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INTRODUCTION |
Transcription termination factor Rho is an RNA-binding protein
that couples the energy derived from ATP hydrolysis to actions that
dissociate a RNA transcript from its biosynthetic complex with DNA and
RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to
DNA through a short hybrid helix in a reaction that is dependent on ATP
hydrolysis and the presence of an attachment site for Rho on the RNA on
the 5' side of the hybrid helix (3-5). This RNA-DNA helicase activity
may mimic the process of removal of the nascent RNA strand from the
transcription elongation complex.
Rho factor is believed to function in vivo as a hexamer
consisting of six polypeptide subunits arranged in a ring-shaped
structure (6, 7). Burgess and Richardson (8) have recently presented a
model for the Rho-RNA complex in which a segment of RNA, called a
rut site (Rho utilization site),
binds to a cleft comprised of the N-terminal RNA-binding domains of the
six individual subunits located at one end of the hexameric structure
(the crown) (9-11). RNA sequence 3' of the rut site then
passes into the hole located at the center of the hexamer. Evidence for
passage of a single-stranded nucleic acid substrate through a
ring-shaped hexameric structure has been observed for other hexameric
helicases, such as DnaB and the T7 gene 4 product (12, 13).
The process by which Rho binds to and captures the 3'-end of an RNA in
the center of the hexamer is not known. Gan and Richardson (14) have
recently presented data indicating that Rho, in vitro, could
form hexamers by partial assembly of subunits on an RNA. Another
mechanism could have the RNA enter into the center of the hexameric
structure through an opening or notch in the ring (7). A third
mechanism may involve Rho threading onto the nascent transcript from
the free 5'-end of the RNA.
To test the model in which Rho requires a free end of RNA to
thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase
activity on an RNA lacking a free end. This can be done by using a
circular RNA to test Rho's ability to disrupt RNA-DNA hybrid helices.
These results indicate that Rho is indeed able to function as a
helicase on an RNA lacking a free end.
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EXPERIMENTAL PROCEDURES |
Materials--
All restriction enzymes, T4 RNA ligase, T4
polynucleotide kinase, and calf intestinal alkaline phosphatase were
purchased from New England Biolabs, Inc. The oligonucleotides were
purchased from Integrated DNA Technologies. All of the ribonucleotides
were purchased from Roche Molecular Biochemicals. Radioactive
nucleotides were purchased from ICN Radiochemicals. RNasin was
purchased from Promega. T7 RNA polymerase and wild-type Rho were
provided by Lislott Richardson (Indiana University).
pBcCro--
pBcCro is a derivative of pIF2 (15) that
contains a 10-base pair insertion sequence at the 5'-end of the
transcription unit for the
cro RNA. This derivative was
prepared by polymerase chain reaction amplification of pIF2 with the
mutagenic primers: primer 1 (5'-CGACTCACTATAGGGATCGTAGAGCCATTACTAAGGAGGTTG-3') and primer 2 (5'-CAACCTCCTTAGTAATGGCTCTACGATCCCTATAGTGAGTCG-3').
The resulting plasmid was sequenced to confirm the presence of the desired insert in the
cro gene.
T7 Transcription of Linearized pBcCro--
The pBcCro plasmid
was prepared for T7 transcription by linearization with the restriction
enzyme TaqI. Subsequent purification of the DNA, followed by
transcription and purification of the RNA product were done as reported
by Burgess and Richardson (8).
Circularization of the Linear pBcCro RNA Transcript--
The
5'-terminal triphosphate of the transcript was removed by treatment of
20 µg of RNA with 2 units of calf intestinal alkaline phosphatase in
50 µl of a solution containing 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM dithiothreitol, and 40 units of
RNasin for 2 h at 23 °C (16). The reaction mixture was then
treated with an equal volume of phenol, followed by an equal volume of chloroform/isoamyl alcohol (24:1). The RNA was precipitated with ethanol, washed with 70% ethanol, dried, and dissolved in water.
The RNA was phosphorylated at its 5'-end by treatment with 20 units of
T4 polynucleotide kinase in a 50-µl reaction volume containing 10 mM Tris acetate, pH 7.5, 10 mM magnesium
acetate, 50 mM potassium acetate, 8.0 µM
[
-32P]ATP (~1500 Ci/mmol), and 40 units of RNasin
for 2 h at 23 °C. The RNA was again purified by treatment with
phenol, chloroform/isoamyl alcohol (24:1), and ethanol precipitation.
The 5'-end labeled RNA was circularized by treatment with 25 units of
T4 RNA ligase in a 50-µl solution containing 25 mM
Tris-HCl, pH 7.6, 10 mM dithiothreitol, 5 mM
MgCl2, 5 mM ATP, and 0.25 mg/ml acetylated
bovine serum albumin, and 40 units RNasin for 15 h at 17 °C.
The RNA was precipitated with ethanol; washed with 70% ethanol; dried;
dissolved in 16 µl of a solution containing 98% formamide, 2 mM EDTA, 0.03% bromphenol blue, and 0.03% xylene cynanol;
and separated by electrophoresis on a 6%, 7 M urea
polyacrylamide gel (acrylamide/bisacrylamide, 19:1) containing
45 mM Tris borate and 1 mM EDTA. The ligated,
circularized RNA ran with a slower mobility than the unligated, linear
form on this denaturing gel. Approximately 30% of the RNA in the
ligation reaction was circularized. The RNA was identified by UV
shadowing and excised using a sterile razor blade. The RNA was
recovered as described by Burgess and Richardson (8). Concentration of
the RNA was determined by its optical absorbance at 260 nm.
Filter Binding Experiments--
Filter binding experiments
involving both the unligated and circular derivatives of the
cro RNA with wild-type Rho were performed and analyzed
according to Gan and Richardson (14). All binding experiments were
performed in a solution containing 40 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 50 mM KCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 1 mM ATP, and 0.25 mg/ml acetylated bovine serum albumin.
Annealing of DNA Oligonucleotides to the RNA--
DNA
oligonucleotides complementary to nucleotides 28-46 (5'
oligonucleotide), 326-348 (rut oligonucleotide), and
327-348 (3' oligonucleotide) of the standard
cro RNA
was chosen for these helicase experiments (see Fig. 1 for sequences)
The annealing reaction contained 50 nM
32P-labeled RNA, 500 nM DNA oligonucleotide, 20 mM Hepes-KOH buffer, pH 7.5, 150 mM KCl, and
0.1 mM EDTA. The annealing mixture was placed in a
thermocycler (Minicycler; MJ Research), incubated at 90 °C for 2 min, followed by a cooling phase of 2 °C/min to 25 °C. The
annealed RNA-DNA hybrids were stored at
20 °C. Under these
conditions, ~85% of the RNA was in the hybrid form.
Rho RNA-DNA Helicase Reaction--
The helicase reactions
contained one-tenth volume of the annealing reaction (50 nM
RNA-500 nM DNA oligomer), which resulted in ~4.5
nM RNA-DNA hybrid. The reactions also contained 32 mM Hepes-KOH buffer, pH 8.0, 50 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 40 units of RNasin, 0.2 mg/ml acetylated bovine serum albumin, and 5 nM wild-type Rho hexamer. The
reaction was heated to 37 °C for 1 min, and the reaction was
initiated by the addition of 1 mM ATP. Samples (6 µl)
were taken at the indicated times and mixed with 3 µl of a buffer
containing 150 mM Tris-HCl, pH 6.8, 4% SDS, 0.2%
bromphenol blue, and 20% glycerol. The helicase reactions were
analyzed on a discontinuous Laemmli polyacrylamide gel (17); stacking
gel contained 5% polyacrylamide (acrylamide/bisacrylamide, 29:1), 125 mM Tris-HCl, pH 6.8, and 0.1% SDS; resolving gel contained
380 mM Tris-HCl, pH 8.8, and 0.1% SDS. The gel was run in
a buffer containing 25 mM Tris, 250 mM glycine,
and 0.1% SDS. These gels were run at low current (11 mA) and
temperature (4 °C) to prevent disruption of the RNA-DNA hybrid
during electrophoresis. Following exposure of the gel to a
PhosphorImager plate, the data were analyzed using ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA).
The data points relating the fraction of RNA-DNA hybrid remaining
versus time for each sample were fit to the following
two-phase decay equation (4),
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(Eq. 1)
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where F is the fraction RNA-DNA hybrid remaining
after time t (t = 0 s was normalized to
1), A1 and k1 are the
amplitude and rate constant (units s
1), respectively, for
the first fast burst (exponential) decay phase, and
A2 and k2 are the
intercept and slope, respectively, of the second slower linear-decay phase.
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RESULTS AND DISCUSSION |
To determine whether Rho could bind to and act on a transcript
lacking a free 5'-end, a circular derivative of
cro RNA, a transcript that is terminated at the well characterized
Rho-dependent terminator tR1 located between genes
cro and cII of bacteriophage
, was prepared.
The method chosen to make a circular RNA (16) involved preparing a
391-nucleotide derivative of the
cro RNA, which
contained an inserted sequence of 10 nucleotides (between nucleotides 4 and 5 in the wild-type cro RNA sequence) that was complementary to a 10-nucleotide sequence near the 3'-end of the RNA
(nucleotides 366-375). With this extra sequence, the RNA can form a
stable stem helix, which would bring the 5'- and 3'-ends into close
proximity. After converting the 5'-triphosphate of the primary
transcript to a monophosphate, treatment of this RNA with T4 RNA ligase
converted ~30% of the RNA to a circular form. The ligated, circular
RNA was separated from the unligated RNA because of its slower mobility
during electrophoresis on a 7 M urea polyacrylamide gel.
Rho Binds to Circular RNA--
To determine whether Rho can bind
the circular
cro derivative RNA, we used the
nitrocellulose filter-binding technique to isolate complexes of Rho
with radiolabeled RNA (15, 18). With saturating levels of Rho, ~60%
of the circular cro RNA was retained on the filter. This
efficiency of retention of the complex was nearly identical to that
found with the standard cro RNA under the same binding
conditions (14). The affinity of Rho for the RNAs was determined by
measuring the fraction of RNA retained on the filter at varying
concentrations of Rho and finding the best fit of these data to the
equation for formation of a simple bimolecular complex between the RNA
and hexameric Rho. As was shown for the standard cro RNA,
the assumption that Rho bound RNA as a hexamer is a good approximation
when ATP was present in the binding mixture (14). The
Kd values determined by this procedure were:
0.03 ± 0.01, 0.07 ± 0.01, and 0.14 ± 0.03 nM for the complex with the standard cro RNA,
the ligated (circular) cro derivative RNA, and unligated
cro derivative RNA, respectively. Thus, Rho does not bind as
tightly to the derivative cro RNAs as to the standard
cro RNA, but it does bind with slightly higher affinity to
the circular RNA than to its unligated precursor.
One possible reason why Rho binds with a somewhat weaker affinity to
the derivative RNAs than to the standard cro RNA is that the
formation of the stem-helix between the 3'- and 5'-ends in the
derivative RNAs has changed their overall tertiary structure. These
structural changes may have made the binding site on the derivative
RNAs less accessible to Rho than on the standard cro RNA. In
any case, these data indicate that Rho can bind to the circular RNA
nearly as well as it binds to the standard cro RNA.
Rho Exhibits ATP-dependent RNA-DNA Helicase Activity on
a Circular RNA Substrate--
Rho can utilize the energy derived from
ATP hydrolysis to translocate along a RNA in a 3' direction and to
disrupt RNA-DNA hybrids that are 3' of the primary Rho attachment site
(3, 4). To determine if Rho can perform this ATP-driven reaction on a
circular RNA, a DNA oligonucleotide complementary to a sequence on the
3' side of the rut site (Fig.
1, 3' oligo) was
annealed to the RNA. When separated on a polyacrylamide gel containing SDS (no urea), the circular RNA-DNA oligonucleotide complex migrated more slowly than did the free RNA (Fig.
2). Incubation of the complex with a
stoichiometric amount of Rho and ATP caused a
time-dependent displacement of 3' oligonucleotide from the
circular RNA. 86% of the oligonucleotide was displaced within 6 min.
In the absence of either Rho or ATP, less than 7% of 3'
oligonucleotide was displaced within 6 min. These results demonstrate
that Rho acts as a helicase on a circular RNA.

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Fig. 1.
Schematic representation of the location and
sequence of the oligonucleotides used to form hybrid helices with the
various cro RNAs.
a, schematic of the standard cro RNA with
locations of the three DNA oligonucleotides. b shows the
formation of the stem structure that brings the 5'- and 3'-ends near
one another and the location of the three oligonucleotides on the
unligated and circular cro derivative RNAs. c,
primary sequence of the three DNA oligonucleotides. Secondary
structural elements other than the introduced stem-helix are not
represented.
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Fig. 2.
ATP dependence of Rho's RNA-DNA helicase
activity. Lane 1, 5 nM circular
cro RNA; lane 2, 4.5 nM circular
cro RNA-3' oligonucleotide hybrid; lanes 3 and
4, 4.5 nM hybrid after incubation with 5 nM Rho hexamer and 1 mM ATP for 60 and 360 s, respectively; lanes 5 and 6, 4.5 nM hybrid after incubation with 1 mM ATP for 60 and 360 s, respectively; lanes 7 and 8, 4.5 nM hybrid after incubation with 5 nM Rho
hexamer for 60 and 360 s, respectively. All samples were separated
by electrophoresis on a 5% polyacrylamide gel with 0.1% SDS at
4 °C. Reactions were analyzed by exposure of the gel to a
PhosphorImager plate and were quantitated using ImageQuant software
(Molecular Dynamics). Percentages were normalized to lane 2 as fraction hybrid remaining = 1.00.
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To rule out the possibility that the displacement occurred because
there was sufficient contaminating nuclease present to nick a majority
of the circular RNA, samples of the reaction mixture were also analyzed
on a polyacrylamide gel containing 7 M urea. The results
revealed that
95% of the circular RNA retained its characteristic
slower mobility on the 7 M urea polyacrylamide gel. Hence,
nearly all of the circular RNA remained covalently sealed throughout
the course of the helicase reaction.
Kinetics of the RNA-DNA Helicase Reaction--
To determine
whether the absence of a free end alters the rapidity by which Rho can
separate RNA from a hybrid complex with a DNA oligonucleotide, the time
courses for the displacement reaction for the various RNAs was
determined. With the standard cro RNA, the displacement
reaction had two phases. About half of the complexes were dissociated
rapidly, while the rest were dissociated at a much slower rate (Fig.
3). With the two cro
derivatives, the initial displacement was less rapid but involved a
larger fraction of the complexes. The two-phase reaction observed for
these RNAs was very similar to results obtained by Walstrom et
al. (5) performing similar experiments using DNA oligonucleotides
complexed with trpt' RNA as the substrate in their helicase
reaction. These reactions can be described in terms of a two-phase rate
expression composed of a rapid, first-order burst phase followed by a
slower linear phase (see Equation 1). The curves in Fig. 3 represent the fit of the data to the equation. The corresponding rate parameters are presented in Table I. For the hybrid
complex with the standard cro RNA, about 38% of the
hybrid complexes were disrupted in the initial burst phase, with a
first-order rate constant (k1) of 0.28 s
1. The remaining complexes were dissociated
with a steady-state rate constant (k2) of 8 × 10
4 s
1. In contrast, for
both of the cro derivatives, about 80% of the complexes
were dissociated in the burst phase; however, the rates (~0.03
s
1) were about 10-fold slower than the rate
determined for the standard cro RNA. The small fraction of
complexes remaining for both of the derivative cro RNAs were
dissociated with a k2 of <5 × 10
4 s
1. These
results indicate that the RNA-DNA hybrid complexes with the derivative
cro RNAs were poorer substrates for helix displacement by
Rho in the initial burst phase reaction than the complexes with the
standard cro RNA.

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Fig. 3.
Kinetics of displacement of the 3'
oligonucleotide from the various cro RNA transcripts
by Rho's helicase activity. Reactions contained 4.5 nM hybrid helix and 5 nM Rho hexamer and were
initiated by the addition of 1 mM ATP at 37 °C. Aliquots
of the reaction were taken at 15, 45, 90, 180, and 360 s and were
immediately mixed with a buffer containing 0.1% SDS, which quenched
the reaction by denaturation of Rho. All samples were separated by
electrophoresis on a 5% polyacrylamide gel with 0.1% SDS at 4 °C.
Open squares, standard cro RNA;
open circles, unligated cro derivative
RNA; closed circles, circular cro
derivative RNA. Reactions were analyzed by exposure of the gel to a
PhosphorImager plate and were quantitated using ImageQuant (Molecular
Dynamics) and Grafit 3.03 (Erithacus Software Ltd.) software.
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Table I
Values of parameters from best fit curves of Rho's RNA-DNA helicase
reaction
Parameters are from data fit to Equation 1. The curves fit to these
data are shown in Fig. 3. The values shown are the parameters from the
data that were averaged from two experiments per RNA.
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One possible explanation for differing rates of the initial burst phase
between the standard cro RNA and the derivative RNAs is that
formation of the stem structure between the 3'- and 5'-ends in the
derivative RNAs, ligated or not, makes the RNA less accessible to Rho
for immediate action. Another possibility is that the overall changes
in the tertiary imposed on the two derivative RNAs due to the formation
of the stem structure alters Rho's ability to efficiently displace
downstream hybrid complexes. The second explanation may be more likely,
since the RNA-DNA complexes were preincubated with Rho for two min at
37 °C prior to the addition of ATP, allowing Rho ample time to bind
to the complexes. Whatever the reason, the ligation of the 3'- and
5'-ends did not make the circular derivative any less reactive than its
unligated counterpart.
Investigation of Rho's Tracking along an RNA Molecule--
The
helicase action of Rho has been shown to be directional. Rho will
disrupt RNA-DNA hybrids located 3' of the rut site but not
hybrids located at the 5'-end of the transcript (3, 4). Another way to
demonstrate Rho's ability to act on a circular RNA is to show that it
can disrupt a hybrid with a DNA oligonucleotide (called 5'
oligonucleotide) that is complementary to a segment of RNA near the
5'-end of the original transcript that was used to make the circular
derivative (Fig. 1). After a 6-min incubation of the hybrid complex
with ATP and a stoichiometric amount of Rho, 44% of the 5'
oligonucleotide was removed from the circular RNA as compared with 71%
of the 3' oligonucleotide from its complex (Table
II). In contrast with the
unligated cro RNA, only 12% of the 5'
oligonucleotide was displaced, whereas again 71% of the 3'
oligonucleotide was displaced. On the ligated, circular cro RNA, the 5' oligonucleotide was located further downstream (54 nucleotides) from the rut site than the 3' oligonucleotide
was in its complex. In addition, the 5' oligonucleotide hybrid was downstream of the stem structure that brought the 3'- and 5'-ends together for ligation. Both of these conditions are likely reasons why
the 5' oligonucleotide was removed less efficiently than the 3'
oligonucleotide from the circular cro RNA. On the other
hand, the much lower displacement of the 5' oligonucleotide from the unligated derivative RNA containing the same stem structure
demonstrates a dependence on the covalent continuity of the RNA across
this segment of RNA for the improved efficiency of displacement. This is thus further evidence that Rho can function on a circular RNA.
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Table II
Characterization of Rho's helicase activity using DNA oligonucleotides
annealed to different locations on the various cro RNA transcripts
All reactions were performed using 4.5 nM RNA-DNA hybrid
and 5 nM Rho hexamer at 37 °C. All reactions were
initiated by the addition of 1 mM ATP to mixtures
preincubated with both the RNA-DNA hybrid and Rho. Less than 2% of the
hybrid complexes listed were displaced upon incubation of that complex
in the presence of either Rho or ATP individually.
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This result with the unligated cro derivative is also
consistent with other observations that Rho will not displace a hybrid that is on the 5' side of the rut sequence. This latter
observation is important because of the unexpected finding that
Rho was able to displace as much as 30% of the 5' oligonucleotide
within 6 min (Table II) from a complex with this oligonucleotide
hybridized to the standard cro RNA. The reason for this
partial but significant level of displacement is unknown. However, the
displacement was dependent on the presence of both Rho and ATP, since
less than 2% of this hybrid was displaced from the standard
cro RNA or either of the cro derivatives when
incubated with either ATP or Rho alone for 6 min (data not shown). In
all of the previous cases in which there was little or no displacement
of an upstream DNA hybrid, the 5'-end of the RNA transcript was in a
double helical structure. In experiments with trpt' RNA (3,
4), the upstream hybrid was at the 5'-end and in our experiment with
the unligated cro derivative RNA, the 5'-end was in a
helical stem with the 3'-end of the transcript. In contrast to those
RNAs, the standard cro RNA had a 5' RNA segment of 28 nucleotides that could provide Rho access to an upstream hybrid from
the 5' side. Because the amount of Rho was stoichiometric with the
amount of RNA under the conditions used, most, if not all, of the Rho
molecules would be bound to the rut segment of the
cro RNA (19). In this case, an interaction of the 5'-end of
an RNA with the hybrid dissociation site in Rho could occur by looping
of the same RNA into the site or by action on a second, separate RNA molecule.
To investigate the importance of the rut site to Rho action,
the rut oligonucleotide was used to block the beginning of
the rut site for all three RNAs. Faus and Richardson (15)
showed that blocking this portion of the rut site with an
oligonucleotide inhibited Rho's ability to bind to cro RNA.
Also, Chen et al. (20) showed that blocking the
rut site with a similar DNA oligonucleotide inhibited Rho's
in vitro termination activity at the tR1 terminator of the
cro gene. As expected from these prior observations, Rho
displaced less than 7% of the rut oligonucleotide from the complexes with any of the three cro RNAs (Table II). These
results also provide further evidence that the helicase activity is a faithful representation of Rho's action in transcription termination.
Implications for Rho's Function in Vivo--
According to a
recent model of Rho-RNA interaction presented by Burgess and Richardson
(8), Rho is believed to initially bind to a loading site located on the
RNA transcript through interactions in a cleft located around the crown
of the hexameric structure, composed of the N-terminal RNA-binding
domains of the individual subunits of Rho. Following this primary RNA
binding event, RNA is then passed into the center of the ring. The
mechanism for this transit is unknown. From these results, a mechanism
that requires Rho to thread onto a free 5'-end of the RNA for function is unlikely. Other possible mechanisms include either passage of the
RNA through an opening of a notched form of the hexamer (as seen in
electron micrographic images of Rho) (6, 7) or through an opening
caused by partial disassembly of the hexamer on the RNA (14).
In its normal context at the end of an operon, a
Rho-dependent terminator would encode a rut
segment that would be located downstream of the translated segment of
the RNA. In order for Rho to load onto this RNA, it must be able to
bind an RNA from which the 5'-end would be blocked by co-translating
ribosomes and the 3'-end would be blocked by the RNA polymerase. Thus,
Rho would have to bind by a mechanism that is not dependent on the availability of a free end. A similar constraint would apply to the
loading of Rho onto an RNA segment downstream of a ribosome stalled on
a nascent RNA during amino acid starvation, as happens for the
functioning of latent intragenic terminators under starvation conditions (21). Thus, these results with the circular RNA are consistent with the expected mechanism of Rho action in the cell.