(Received for publication, December 26, 1996, and in revised form, March 26, 1997)
From the Public Health Research Institute, New York,
New York 10016 and the ¶ ABL-Basic Research Program, NCI-Frederick
Cancer Research and Development Center,
Frederick, Maryland 21702-1201
Important regulatory events in both prokaryotic
and eukaryotic transcription are currently explained in terms of an
inchworming model of elongation. In this model, RNA extension is
carried out by a mobile catalytic center that, at certain DNA sites,
advances within stationary RNA polymerase. This idea emerged from the
observation that footprints of individual elongation complexes, halted
in vitro at consecutive DNA positions, can remain fixed on
the template for several contiguous nucleotide additions. Here, we
examine in detail the structural transitions that occur immediately
after the enzyme stops at sites where discontinuous advancement of RNA polymerase is observed. We demonstrate that halting at such special sites does not "freeze" RNA polymerase at one location but induces it to leave its initial position and to slide backward along the DNA
and the RNA without degrading the transcript. The resulting loss of
contact between the RNA 3-hydroxyl and the enzyme's catalytic center
leads to temporary loss of the catalytic activity. This process is
equilibrated with enzyme return to the original location, so that RNA
polymerase is envisaged as an oscillating object switching between
catalytically active and inactive states. The retreated isoform
constitutes a principal intermediate in factor-induced endonucleolytic
RNA cleavage. These oscillations of RNA polymerase can explain its
apparent discontinuous advancement, which had been interpreted as
indicating flexibility within the enzyme.
Ternary elongation complexes consisting of RNA polymerase
(RNAP),1 DNA, and nascent RNA are the
essential structures for transcription regulation in both pro- and
eukaryotes. Distinct properties of these complexes determine the
response of RNAP to pausing and termination signals and to the action
of regulatory factors (1-3). The traditional view of the process of
transcription has been based on thermodynamic analysis and structural
studies of diverse populations of elongation complexes. The entire RNAP
molecule was considered to translocate along the template monotonously as each new nucleotide was added to the RNA (4). However, DNA footprinting studies of ternary complexes stopped at one point along
the sequence revealed that at certain short sequence intervals, RNAP
appeared to remain fixed on the template while the RNA chain increased
a few nucleotides in length, leading to a relative contraction of the
distance in nucleotides between the 3 RNA terminus and the front end
footprint of RNAP on the DNA (5-11). These special sites where
contraction occurs are associated with an increased sensitivity of the
RNA near the 3
end to endonucleolytic cleavage by factor GreB (SII in
eukaryotes) and with a predisposition for collapse of the ternary
complex into a nonproductive or "arrested" state from which
polymerase neither transcribes RNA nor dissociates (7-9).
Re-establishment of the catalytic competence of the arrested complexes
requires cleavage of the RNA by Gre factors (5, 12, 13). This unusual
capability of RNAP to synthesize RNA within the stationary enzyme was
recently shown to participate in transcription pausing and termination
(9, 10).
To account for the perplexing way elongation and discontinuous movement
of RNAP are coupled, an "inchworming" model based on the idea of
structural transitions in enzyme consisting of flexibly connected parts
was developed (3, 6-9, 14). In this model, at specific DNA sites RNA
chain elongation, carried out by a mobile catalytic center advancing
within the fixed enzyme, alternates with threading of the transcript
through the RNAP in concert with the forward translocation of the
enzyme on the DNA. If the translocation goes awry, the catalytic center
is supposed to become disengaged from the RNA 3 terminus and to slide
backward within the fixed enzyme, thus causing arrest of elongation.
Cleavage of the transcript is thought to return the complex to the
active state by producing an appropriate 3
-OH group near the new
position of the catalytic center. Thus, the inchworming model was the
first insight into detailed structure of elongation complexes, which was shown to depend on a particular sequence which RNAP
transcribes.
However, the inchworming model is based solely on data obtained when
RNAP is stalled, raising the possibility that such ternary complexes
are not necessarily related to the structure of true intermediates in
RNA chain extension (6, 7, 15). Indeed, some approaches do reveal
differences between stopped and moving RNAP. Transiently halted RNAP is
distinguished by bacteriophage T4 factor Alc, since the factor to loses
its capacity to terminate host transcription in the presence of low
concentration of NTPs (15). In addition, kinetic studies of nucleotide
misincorporation by Escherichia coli RNAP suggest that a
reduced rate of transcription elongation causes a switching between
active and inactive conformations at each DNA position (16). Similar
conformational changes were proposed for eukaryotic elongation;
mathematical modeling of RNA chain growth desynchronization implied
that yeast RNAP III may flip between a "fast-step" state and a
"slow-step" state at each nucleotide of the template (17). The
most apparent consequence of halting of transcription is the
aforementioned arrest of RNAP in sites where unimpeded elongation would
proceed normally (5, 8, 9, 18). Using a combination of DNA and RNA
footprinting technique, we recently demonstrated that the loss of
catalytic activity during arrest is accompanied by an isomerization of
the ternary complex; the RNAP disengages from the 3 end of the
transcript and moves backward along the DNA, while the intact RNA
threads through the enzyme leaving its 3
end free (32). Taken
together, these observations suggest that RNAP may adopt multiple
conformations, only one of which needs to be capable of forming a
phosphodiester bond.
The goal of the present study was to examine in detail the structural transitions that occur immediately after the enzyme stops at sites where discontinuous advancement of RNAP is observed. A combination of approaches, including DNA and RNA footprinting with fast-acting chemical and enzymatic agents and time-resolved correlation of catalytic function and structural parameters, were used. It was revealed that at these sites RNAP repeatedly switches between inactive and active states as it moves back and forth using the DNA and RNA as tracks. The observations give rise to a new interpretation of the discontinuous phase of elongation.
The
standard template for transcription was the purified on PAGE polymerase
chain reaction-amplified 386-base pair DNA fragment carrying the T7A1
promoter. Its transcribed sequence (non-template strand) is
A1TCGAGAGGG10ACACGGCGAA20TAGCCATCCC30AATCGACACC40GGGGTCCGGG50ATCTGGATCT60GGATCGCTAA70TAACATTTTT80
ATTTGGATCC90CCGGGTGAAT100TCACT.
The permanganate-reactive single-stranded pyrimidine residues involved into transcriptional bubble in EC50,
EC56, or EC57 are underlined (see Fig.
1D for details).2
E. coli RNAP bearing a hexahistidine tag genetically fused
to the carboxyl terminus of the RNAP subunit was reconstituted from the isolated subunits and immobilized on Ni-NTA-agarose (QIAGEN) as described (19). Stable ternary complexes stalled at position 11 were
obtained by preincubating 2 pmol of the template with 2 pmol of the
immobilized enzyme in transcription buffer (TB; 20 mM
Tris-HCl, pH 7.9, 40 mM KCl, 5 mM
MgCl2, 1 mM
-mercaptoethanol) for 5 min at
37 °C and subsequently adding 5 µM tetraribonucleotide primer CpApUpC and 25 µM both ATP and GTP for 5 min at
25 °C. The complex was then walked to a desired position by repeated alterations of washing (five cycles of centrifugation and resuspension of the resin pellet in 1 ml of fresh TB) and chain extension
(incubation with a subset of NTPs (5 µM each) for 3 min
at 25 °C in 100 µl of TB). The transcripts were labeled by
incorporation of [
-32P]NTP (DuPont NEN, 40 µCi of
the labeled NTP (3000 Ci/mmol), 5 min at 25 °C). For the experiments
of Figs. 1, 2, 3, and 5, all ternary complexes were labeled by
incorporating [
-32P]CTP into position 12, followed by
"walking" of the enzyme to the desired site.
In the experiments testing the catalytic activity of halted RNAP, 5 µM each of ATP and UTP were added to EC56 at 16 °C for 5 s, unless otherwise indicated. In this and further experiments, reactions were stopped with the gel loading buffer (50 mM EDTA, 10 M urea) and the products were separated on 6% denaturing PAGE, unless specified otherwise.
Potassium Permanganate and ExoIII FootprintingThe DNA
template for KMnO4 and ExoIII footprinting was labeled in
10 µl of immobilized EC20 with 10 units of T4
polynucleotide kinase (New England Biolabs) and 50 µCi of
[-32P]ATP (7000 Ci/mmol; ICN Biomedicals, Inc.) in an
exchange reaction for 10 min at 25 °C, and the complex was used as
starting material for the walking reaction.
The labeled complexes were treated with 10 mM
KMnO4 at 16 °C. The reaction was stopped after 2 s
by adding 1 µl of -mercaptoethanol. The complexes were eluted from
Ni-NTA-agarose with 50 mM EDTA; the DNA in the supernatants
was precipitated in the presence of 4 µg of the carrier plasmid DNA,
cleaved with 10% piperidine for 15 min at 90 °C, reprecipitated,
lyophilized, dissolved in gel loading buffer, and separated on 6%
denaturing PAGE.
10 µl of the immobilized labeled complex was incubated with either 1 unit of the ExoIII (New England Biolabs) for 10 min at 25 °C or with 100 units of the nuclease for 5 min at 16 °C (as specified in figure legends).
Cleavage of the RNA by RNase T1 and GreB-induced RNA Cleavage Reaction10 µl of immobilized EC52-58 labeled by
incorporating [-32P]ATP in position 51 of the
transcripts, and then "walked" to the desired position, were
incubated for 10 min with 10 units of RNase T1 (Sigma) at 25 °C. The
cleavage was stopped by adding 3 µl of phenol, the products were
dissolved in the gel loading buffer and separated on 20% denaturing
PAGE.
GreB protein was purified as described (14). The cleavage reaction was
performed in 10 µl of TB under conditions specified in the figure
legends. The 56-nt transcript subjected to the treatment with GreB was
labeled in both 12C and 54U positions, which allowed visualization of
both 5 and 3
products of cleavage.
The immobilized EC halted at positions 27, 56, or 80 were obtained from EC26, EC54, and EC75, respectively, as described above in the presence or absence of 200 µM specific oligonucleotide at 16 °C for 10 min. The nonhybridized oligonucleotides were then removed by washing five times with 1 ml of ice-cold TB. 10 µl of each sample were then treated with ExoIII, GreB, or NTPs as described above or specified in figure legends. To melt the oligonucleotides off the RNA, the samples were incubated 5 min at 37 °C, washed once with warm (37 °C) and four times with cold TB and then the assays were performed as described above.
In
this work a solid-phase transcription system exploiting
hexahistidine-tagged RNAP from E. coli immobilized on
Ni-NTA-agarose beads was employed. The method allows one to obtain
elongation complexes stalled at any site of the DNA by alternating
limited RNA extension with washing of the beads (8-10, 19). We focused our study on the complex halted in position 56 of a template containing the T7A1 promoter (EC56). EC56 represents the
discontinuous phase of elongation (8). A fraction of EC56
complexes fails to resume transcription after the addition of the
missing NTP (in this case ATP), which in our previous study was
interpreted as arrested state formation (8). Indeed, the fraction of
EC56 not responding to 5 s incubation with 5 µM ATP and UTP retains the RNA after washing of the
beads, which rules out the dissociation of the complex (Fig.
1A, lanes 7 and 8; see
also Ref. 8). The unimpeded elongation through position 56 as well as
the halting of transcription in the neighboring positions 54, 58, and
60 do not reveal any inactivation of RNAP in the same test (Fig.
1B). Lanes 1-7 of Fig. 1A disclose a
distinctive feature of EC56 inactivation; within the first
2 min of the halted state, the complex loses its catalytic competence
gradually, but then the inactive fraction maintains a constant value.
It is important to notice that the incorporation of ITP instead of GTP
in two 3-terminal positions of the RNA, which weakened the
Watson-Crick pairing of the transcript terminus with the template,
dramatically increased the rate of catalytic inactivation of
EC56 (Fig. 1A, lanes 9 and
10). Unexpectedly, the inactive EC56 turned out
to be able to re-enter elongation directly upon incubation with NTPs,
bypassing internal cleavage of the transcript. In the experiment shown
in Fig. 1C, the fraction of EC56 capable of RNA
extension was removed by a 2-min chase with 500 µM each
of all four NTPs. After removal of the substrates, the rest of the
complex (lane 1) was incubated with 5 µM each
of ATP and UTP. As can be seen from lanes 2-4, the
remaining EC56 slowly resumes transcription. The rate of
NTP addition substantially increased in high salt (lane 5),
but it is still far below that in the unimpeded elongation. However,
prolonged incubation with the NTPs ultimately leads to complete
reactivation of the complex. The omission of GTP from the added NTPs
rules out participation of internal RNA cleavage in the activation,
since the 3
-terminal nucleotide in EC56 is guanine. Here
and in all further experiments involving halted EC56 and
all other ternary complexes, the 3
-terminal labeling of the
transcripts was used to exclude the possibility that shortening of the
RNA was responsible for all observed effects of halting (data not
shown). Below, the inactivated form of EC56 is defined as
temporarily arrested on account of the remarkable reversibility of the
inactivation, to distinguish it from irreversible arrest described
elsewhere (20, 32). Thus, the fact that after 2 min of halting no
further net change in the system occurs reflects an equilibrium between
the active and inactive forms. After removal of the active fraction by
incubating with NTPs, the equilibrium soon restores at the level
typical for halted EC56 (compare lane 7 in Fig.
1A with lane 2 in Fig. 1C).
Because of the
extremely brief appearance of the fully active form of EC56
(Fig. 1A), a fast acting footprinting agent was employed to
examine the structural difference between the active and the
temporarily arrested isoforms. GTP was added to EC54 to
synthesize EC56 which completed in 2 s. After
different periods of time following the addition of GTP (ranging from
2 s to 5 min), the complex was exposed for 2 s to potassium
permanganate, which allowed visualization of the transcriptional bubble
by modifying single-stranded pyrimidine residues of the nontemplate DNA
strand. Lanes 3 and 4 of Fig. 1D show
that within the first 10 s after the halting, the leading edge of
the bubble in EC56 occupies a position 1-3 nucleotides
downstream of the 3 tip of the RNA, corresponding to the normal
location for the bubble in the majority of the complexes tested so far
(11). However, while the halted state is maintained, the bubble
steadily shifts backward to a position similar to that in
EC50 (lanes 1 and 5-9). In the
control experiment, RNA in EC56 was labeled at the 3
end
by incorporating [
-32P]GTP to positions 55 and 56 of
the transcript. This labeled fraction maintained a constant value
during 10 min of incubation of the complex at room temperature, which
excluded the possibility that backward shift of the bubble was caused
by shortening of the RNA in the complex (data not shown). Remarkably,
the translocation of the bubble in EC56 coincides in time
with the RNAP inactivation both ceasing at about 2 min after the
halting. The substitution of two 3
-terminal guanosines with inosines
in the RNA increased the rate of bubble isomerization (lane
10) in accord with the much higher rate of inactivation of
inosine-substituted EC56 (Fig. 1A, lane
9). The transcriptional bubbles in EC50 and
EC57, which we took as representative of the monotonous
mode of transcription, have the normal locations relative to the 3
ends of their transcripts (lanes 1 and 11).
The irreversible arrest of E. coli RNAP
is associated with a similar retreat of the transcriptional bubble,
which involves backward sliding of the whole enzyme along the DNA (32).
We suggested that an analogous retreat of RNAP accompanies the
appearance of the temporarily arrested form. But, in our previous
report (8), the mapping of protection of the nontemplate DNA strand using 3 to 5
degradation with ExoIII did not discriminate between the
active and inactive isoforms of EC56 and detected only a
single position of the enzyme front edge coinciding with that in
EC50. Nevertheless, the recently reported ability of the
processive exonuclease to push RNAP upstream may have eliminated the
footprint of the active form (9, 10). Our own preliminary results also showed that prolonged incubation of some ECs with ExoIII led to the
catalytic inactivation of RNAP without dissociating it from template.
These observations, taken together, suggested that ExoIII may affect
both structure of ternary complex and catalytic activity of
RNAP.3 To weaken these side effects of
ExoIII, in the present study we carried out the footprinting at a lower
temperature, which was shown to decrease ExoIII processivity and was
believed to eliminate "pushing" activity of the exonuclease (21).
At 16 °C ExoIII does reveal an additional front end footprint in
EC56, located 6 nucleotides downstream from the position
observed previously at room temperature (lane 3 of Fig.
2, top panel). We designate this new
downstream boundary "regular" to highlight that in EC56
it is located at the same distance from the 3
tip of the RNA as in the
majority of the complexes, which advance monotonously along the
template, and we term the previously identified boundary "retarded"
to underline that it does not move with the growth of the transcript
from 50 to 56 nucleotides. However, it cannot be completely excluded
that RNAP itself acquires slightly different conformation at different
temperatures, which may affect front end footprint of EC56
in a similar way.
The following experiment proves that the appearance of the regular boundary is explained by the presence of a distinct catalytically competent isoform of EC56 rather than by the partial protection of the DNA by RNAP. EC56 was incubated for different periods of time with the subset of NTPs allowing RNA extension to 60 nt, and then was treated with ExoIII at 16 °C (lanes 4-8 of Fig. 2, top panel). The addition of the NTPs immediately shifts the regular EC56 footprint forward to the position specific for EC60 (compare lanes 3, 4, and 14) imitating the behavior of fully active EC57 (lanes 11 and 12). In contrast, the retarded boundary of EC56 accomplishes this transition slowly, matching the overall low rate of RNA extension characteristic for this complex (compare lanes 4-8 of the top panel with lanes 16-20 of the bottom panel). Since the active fraction of halted EC56 responds to the NTPs within 5 s (see Fig. 1A, lane 1), the "retreated" boundary remaining after the chase belongs to the catalytically incompetent form. Thus its delayed shift in the presence of the NTPs reflects the process of relieving the temporary arrest.
To visualize the rearrangement of the DNA/protein contacts associated with RNAP reactivation, we chased EC56 with all four NTPs and then quickly removed them by washes with the ice-cold transcription buffer. Although footprinting performed immediately after NTP removal ceases to detect the regular boundary in EC56, ExoIII added after several minutes of incubation without NTPs reveals that the remaining retreated boundary reproduces the regular one at the preexisting level (lanes 9, 10, and 3). The reorganization of the front end footprints correlates with the restoration of the limited catalytic activity; the fraction of EC56 that is catalytically inactive right after removal of the NTPs regains the elongation competence characteristic for the complex within 5 min (lanes 22 and 23 of the bottom panel). It indicates that EC56 comes out of the arrested state slower than it enters. Thus, the reactivated form of EC56 originates from the retreated inactive intermediate, and this transition is accompanied by the forward translocation of RNAP along the DNA by 6 nucleotides.
Active-to-arrested Transition Involves Backward Sliding of RNAP along the DNAThe reverse process, backward translocation of RNAP
during the development of the arrested fraction, cannot be detected
with exonuclease III directly after halting of EC56 because
the equilibrium between the temporarily arrested and active isoforms is
established faster than the relatively slow-acting nuclease reaches the
RNAP location by processive digestion of the linear template from the
3 end. In a separate experiment, we demonstrated that in the applied
conditions it took 5-7 min for ExoIII to reach location of
EC56 by progressive degradation of nontemplate DNA strand
from the 3
end and this time cannot be decreased by adding more ExoIII to the reaction.3 It suggests that acquisition of the
arrested state is faster than ExoIII can probe it. To visualize the
process of reverse translocation, we employed an alternative approach
utilizing short deoxyoligonucleotides complementary to the 56-nt
transcript immediately behind RNAP (Fig. 3A).
The analogous oligonucleotides were previously shown to suppress the
irreversible arrest of transcription by blocking enzyme retreat (32).
In the experiment of Fig. 3B, EC56 was
synthesized from EC54 in the presence of the
oligonucleotides, and the arrested fraction was then evaluated by
adding ATP and UTP. As expected, oligonucleotides 12-14, complementary
to the RNA at a distance of about 14 nucleotides from the 3
end (which
corresponds to the end of the zone of tight contact between the enzyme
and the transcript, see below), preserve the catalytic activity of the
complex (lanes 4-6), whereas the oligonucleotides
complementary to other parts of the RNA have no effect (lanes
3, 7, and 8). Although ExoIII footprinting
of EC56 performed at room temperature reveals only the
retarded position of the RNAP front edge (compare lane 3 of
Fig. 3C and lane 3 of Fig. 2; see also Ref. 8),
the arrest-suppressing oligonucleotides completely shift this boundary
forward to the regular position characteristic for the active form of
the complex (lanes 5, 7, and 8). The
control experiments performed with the other oligonucleotides and with
EC50 and EC57 establish the specificity of this
effect (lanes 1, 2, 4, and 9-12). Oligonucleotide 12 protects the corresponding region
of the transcript in EC56 and EC57 from
degradation with single-strand specific ribonucleases, indicating that
it forms a hybrid with the RNA (data not shown). Oligonucleotides 12-14 even added to already formed EC56 can switch it to
the active state and shift the footprint forward on the DNA (data not
shown).
Since octanucleotides can be easily melted off the RNA by increasing the temperature, one can launch RNAP inactivation by washing the arrest-suppressing oligonucleotide off the immobilized complex at 37 °C (lane 9 of Fig. 3B). Lanes 5 and 6 of Fig. 3C demonstrate the crucial result of the present experiment; the loss of the catalytic activity upon oligonucleotide departure coincides with the restoration of the retarded position of the enzyme front end. This observation shows that RNAP shifts backward by 6 nucleotides in the course of catalytic inactivation, which reveals the missing element in the cycle of the RNAP active-to-arrested interconversion.
The Rear Edge of Temporarily Arrested RNAP Moves along the DNA in Synchrony with Its Front EdgeWe used the arrest-suppressing oligonucleotides to confirm that the position of the rear edge of the temporarily arrested RNAP is also shifted backward compared with its position for the active enzyme. ExoIII footprinting performed in the standard conditions reveals that the rear edge of the protein in EC56 occupies the same position as in EC50, whereas the rear edge in EC57 is located 7 nucleotides downstream (Fig. 3C, lanes 13, 14, and 16). At the same time, EC56, maintained in the active state by oligonucleotide 12, has the rear edge shifted forward at the distance of 6 nucleotides (lane 15). Thus, in halted EC56, the whole RNAP mainframe seems to move back and forth along the DNA.
Reverse Threading of the Transcript in the Temporarily Arrested Ternary ComplexUsing ribonuclease RNA footprinting, we
previously showed that the irreversible arrest of RNAP involved the
reverse threading of the transcript through the enzyme (32). Fig.
4A represents the results of RNA footprinting
in a series of halted EC52-58, labeled near the 3 end,
with ribonuclease T1 which is specific to single-stranded G residues.
In the majority of the complexes tested so far, RNAP protected about 14 nt of the 3
-proximal RNA from degradation with different
ribonucleases. This suggests that tight contacts between the enzyme and
the transcript are limited to the 14 nucleotides from the 3
end.4 In the complexes we studied in this
experiment, a stretch of four adjacent guanines in positions 41-44 of
the transcript allows tracing the coverage of its 3
-terminal part with
RNAP. In EC56, as well as in EC52, all four G
nucleotides are completely inaccessible to the RNase (lanes
1-3). However, the addition of just one nucleotide to the 56-nt
transcript causes the contraction of the protected region by at least
four nucleotides at once, as very extensive cleavage in the stretch of
guanines in EC57 and EC58 testifies
(lanes 4 and 5). The expansion of the zone of
transcript protection in the 5
direction in EC56 conforms
with the idea of reverse threading of the RNA in the temporary inactive
fraction of the complex.
Temporarily Arrested Retreated RNAP Is the Obligatory Intermediate in GreB-induced Transcript Truncation
One of the principal
attributes of EC56 as a representative of the discontinuous
phase of elongation is its high sensitivity to internal RNA cleavage
induced by factor GreB. The initial cleavage event proceeds very fast
and releases a 3-terminal fragment 4 nucleotides long from the
complex; further truncation of the 5
fragment, which remains bound to
the RNAP, requires much longer incubation with the cleavage factor and
proceeds in mononucleotide increments (8). To test the active and
temporarily arrested forms of EC56 for sensitivity to GreB,
we subjected the complex containing the RNA simultaneously labeled at
12C and 56G positions to brief (5 s) treatment with GreB at different
times after halting and followed the amounts of 5
and 3
cleavage
products. In these conditions, 56-nt transcript receives only one cut
between positions 52 and 53 but further cleavage does not occur yet.
This makes it possible to visualize both labeled products of the
primary cleavage and to avoid any interference from the secondary cuts, which appear later in time as a result of further shortening of the
52-nt RNA from the 3
end. Fig. 4B shows that newly formed EC56 is first resistant to GreB, but its sensitivity
increases as the halted state continues, matching the accumulation of
the temporarily arrested form (compare with Fig. 1A). In the
experiment shown in Fig. 4C, EC56 maintained in
the active state by oligonucleotide 12 is even more resistant to GreB
than EC57, which represents the monotonous state of
elongation (compare lanes 1-3, 4-6, and
9 and 10). This effect ceases after removal of
the oligonucleotide (lanes 11 and 12);
oligonucleotide 10, which does not display arrest-suppressing activity,
does not affect sensitivity to GreB (lanes 7 and
8). These results strongly suggest that the temporarily
arrested, retreated form of RNAP represents an obligatory intermediate
in the endonucleolytic cleavage of the RNA, whereas the active form of
the enzyme is resistant to GreB.
The data of Fig.
5 argue that the ternary complexes stalled at other
known sites of discontinuous movement (6, 8, 9) undergo the same
isomerization as EC56 does. EC80, halted at the
oligo(T) sequence of a phage TR2 terminator derivative
from which the hairpin has been deleted, demonstrated the dominance of
the retarded position of the enzyme's front end on the DNA (compare
lanes 2-4 and 8-10 of Fig. 5A), high
sensitivity to GreB (lanes 11 and 12) and partial
loss of catalytic activity upon halting (see Ref. 9). Inactivation of
EC80 turned out to be reversible (data not shown), and the
complex, obtained in the presence of the oligonucleotide complementary to the RNA behind RNAP, has the downstream boundary shifted forward, decreased sensitivity to GreB (Fig. 5A, lanes 4 and 5 and lanes 12 and 13,
respectively), and normal catalytic activity during the whole period of
halting (data not shown). All these effects can be completely reversed
by the removal of the oligonucleotide from its target in the RNA
(lanes 6 and 14). The analogous oligonucleotide acts similarly on RNAP front end footprint in EC27 (Fig.
5B), where the phenomenon of discontinuous elongation was originally described (6, 8). The control oligonucleotides do not affect
the parameters of the complexes (lane 7 in Fig. 5A and lane 3 in Fig. 5B).
In the studies of elongation, pausing, and termination utilizing the method of halted transcription, the retention of catalytic activity and stability were generally accepted criteria for considering halted complexes as true representatives of actively transcribing RNAP (6-11). The results of the present research demonstrate that the apparent ability to catalyze RNA chain extension does not necessarily signify that the halted enzyme remains permanently in the original elongation prone conformation.
Here we have studied the effect of halting using complexes of E. coli RNAP stopped at three DNA regions where discontinuous advancement of the enzyme was previously detected (6, 8, 9). Stopping
of the enzyme in position 56 of the template, which was selected for
the most comprehensive analysis, does not affect the ternary complex
stability but causes its fast arrest-like inactivation. The fraction of
inactive EC56 remains constant after 2 min of halting.
Since the inactivated fraction turned out to be able to re-enter normal
elongation directly, bypassing internal transcript cleavage, we defined
it as temporarily arrested. The inactivation of the enzyme proceeds in
good synchrony with backward translocation of the transcriptional
bubble as revealed by the footprinting with potassium permanganate.
ExoIII maps the front end boundary of the active form of
EC56 as 6 nucleotides downstream from the front edge of its
temporarily arrested counterpart. Remarkably, the footprint of the
active form is situated at the same regular distance from the 3 end of
the RNA as in the majority of elongation complexes that advance monotonously along the template. We demonstrate that this footprint shifts backward in the process of EC56 inactivation, and
that reactivation is accompanied by its return to the original
location. The rear end footprint in the active form of EC56
translocates in concert with its front end. In that view, results of
ribonuclease T1 RNA footprinting, showing that the protected segment of
the transcript is more extended to the upstream direction in
EC56 than in the closely located complexes representing the
monotonous mode of elongation, can be also explained by the reverse
translocation of RNAP along the RNA chain. The sensitivity of the
complex to transcript cleavage induced by factor GreB substantially
increases in line with enzyme inactivation. Only the temporarily
arrested, retreated form of RNAP is susceptible to the cleavage, while
its catalytically competent form is resistant. All other complexes of
the discontinuous phase of elongation studied in this work display
similar halting-dependent rearrangements, whereas the complexes in the monotonous state do not.
In combination with our data that RNAP disengages from the 3 end of
the transcript and moves backward along the DNA and the RNA in the
process of irreversible arrest (32), the most reasonable interpretation
of the present results is that RNAP, halted at sites of discontinuous
elongation, moves back and forth along the DNA and RNA, repeatedly
switching between inactivated and activated states. These
"oscillations" proceed without breaking down the RNA and do not
alter the correct alignment of RNA-DNA base pairing. Thus, such halted
complexes are represented by at least two convertible isoforms
co-existing in a state of dynamic equilibrium (see Fig.
6A).
The fact that incorporation of IMP instead of GMP into the 3 terminus
of the transcript drastically facilitates backward translocation (Fig.
1, A and D) supports the idea that nucleic acid
rather than protein-nucleic acid interactions primarily determine the
direction of lateral motion. Clearly, the shift of the bubble and the
reverse threading of the transcript are associated with spreading apart
of the DNA and RNA chains ahead of the catalytic center and with
restoring the DNA duplex at the leading edge of the bubble. In all
catalytically active ECs, the 3
end of the transcript is always
positioned right next to the downstream DNA branching point, suggesting
that the elementary step in backward sliding may involve a direct
competition between DNA/DNA and DNA/RNA pairing at the leading edge of
the bubble. Since the relative stability of each DNA-DNA
versus DNA/RNA base pair varies significantly, there must be
template positions where stronger DNA/DNA hybrid can easily displace
the 3
end of the RNA from the template, thus dragging RNAP backward
and causing its inactivation. Although in this work we describe two
extreme states of RNAP, the "zipping" model of lateral sliding
suggests that the oscillating enzyme has the option to move back or
forth at each nucleotide of the DNA and RNA it passes by in the course
of isomerization.
It is necessary to discuss our unexpected findings in the relation to
the inchworming model of elongation. That novel view was initially
proposed based on the pioneering Krummel and Chamberlin (6), who
analyzed deoxyribonuclease I footprints in a series of successive
complexes of E. coli RNAP. In those complexes, the front end
of the enzyme appeared to advance in a leap of several nucleotides,
whereas the rear end moved forward steadily with the growth of the RNA
(see Ref. 7). Subsequent studies of pro- and eukaryotic RNAPs
exploiting ExoIII and hydroxyl radical as footprinting agents
demonstrated that the size of the footprints and their positions
relative to the 3 end of RNA substantially varied, depending on the
particular complex and on probe applied (10, 11, 22, 23). Extensive
studies with ExoIII of a great number of halted complexes detected
variations in footprint size only in short template regions, some of
which were involved in transcription termination and pausing, whereas
in the majority of complexes the RNAP progressed monotonously (8, 9,
24). From all these results emerged the principal idea of the
inchworming model that a mobile catalytic center is capable of
synthesizing RNA within the stationary enzyme (3, 7). Our new findings introduce an alternative interpretation of the footprinting data without the need to assume unusual flexibility of RNAP, since the
footprint of the complexes halted in the sites of discontinuous synthesis is determined by at least two convertible isoforms. In such a
dynamic system, the size and location of the DNA segment protected by
the protein and the momentary location of the active center relative to
the 3
end of the transcript should depend primarily on such parameters
as the amplitude and the frequency of oscillations and the equilibrium
concentrations of the isoforms as well as on the nature of footprinting
agent (see Fig. 6B). In this view, the characteristic
leaplike forward translocation of the footprints in some complexes,
earlier interpreted as actual "jumps" (see also Introduction), may
signify RNAP passage to a site where it does not oscillate.
The idea of RNAP internal plasticity is supported by cross-linking
experiments where linking of the priming substrate to the 5 "face"
of RNAP active center in the promoter complex did not interfere with
the synthesis of the RNA up to 9 nucleotides long (25). However, the
authors themselves agreed that the extension of the cross-linked
nucleotide might proceed through the RNA looping between the 5
and 3
faces of a nonflexible catalytic center. The extremely slow rate of RNA
synthesis observed in those experiments (it required 1 h of
incubation with 1 mM NTPs to synthesize 9 nt of RNA chain),
suggests that cross-linking of the primer might induce temporary
inactivation of RNAP via shifting backward.
The view of RNAP as a flexible protein appeared independently as the
explanation of internal transcript cleavage in ternary complexes
induced by the protein factors GreB or SII (8, 14, 26-28). According
to the inchworming model, the point of the cleavage demarcates two
operationally defined product binding sites, different in their
affinity for RNA: a loose site from which the 3-terminal fragment
falls out and a tight site where the 5
fragment is held. In the series
of successive complexes having the invariant position on the DNA, GreB
removes the 3
RNA increments of increasing length without any
translocation of the enzyme. Leaping along the DNA, which
re-establishes the monotonous mode, coincides with a drop in
sensitivity to cleavage, which thereafter results only in removal of
mono- and dinucleotides from the 3
end. This much less efficient RNA
cleavage was shown to causes the retreat of the enzyme along the
template (8, 23, 29). The inchworming model considers internal cleavage
as indication of a two-stroke process of RNA extension in the
discontinuous phase of elongation, where filling of the loose site
within the stationary RNAP alternates with threading of the recently
synthesized RNA increment into the tight site when the enzyme leaps
ahead (7, 8, 14). The resistance of monotonous complexes to cleavage
was attributed to difficulty in pushing the enzyme backward (8).
Most recent observations imply that the cleavage function belongs to
the catalytic center of RNAP (30, 31). This could be incorporated into
the inchworming model by assuming that a mobile catalytic center
temporarily draws back within the enzyme to execute cleavage. However,
at the moment of cleavage in the irreversibly arrested complexes, the
active center is located at an internal RNA position due to backward
translocation of the whole enzyme along the DNA and RNA (32).
Additionally, the data of the present paper show that for the cleavage
to occur in the complexes of discontinuous phase, RNAP must be
temporarily inactivated through the same kind of retreat. The shifting
backward prior to the cleavage explains why transcript truncation does
not affect the position of the enzyme halted in the sites of
discontinuous movement (see Fig. 6A). Since such a retreated
complex has the 3 end of the transcript extruded out of the RNAP, it
is not necessary to propose a special 3
-proximal loose product binding
site to explain why the terminal fragment dissociates after cleavage. The above model can be extended to explain cleavage in the monotonous phase, if we assume that these complexes still can temporarily retreat
by 1-2 nucleotides from their original positions even though they
display neither unusual footprints nor loss of catalytic activity.
Cleavage alone makes this otherwise hidden retreat visible by fixing
the shifted conformation and prohibiting enzyme from returning to the
original position. Thus, GreB sensitivity, the pattern of the cleavage,
and its effect on RNAP footprint must be determined by specific
parameters of the oscillations in various complexes: the amplitude of
the translocations and the ratios of the active and inactive forms.
Since the same parameters will determine both the catalytic activity of
the complex and the DNA footprinting, the elongation complexes will be
misclassified as persisting in monotonous (regular) or discontinuous
(strained) phase in all their characteristics.
The reversible switching between the alternative states may have an important implication to a not yet understood phenomenon of transcriptional pausing. It is easy to imagine that during transcription of template positions where the time interval required for RNAP to start moving backward is less than that required for the next NTP addition, the ternary complex can momentarily fall into the temporarily arrested conformation. The retreated RNAP has much less chance to escape quickly, even when supplied with substrates, which may explain general pausing of transcription by the kind of feedback mechanism. Favoring that idea, strong intrinsic pausing at some DNA positions is completely suppressed by the oligonucleotide complementary to the transcript immediately behind RNAP.3
The dynamic rearrangements discovered in the halted transcription can
also be relevant to the proofreading function of RNAP. Data obtained
from an in vitro study of misincorporation kinetics of
E. coli RNAP suggest that elongation complexes may exist in equilibrium between active and inactive states at each template position (15). The retreat of RNAP can comprise the central component
in this mechanism. In accord with this view, replacement of 3-terminal
guanines with inosines, which weakens DNA/RNA pairing at the 3
end of
the transcript, may be considered as a prototype for the
misincorporation of an NMP into the 3
end of the RNA. As observed with
inosines, such a mismatch may trigger fast temporary isomerization,
facilitating the displacement of the transcript 3
terminus from the
template by invasion of nontemplate DNA strand. Suspension of
transcript extension may provide a chance for RNAP to remove the
mismatch by pyrophosphorolysis or endonucleolytic cleavage.
Lateral oscillations at the run of thymidines taken from the normal
bacterial terminator (Fig. 5A, TR2 terminator of
bacteriophage ) suggest that sliding could be a step in the normal
termination process. The backward sliding of RNAP at a full terminator
sequence, which always contains a region of dual symmetry immediately
preceding the oligo(T) tract, may proceed amiss due to formation of the hairpin-like structure in the RNA near the upstream edge of the transcriptional bubble. Since such structure may compete with DNA/RNA
rehybridization necessary for the backward translocation, it may
initiate simultaneous closing of the bubble from the both sides,
leading to collapse of the bubble, and to release of RNAP.
We thank Alex Goldfarb for providing resources for our research and Loren Day and Elizabeth Kutter for the critical reading of the manuscript. We are especially grateful to Sergey Borukhov for GreB protein and in vitro reconstituted His-tagged RNAP.