to provide an entry site for the RNAP. The transcription reaction contained 40 fmol of template DNA and 400 or 800 fmol (10-20-fold molar excess) of RTP. Reaction conditions were same as described for transcription blockage experiments with Reb1p.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Orientation-dependent Blockage of T7 and E. coli RNAP
by Tus-Ter () Complex--
We have shown previously that RTP
impedes RNA chain elongation catalyzed by E. coli and T7
RNAPs in an orientation-dependent manner (30). The same
orientation of the RTP-TerI (BS3) complex impedes both RNA and DNA
chain elongation. We wished to generalize and extend our previous
observation by investigating the effect of Tus-Ter (
)
complex on chain elongation catalyzed by both T7 and the E. coli RNA polymerases. We linearized pET22b-
DNA carrying the
TerB (see Fig. 1) site of
E. coli in the orientation that impedes replication forks
(11, 12), and initiated transcription from a T7 promoter by T7 RNAP in
the presence of 0-, 2-, 4-, or 8-fold molar excess of Tus over DNA
substrate (100 fmol of DNA and 0, ~7, 14, and 28 ng of Tus). A
full-length transcript of ~450 nt (arrow showing F) and
two very closely spaced truncated transcripts of ~290 nt
(arrow showing T) were generated (Fig. 2A, lanes 1-4).
Transcription of the template pET22b-
rev. that contained the Ter
(
) site in the reverse orientation under identical conditions did
not show impedance of the transcription, and thus only full-length
run-off transcripts were observed even if an 8-fold molar excess of Tus
was used (Fig. 2A, lanes 6-8). Transcription from a control DNA fragment that also had a T7 promoter but no downstream Ter (
) site generated only full-length run-off
transcripts (marked C in Fig. 2A). Thus, the
Tus-Ter (
) complex blocked up to a maximum of 60% of the
transcripts in the replication blocking orientation.
|
|
E. coli RNAP Is Arrested but Not Terminated by Tus-Ter () and
RTP-BS3 Complexes--
The observed impedance of RNAP by the
Tus-Ter (
) complex could be explained as (i) a transient
pause, (ii) arrest of the transcripts for a longer period without the
dissociation of the RNAP from the DNA, or (iii) an authentic
termination of transcription caused by the dissociation of RNAP from
the DNA and release of the transcripts. We wished to distinguish among
these possibilities as follows, using two different approaches. The
first approach was based on the assumption that, if RNA chain
elongation by RNAP is arrested but not terminated at the terminator
protein-DNA complex, a truncated transcript should be generated and
removal of the terminator protein from its binding site should allow
the RNAP to resume transcription leading to the extensions of the
truncated products into full-length transcripts. We decided to use
E111Q mutant form of EcoRI restriction endonuclease as a
positive control, with which we compared the behavior of Tus. Other
investigators have shown that E111Q binds to DNA with a relative
Kd of 10
15 mol/liter without cleaving
the DNA (36). The bound E111Q protein arrests but does not terminate
transcription initiated from an upstream promoter (37). It has been
shown that high salt concentrations do not disrupt a DNA-RNAP-RNA
ternary complex or inhibit an elongating E. coli RNA
polymerase from template DNA (38). The experimental strategy consisted
of initiation of transcription from the designated promoter by E. coli RNAP and blockage of the transcripts by E111Q, Tus, or RTP.
The blocking protein was then dissociated from the template by adding
KCl to 0.5 or 0.7 M, and the RNA transcript was resolved on
denaturing polyacrylamide gels. Fig.
3B shows the fate of E. coli RNAP blocked by E111Q and RTP. Transcription was initiated
from the T7A1 promoter (that binds E. coli RNAP) downstream
of which was positioned a BS3 sequence (TerI site of B. subtilis; see Fig. 1) flanked by two EcoRI sites.
Fifteen minutes after transcription was initiated in the absence or the
presence of E111Q or RTP proteins bound to the cognate sites, the
reaction mixture was divided into two equal halves and KCl was added to 0.5 or 0.7 M to one half of the reaction mixture, whereas
to the other half only buffer was added and the reaction was continued for another 30 min. Transcription by E. coli RNAP in the
presence of E111Q protein generated two truncated transcripts in
addition to the full-length transcript (Fig. 3B, lane
3). In the presence of RTP bound to the single BS3 site, a
truncated and a full-length transcript were generated (lanes
5 and 7). Addition of 0.5 or 0.7 M KCl
caused the truncated transcripts to be almost quantitatively extended
into full-length transcripts (lanes 4, 6, and
8). Presence of high salt did not diminish the amount of
transcription by RNA polymerase (Fig. 3B, compare lane
1 with lane 2).
|
Arrest of E. coli RNA Polymerase at the Replication Terminus as
Shown by Gel Mobility Shift Assay--
To confirm the arrest of
E. coli RNAP by Tus-Ter () complex as
described above, we performed a gel mobility shift assay of the
transcribing RNA polymerase (Fig. 4). The
experimental strategy is shown in Fig. 4 (top). A
single-round transcription of the templates containing the T7A1
promoter and the downstream Ter (
) site, present in
either orientation, was carried out and analyzed as described under
experimental procedures. In the template having the Ter
(
) site in the blocking orientation, the transcribing RNAP arrested
by Tus showed a mobility shift of the DNA-Tus-RNAP-RNA complex (shown
by arrow in lane 5, Fig. 4, bottom
panel). However, in the absence of Tus, there was no corresponding
gel shift (lane 6, Fig. 4, bottom panel),
suggesting that all RNAPs had completed transcription and had fallen
off the template. Rebinding of the enzyme to the template was prevented
because of the presence of rifampin. In comparison, the template with
the Ter (
) site in the nonblocking orientation did not
show any gel shift of the transcribing RNA polymerase in the presence
or absence of Tus (compare lanes 5 and 6 with
lanes 11 and 12, respectively, Fig. 4,
bottom panel). The control experiments included gel shifts performed in the absence of Tus (lanes 1 and 7),
in the presence of Tus (lanes 2 and 8), in the
presence RNA polymerase (lanes 3 and 9), or in
the presence of both Tus and RNA polymerase (lanes 4 and
10). Gel shifts with RNA polymerase alone or with RNA
polymerase and Tus in the control lanes were done without
MgCl2, NTPs, and rifampin to prevent the occurrence of
transcription (lanes 3 and 4 and lanes
9 and 10). The gel shift caused by RNA polymerase without transcription was negligible probably because of the
instability of the RNA polymerase-promoter complex. Thus, these
experiments confirmed the observation that RNA polymerase is arrested
at the Tus-Ter (
) complex but does not dissociate from
the DNA.
|
Fate of Tus Protein When RNAP Invades Tus-Ter () Complex from
the Nonblocking End--
We have reported previously that passage of
an RNA transcript through the replication terminus causes the release
of the arrested replicative helicase and the replication fork (30, 31).
There are at least two possible mechanisms that could explain the
functional inactivation of the replication terminus by transcriptional
invasion: (i) the passage of the transcript could alter the
conformation of the DNA-protein complex at the replication terminus
without dissociating the bound form of Tus (RTP) or (ii) it could
dissociate the Tus (RTP) from the cognate DNA sequences. We wished to
distinguish between these two possibilities by performing the following
experiments. The experimental design is shown in Fig.
5 (top panel). A
32P-labeled, 177-bp-long DNA fragment containing the
Ter (
) site was used to trap the Tus protein that might
be dislodged from the pET22b-
and pET22b-
rev. templates during
transcription by T7 RNAP. Templates containing Tus-Ter (
)
complex (with protein:DNA ratio of 0.65:1.0, so that there was little
or no unbound protein) were transcribed by T7 RNAP. When pET22b-
rev. was transcribed, the bound Tus dissociated from the complex and
trapped by the 177-bp labeled fragment showing a gel shift (Fig. 5,
bottom panel, lane 8). Transcription of
pET22b-
did not show a gel shift, thus suggesting that RNAP was
arrested at the Tus-Ter (
) complex and thus could not
dissociate the bound Tus from the Ter (
) site. Addition
of only RNAP or NTPs alone did not show gel shifts either with
pET22b-
rev. (Fig. 5, bottom, lanes 6 and
7) or with pET22b-
(Fig. 5, bottom, lane
1 and 2) templates. The 177-bp fragment bound to Tus
alone showed gel shift (Fig. 5, bottom, lanes 4 and 5). No gel shift was observed when the same 177-bp
fragment (that lacked a promoter) was incubated with T7 RNAP
(Fig. 5, bottom, lane 9). These experiments
support the conclusion that passage of an RNA transcript through the
Tus-Ter (
) complex from the permissive end causes the
dissociation of the Tus protein from the complex, thus explaining the
mechanism of transcriptional inactivation of replication terminus.
|
Mapping of in Vivo Arrest Sites for Leading and Lagging DNA Strand
in E. coli--
Since the replication terminator proteins arrest both
replication and transcription, we wanted to compare the sites of
replication and transcription arrest with the hope that this
information might contribute to the understanding of the mechanism of
arrest. The experimental strategy for mapping of the arrest sites of
the leading and the lagging strand is schematically shown in Fig.
6A. We reasoned that the
prolonged arrest of the replication fork would allow the processing and
ligation of the Okazaki pieces, thus generating a continuous stretch of
the lagging strand of the unidirectionally replicating pUC18-
template. The location of the point of arrest of the lagging strand can
be determined by unidirectional primer extension by PCR using an
appropriately chosen end-labeled primer.
|
Mapping of Transcription Arrest/Block Sites and Comparison with the
Sites of Arrest of DNA Strands--
The 3 ends of the RNAs generated
by transcription blockage experiments were mapped by S1 nuclease
mapping technique using 32P-labeled DNA probes. Fig.
7 shows S1 nuclease mapping data on transcription stop sites of E. coli RNAP and T7 RNAP at
Tus-Ter (
) and RTP-BS3 complexes, respectively. A
comparison of the E. coli and T7 RNAP block sites and
leading and lagging strand block sites is shown in Fig. 7E
(top panel). The data showed that both the RNA polymerases
and DNA polymerase (for leading strand) stop at different positions
from the terminator site though not very far from one another. As
expected, the 5
end of the last Okazaki fragment was 63-65 nt
upstream of leading strand arrest site (39).
|
RTP-BS3 Complex Blocks Yeast RNAP I and II, but Reb1p Does Not Block T7 RNA Polymerase and DnaB-- Replication terminator proteins are known to arrest several replicative helicases but not those involved in rolling circle replication, conjugational transfer, and DNA repair (11, 12, 19, 31). RNAPs of yeast and mammalian cells are blocked by transcription terminator proteins that bind to specific DNA sequences (33, 40). We were curious to test the specificity of blocks mediated by the yeast Reb1 protein since the topic is relevant to the possible mechanism of polar arrest of DNA and RNA chain elongation. We wished to test if the yeast transcription terminator Reb1p, which blocks all the three RNAPs of yeast (33), could also block a prokaryotic phage RNAP, and if RTP, which impedes prokaryotic and phage RNAP, could also block eukaryotic RNAP. Fig. 8A shows that, whereas Reb1p, as reported previously (33), blocked yeast RNA polymerase II in an orientation-dependent manner (Fig. 8A, lanes 1-4), it could not block T7 RNAP in any orientation (Fig. 8A, lanes 5-8). We have also observed that Reb1p did not block DnaB replicative helicase of E. coli (data not shown). Fig. 8B shows that yeast RNA polymerase II was blocked by RTP in an orientation-dependent manner (Fig. 8B, compare lanes 1-3 with lanes 4-6). Similarly, RTP was also found to block yeast RNA polymerase I in an orientation-dependent manner (Fig. 8C, compare lanes 1 and 2 with lanes 3 and 4). In summary, although Reb1p could not block either T7 RNA polymerase or DnaB helicase, RTP was capable of blocking both yeast RNAP I and II.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The spatial and temporal overlap of replication and transcription
in the chromosome has stimulated investigations not only of the
possible mechanistic impact of transcription on the three steps of
replication but also of the impact of replication on transcription
(26-30, 41, 42). Transcriptional activation of replication origin of
bacteriophage and of yeast have been reported (26, 27). The impact
of transcription on the elongation step of replication has been
elegantly investigated, and the results show that transcription does
not have much impact on fork movement other than causing a transient
arrest of the fork when the replication fork and the transcriptional
apparatus encounter each other while approaching from opposite
directions. Conversely, passage of a replication fork did not dislodge
RNAP from a promoter (28, 29).
In eukaryotes, genes that are transcribed early in S phase also replicate early (41) and chromosomal segments that are heterochromatic tend to replicate late (43). Orc proteins, which have been implicated in initiation of chromosome replication, also contribute to silencing of transcription of yeast mating type cassettes (42). Thus, transcription and DNA replication seem to have a significant influence on each other.
We have investigated the impact of transcription on the termination step of DNA replication and vice versa and have noted that replication terminator proteins not only arrest replication forks and replicative helicases but also transcription elongation catalyzed by several prokaryotic RNA polymerases in a polar mode. When transcription is allowed to pass through a replication terminus from the permissive direction, the ability of the replication terminus to arrest replication forks is abrogated (30). Since transcription, in some cases, can pass through a protein-DNA complex without dissociating the bound protein (44-46), it was necessary to work out the mechanism of transcriptional inactivation of replication termination. The evidence presented in this article showed that the transcriptional passage dissociated the replication terminator proteins from the replication termini. Thus, in principle, inducible transcription should provide a mechanism for regulation of replication termination.
In B. subtilis, replication forks under stringent conditions
are arrested at conditional termini ( sites) located approximately 200 kilobase pairs on either side of the replication origin and the
arrest requires RTP. Under relaxed conditions, the forks are released
and proceed to the normal replication termini before being terminated
(32). Our recent work shows that transcription modulated by the
alarmone ppGpp seems to be responsible for the conditional derepression
of the
sites.3 The arrest
of transcriptional elongation has also been implicated in the
autoregulation of Tus protein of E. coli (47).
The elongation phase of transcription in both prokaryotes and
eukaryotes can be subject to regulation of three types: pausing, arrest, and authentic termination (48). The pausing is due to the
interaction of RNA polymerase with specific DNA sequences and is
transitory in duration, and the RNA polymerase continues to elongate
the RNA chain after the brief period of pausing. Arrest of
transcription is of longer duration and is caused by the encounter of
RNAP with proteins bound to DNA, although not all DNA-binding proteins
arrest RNAPs (37, 48). The arrested RNAP remains bound to the DNA and
can continue to elongate the RNA chain once the bound protein is
removed by some factor. Authentic termination of transcription can be
induced by DNA sequences and transcription terminator proteins,
resulting in the dissociation of RNAP and release of the RNA chain. The
evidence presented in this article showed that replication terminator
proteins of E. coli and B. subtilis arrested but
did not terminate RNA chains. In this regard, the replication
terminator proteins behaved very much like the EcoRI E111Q
mutant protein, which binds very strongly to EcoRI sites
with a Kd of 1015 mol/liter and
arrests (or pauses) but does not terminate transcription (36, 37, 48).
The DnaA initiator protein is also known to arrest transcription in a
somewhat polar mode (49).
Does the arrest of RNAPs by the replication terminator proteins involve a nonspecific barrier created by the interaction of the proteins with the replication terminus, or does it also involve some protein-protein interaction between the arresting and the arrested proteins? On one hand, the diversity of primary sequences of the arrested proteins that include several replicative helicases, prokaryotic RNAPs and, as reported here, RNAPs I and II of yeast by RTP (and Tus) might suggest a lack of specific protein-protein interaction (2, 30, 31, 37). On the other hand, replication terminator proteins do not arrest all helicases and, therefore, show some specificity. For example, helicases involved in rolling circle replication are not arrested either in vitro or in vivo (19, 31, 50). It is likely that there may be protein-protein interactions between the arrested and the arresting proteins. Even if such interactions turn out to be relatively nonspecific, these interactions probably are critical for the polar arrest of DNA and RNA chains by replication terminator proteins.
Such essential but nonspecific protein-protein interactions have been
reported to occur between transcriptional activator proteins and RNAPs
in both eukaryotes and prokaryotes (51). Transcriptional activator
proteins contact different subunits of E. coli RNAP. The
single strand binding protein of phage N4 called N4SSB contacts the
subunit (52), whereas
repressor contacts the carboxyl terminus
of the
subunit of RNAP. Although mutations at the contact points of
either the activator or RNA polymerase can cause loss of transcription
activation, the loss of one contact can be relieved by the
establishment of a different contact with a different subunit of RNAP
(51-54). Thus, these interactions, although biologically important,
need not be very specific.
The observation reported here that replication forks and RNAPs are
arrested at different locations of the Ter () sequence may be due to protein-protein interaction in addition to the
interaction of Tus and RTP with their cognate sites on DNA. The
differences might suggest different locations of the active sites of
DNA and RNA polymerases from the point of arrest or from the point of contact between these enzymes and the terminator protein-replication terminus complex. The observation that the Reb1 protein of yeast while
arresting RNAP I of yeast was incapable of arresting either T7 RNAP or
DnaB helicase, as reported here, would suggest that the eukaryotic
transcription terminator protein probably also interacts with the RNAPs
that it arrests (40).
Finally, future work on the interaction between transcription and replication termination will be directed toward the isolation of mutants of Tus and RTP that bind normally to DNA but do not arrest RNAPs. Preliminary work shows that the Y33A mutant of RTP fails to arrest RNAPs.4 If protein-protein interaction is involved, future work will also be directed toward mapping physically the contact points between RNAPs and Tus and RTP by photo-cross-linking procedures (52, 54).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Paul Modrich, Walter Lang, and
Alex Goldfarb for gifts of E111Q, yeast RNAP I and II, and 6His-tagged
subunit overproducer of E. coli RNAP, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported by a Merit Award from NIAID and a grant from NIGMS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed. Tel.:
919-684-3521; Fax: 919-684-8735; E-mail:
bastia{at}abacus.mc.duke.edu.
1 The abbreviations used are: bp, base pair(s); nt, nucleotide(s); RTP, replication terminator protein; RNAP, RNA polymerase; PCR, polymerase chain reaction; DTT, dithiothreitol; TE, Tris·HCl plus EDTA.
2 A. Gautam, B. K. Mohanty, and D. Bastia, unpublished results.
3 A. Gautam, B. K. Mohanty, and D. Bastia, manuscript in preparation.
4 D. Bastia, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|