Cell, Vol. 89, 33–41, April 4, 1997, Copyright 1997 by Cell Press
The RNA–DNA Hybrid Maintains the Register
of Transcription by Preventing Backtracking
of RNA Polymerase
Evgeny Nudler,* Arkady Mustaev,*
Evgeny Lukhtanov,† and Alex Goldfarb*
*Public Health Research Institute
New York, New York 10016
† Epoch Pharmaceuticals, Inc.
Bothell, Washington 98021
Summary
An 8–9 bp RNA–DNA hybrid in the transcription elongation complex is essential for keeping the RNA 39 terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to
detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 39 segment of RNA can be removed
through transcript cleavage. At certain sites, cycles
of unwinding–rewinding of the hybrid are coupled to
reverse–forward sliding of the transcription elongation
complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inchworming). Thus, the 39-proximal RNA–DNA hybrid
plays the dual role of keeping the active site in register
with the template and sensing the helix-destabilizing
mismatches in RNA, launching correction through
backtracking and cleavage.
Introduction
The basic features of the ternary elongation complex
(TEC) are conserved in all living organisms (Sweetser et
al., 1987; Puhler et al., 1989; Darst et al., 1991; Polyakov
et al., 1995), but its detailed structure is unknown. Over
the past five years, models of elongation offered two
alternative views on the extent and functional significance of the RNA–DNA hybrid in the transcription bubble. In the classic view, the hybrid is 12 bp long and
plays a principal role in holding TEC together (Farnham
and Platt, 1980; Gamper and Hearst, 1982; Hanna and
Meares, 1983; Yager and von Hippel, 1986, 1991). In the
revisionist view, the hybrid is 2–3 bp long and serves
no other purpose than templating (Rice et al., 1991;
Chamberlin, 1995). The resolution of this dilemma, which
has been the subject of a lively debate (Kainz and Roberts, 1992; Altman et al., 1994; Johnson and Chamberlin,
1994; Zaychikov et al., 1995), has far-reaching implications for RNA polymerase (RNAP) structure and regulation at the level of elongation and termination (reviewed
by Das, 1993; Chan and Landick, 1994; Platt, 1997; Richardson and Greenblatt, 1996). In this work, we employed
RNA–DNA cross-links to measure directly the length of
the hybrid and used nucleotide analogs that strengthen
or weaken the hybrid to assess its functional role in TEC.
the aromatic bis(2-iodoethyl)amino group attached to
the fifth position of pyrimidine through a spacer arm.
Normally, this group is not reactive, but upon addition
of NaBH4, a reactive aziridinium intermediate is formed
that can cross-link to DNA or protein (Figure 1).
The extended length of the probe’s arm was z12 Å. A
flexible arm of this length can in principle reach multiple
sites, including the nontemplate strand of DNA. However, a U• -DNA cross-link in TEC mapped by piperidin
cleavage went exclusively into a single complementary
adenine in the template strand (E. N., unpublished data),
which is indicative of base pairing. In the base-paired
configuration, because of the need to bend across the
heteroduplex major grove, the length of the arm is just
enough to reach the adenine base paired to U• or an
adjacent purine. In the experiments described below,
the U• probe was incorporated in two different positions
of the transcript. In one case, there were no purines
adjacent to the adenine in the template DNA strand. In
the other case, there was an adjacent guanine, but its
involvement in the cross-link was excluded by piperidine
mapping (E. N., unpublished data). The reactive moiety
of the U• probe could also rotate away from the template
DNA and cross-link to the protein.
The cross-linking of U• was analyzed in a series of
consecutive TECs obtained by using immobilized RNAP
(Kashlev et al., 1993; Nudler et al., 1994). This solidstate transcription system permits walking of RNAP
along DNA in discrete controlled steps. RNA transcript
was 32 P-labeled near its 59 terminus, so that the crosslinking could be visualized by the appearance of protein–[ 32P]RNA or DNA–[32 P]RNA species on the autoradiogram of PAGE slabs after the complex has been
denatured.
The U• probe was incorporated either in position 121
or in position 145 (Figure 1B), and the complex was
walked from 121 to 144 (Figure 1B, lanes 3–10) or from
145 to 162 (Figure 1B, lanes 12–18), respectively. In
the nomenclature used, positive numbers refer to the
sequence position relative to the transcription start site,
whereas negative numbers indicate position in relation
to the RNA 39 terminus.
The results demonstrate that the RNA–DNA cross-link
is readily formed in those TECs where U• is located from
22 to 28, relative to the RNA 39 terminus. At the 210
position and beyond, the cross-linking to DNA is dramatically decreased. Thus, the RNA–DNA hybrid in TEC
extends for the distance of 8–9 nt behind the growing
tip of RNA. It should be noted that RNA–protein crosslinking displays a pattern opposite to RNA–DNA. The
yield of the protein cross-link increases dramatically at
210 and beyond, suggesting that RNA–protein association becomes particularly tight upstream of the RNA–
DNA hybrid region.
Results
The Eight to Nine Base Pair Hybrid Is Detected
by RNA–DNA Cross-Linking
To determine the length of the hybrid by RNA–DNA
cross-linking, we used an analog of UTP, U• , that carried
Unwinding of the 39-Proximal RNA–DNA
Hybrid during Transcriptional Arrest
TEC stalled at 127 (TEC27) gradually loses ability to
resume elongation without releasing RNA (Markovtsov
et al., 1996). This phenomenon is illustrated by the fact
Cell
34
Figure 1. RNA–DNA Cross-Linking in TECs
(A) The U• -A base pair. The arrow symbolizes
the attack of aziridinium intermediate at N8
of adenine, leading to a cross-link.
(B) Cross-linking in the walking TEC. The autoradiogram shows free [32P]RNA transcripts
(bottom panel) and cross-linked protein–
[32P]RNA or DNA–[ 32P]RNA products from TEC
walked to the positions indicated by the RNA
39 end. The U• probe was incorporated in
positions 120 (lanes 3–10) or 145 (lanes 12–
18) as indicated by negative numbers showing distance from the 39 end. The bottom part
of the gel was underexposed to compensate
for low yield of the cross-linked species. The
length of transcript is designated as “ RNA 39
end” ; an asterisk denotes the presence of the
U• probe. The incorporation of U• resulted in
slight retardation of the transcript in the gel
(compare RNA 22 with 22 * and 44 * with 44).
The doublet transcript observed at 126 and
127 (lanes 7 and 8) reflects the fact that these
positions are particularly prone to spontaneous elongation arrest.
(C) Effect of arrest in TEC27 on cross-linking.
Free RNA (bottom) and cross-linked species
(top) were obtained before and after 20 min
incubation, with or without NTP chase as indicated.
that the addition of NTP substrates to TEC27 (NTP
chase) results in z90% RNA extension to 134 at 0 min
but has no effect after 20 min of TEC incubation at 378C
(Figure 1C). The arrest is accompanied by the loss of
RNA–DNA cross-linking by the U• probe at 121 (27 and
214 in TEC27 and TEC34, respectively), suggesting that
the hybrid unwinds during arrest. To explore further the
relationship between arrest and unwinding of the hybrid,
the kinetics of formation of the arrested complex were
determined in TEC27, in which the hybrid was either
strengthened or weakened by incorporation of ribonucleotide analogs (Figure 2A). When RNA in the hybrid
contained standard nucleotides (Figure 2A, lanes 1–5),
z50% of the complex was arrested after 1 min incubation at 378C and z65% after 3 min. Substitution of CMP
at 23 and 24 with 5-iodoCMP (iC), an analog that stabilizes the duplex, resulted in a dramatic decrease in the
rate of arrest, yielding z15% and z25% of arrested
complex at 1 and 3 min, respectively (Figure 2A, lanes
6–10). Placement of another duplex-stabilizing analog,
5-bromo UMP (brU) at 27 had a similar albeit less profound effect, apparently because only one position in
the duplex was substituted (Figure 2A, lanes 11–15). In
contrast, helix-destabilizing substitutions, such as inosine (I) at 25 or 4-thiouridine (sU) at 27, dramatically
increased the rate of arrest (Figure 2A, lanes 16–20 and
21–25, respectively). However, when inosine was placed
at 210, i.e., upstream of the presumed RNA–DNA hybrid,
the rate of arrest slightly decreased (Figure 2A, lanes
26–30). In general, incorporation of analogs upstream
of 28 had the opposite effect as compared to the same
substitutions in the 39-proximal region (unpublished
data). The linkage between stability of the 39-terminal
RNA–DNA hybrid and transcriptional arrest indicates
that the hybrid is essential for maintaining the active
state of TEC.
The RNA–DNA Hybrid Is Needed to Keep
the RNA 39 Terminus in the Active Site
We examined the arrest phenomenon in relation to the
RNA–DNA hybrid at three known arrest sites that occur
in the transcription template used, i.e., TEC27 (Figure
2), TEC56 (Figure 3), and TEC80 (Figure 4). Each of these
sites has a particular characteristic: the arrest at 127
is irreversible, but arrest at 156 and 180 is transient
(E. N., unpublished data), whereas TEC80 carries a
39-proximal oligo(T) track that is the generic arrest signal
in prokaryotes and eukaryotes (Kerppola and Kane,
1991; Gu and Reines, 1995; Nudler et al., 1995).
According to the current hypothesis, transcriptional
arrest involves disengagement of the RNAP-active site
from the 39 terminus of RNA. This view is based on
the argument that transcript cleavage in the arrested
complexes, which in E. coli is effected by the GreB
factor, is actually performed by the active site that has
moved to an internal position within the transcript (Izban
and Luse, 1992, 1993; Borukhov et al., 1993; Nudler et
al., 1994; Reines, 1994; Rudd et al., 1994; Chamberlin,
1995; Orlova et al., 1995). To test the disengagement
hypothesis directly, we took advantage of the recent
observation that the Fe21 ion substituting for Mg 21 in
the RNAP active site in the binary promoter complex
The RNA–DNA Hybrid during Transcription
35
Figure 2. Elongation Arrest in TEC27 as a Function of RNA–DNA Hybrid Stability
(A) Effect of hybrid stability on arrest kinetics. TEC25 carrying [32P]RNA and, where indicated, substituted ribonucleotide analogs was walked
to 127 and then chased to 134 after incubation for indicated time periods. Note the effect of analogs on RNA bend migration. Failure of RNA
to extend from 127 to 134 reflects accumulation of arrested complex. The graphs on the right show quantitation of the data; the bold curve
represents TEC27 carrying natural nucleotides.
(B) Fe2 1- and GreB-induced RNA cleavage. TEC27 carrying [32P]RNA was prepared with standard substrates (lanes 1–5) or NTP analogs (lanes
6–10) and exposed to Fe21 and GreB before or after 5 min incubation. The faster mobility of the Fe2 1 cleavage products is due to 39-terminal
phosphate.
(C) Front-edge mapping. The autoradiogram shows protection of 32P-labeled template DNA fragment from degradation by ExoIII in TECs
identified. On the table, the left column shows the position of the front edge (F) deduced from the actual size of protected DNA fragment,
which is indicated on the right.
(D) Schematic representation of the three states of TEC27. The vertical bar represents the front edge; the circle represents the active site.
causes highly localized cleavage of DNA near the transcription start site. The cleavage occurs through local
generation of free hydroxyl radicals (Zaychikov et al.,
1996). We reasoned that in TEC, the postulated repositioning of the active site would be reflected by an upstream shift of Fe2 1-induced localized cleavage of RNA.
We also determined how GreB- and Fe 21-induced cleavage respond to changes in the hybrid stability.
In the arrested TEC27, substitution of Mg 2 1 with Fe 21
led to cleavage of the transcript at 111/112, which was
also the major site of GreB cleavage. Strengthening of
the 39 proximal hybrid with nucleotide analogs brU and
iC suppressed cleavage both by Fe 21 and GreB (Figure
2B). In TEC56, GreB cleavage strictly correlated with the
stability of the hybrid and with the extent of arrest (Figure
3A). In TEC80, strengthening of the oligo(U:A) hybrid
with brU inhibited cleavage by both GreB and Fe 21, while
weakening of the hybrid with sU increased the GreB
cleavage (Figure 4A). These results provide strong support for the disengagement model and demonstrate that
the RNA–DNA hybrid is essential for keeping the RNA
39 terminus in the polymerase active site.
Is the unwinding of the 39-proximal hybrid accompanied by rewinding of the RNA–DNA hybrid in the upstream region? To answer this question, the cross-linking of the U• probe at 145 was determined in TEC56
before and after arrest (Figure 3B). In the productive
TEC56, the cross-linking was small, apparently because
the probe was located outside of the presumed hybrid
(212) (Figure 3B, lanes 3 and 4). In the arrested TEC56,
however, cross-linking at 212 became prominent (Figure 3B, lanes 5 and 6), indicating that the hybrid in this
position had reformed. These experiments suggest that
elongation arrest and repositioning of the active site are
associated with shifting of the RNA–DNA hybrid upstream from the 39-proximal region.
Cell
36
Figure 3. Elongation Arrest in TEC56
(A) Effect of ribonucleotide analog substitution on arrest rate and
GreB-induced transcript cleavage. TEC56 carrying [32P]RNA was
prepared with standard rNTP (lane 1) or with the indicated substitutions (lanes 6, 11, and 16) and was challenged with the mixture of
NTP (A, C, U-chase; lanes 2, 7, 12, and 17) for 2 min to determine
the extension of RNA in productive complexes from 156 to 160.
Cleavage of RNA was determined by exposing each complex to
three increasing doses of GreB.
(B) Cross-linking of U• in position 145 in productive and arrested
TEC56. The bottom panel shows [32P]RNA in the TEC50 control and
in the productive (lanes 3 and 4) and arrested (lanes 5 and 6) TEC56
before and after NTP chase. The upper panel shows respective
cross-linked protein–[32P]RNA or DNA–[32P]RNA products. The productive TEC56 (lanes 3 and 4) carried 23BrU and 24iC, and the
arrested TEC56 (lanes 5 and 6) carried 21 and 22 I substitutions,
respectively.
(C) Schematic representation of the cross-linking experiment (B).
Backward repositioning of the hybrid during TEC56 arrest allows
the RNA–DNA cross-link.
Discontinuity of Elongation Is Suppressed
by Hybrid Strengthening
In our previous work (Nudler et al., 1994, 1995), it was
shown that DNA sites that induce arrest also cause characteristic irregularities of the TEC footprints. This result
was interpreted as evidence of contraction followed by
saltatory expansion of RNAP protein. The three characteristic features of the contracted, or strained, TEC were
as follows: (i) predisposition to arrest; (ii) sensitivity to
GreB-induced cleavage; and (iii) shortened distance between the 39 end of the transcript and the front boundary
of protection of DNA from degradation with exonuclease
III (Nudler et al., 1994). The latter parameter was thought
to reflect the distance between the active site and the
front edge, or CzF distance, in the RNAP molecule. The
apparent variability of the CzF value at certain DNA
sites was the principal basis for the inchworming metaphor used to describe the movement of RNAP.
Since predisposition to arrest and sensitivity to GreB
were directly linked with the stability of the 39-proximal
RNA–DNA hybrid, it seemed important to assess the
relation of the hybrid to the CzF value. This question
was particularly intriguing because both the presumed
contracted arrest-prone TEC27 and relaxed arrest-proof
TEC52 contained an RNA–DNA hybrid of about the same
length (Figure 1B), whereas the apparent CzF value in
the two complexes had been determined to be 11 and
18, respectively (Nudler et al., 1994).
In the experiment of Figure 2C, the front edge of
freshly stopped TEC27 (Figure 2C, lane 3) maps at 138
(apparent CzF 5 11). Irreversible arrest of TEC27 was
accompanied by backward translocation of the front
edge to about 127 (apparent CzF 5 0; Figure 2C, lane
4), reflecting a long-distance reverse translocation of
the whole RNAP (Komissarova and Kashlev, 1997a). Surprisingly, incorporation of hybrid-stabilizing brU and iC,
the substitutions that suppress arrest (Figure 2A), led to
forward translocation of the front edge to 145, yielding
apparent CzF 5 18, which is the characteristic value
of normal relaxed TEC. To interpret this observation,
one has to assume that TEC27 exists in three alternative
states, 145, 138, and 127, corresponding to the three
observed positions of the front edge (Figure 2D). Of the
three states, only 145 is truly elongation competent,
because it contains the intact 39-terminal hybrid and the
active site attached to the 39 terminus. The 145 and the
138 states are in equilibrium associated with unwinding–rewinding of the hybrid, whereas the conversion
of 138 into 127 is irreversible. The 138 and the 127
locations of the front edge apparently correspond to
relocation of the active site to 120 (28) and 1 11 (217),
respectively, as revealed by the Fe 21 and GreB cleavage
pattern (Figure 2B). Thus, in each of the three states, the
actual CzF always equals 18, even though the apparent
distance between the RNA 39 terminus and the front
edge is variable. Our earlier conclusions (Nudler et al.,
1994) were in error because we equated the position of
the active site with the 39 terminus, which is not the
case in complexes where the active site has relocated
to an internal position in RNA.
These conclusions were confirmed by analysis of
TEC80, another arrest-prone complex, containing 39-proximal oligo(T) track (Nudler et al., 1995). In most of the
The RNA–DNA Hybrid during Transcription
37
Figure 5. Induction of Discontinuous Elongation at 152 with Inosine
(A) Fe21- and GreB-induced cleavage of [ 32P]RNA transcript in TEC52
carrying guanine (lanes 1–3) or inosine (lanes 4–6) at 148, 149,
and 150.
(B and C) Front-edge mapping by ExoIII and transcriptional arrest.
For details, see legends to Figures 2B and 2C.
(D) Schematic representation of the two states of inosine containing
TEC52.
Figure 4. Elongation Arrest in TEC80
(A) Effect of brU and sU substitutions in positions 176 to 180 on
Fe21- (lanes 11 and 12) and GreB-induced (lanes 3–8) cleavage of
[32P]RNA transcript in TEC80. Control experiments with TEC75 (lanes
1 and 2), and TEC88 (lanes 9, 10, and 13) are presented.
(B) Front-edge mapping in TEC73 (lane 1), TEC75 (lane 2), TEC80
at different analog substitutions (lanes 3, 4, and 5), and TEC88 (lane
6). The autoradiogram shows protection of terminally labeled nontemplate DNA strand from ExoIII degradation. For details, see legend
to Figure 2C.
(C) Effect of brU and sU substitutions on elongation arrest. Autoradiogram shows [32P]RNA in various complexes before and after NTP
chase.
(D) Schematic representation of the two states of TEC80.
complex, the front edge maps at 190, with apparent
CzF 5 10 (Figure 4B). Only a small fraction of the complex displayed the front edge at 198 (CzF 5 18). Stabilization of the hybrid with brU shifted the front edge in
most of TEC80 to 198, while weakening of the hybrid
with sU completely removed the 198 signal. Thus, the
190 and 198 states of TEC80 are interconvertible and
are associated with unwinding–rewinding of the hybrid.
Only the 198 state is elongation proficient, while the
190 state is reversibly arrested (Figure 4C).
The above experiments explain sequence-specific irregularities of ExoIII footprints as the consequence of
the 39-proximal hybrid melting. It can be expected, then,
that destabilization of the 39-proximal RNA–DNA hybrid
at any site would lead to front-edge backtracking. To
test this prediction, we replaced three successive RNA
guanines at 148, 149, and 150 with inosine and determined the parameters of TEC52, which normally displays no footprint irregularities (Nudler et al., 1994). As
was shown above, this complex contains a 39-terminal
RNA–DNA hybrid of at least 8 bp (Figure 1B).
As can be seen from Figure 5, incorporation of inosine
into RNA leads to backtracking of the active site by z6
nt, as revealed by GreB- and Fe21-induced cleavage
(Figure 5A). This is accompanied by retardation of the
front edge for about the same distance, from 170 to
165 (Figure 5B), and elongation arrest (Figure 5C), which
is reversible because most of the backtracked complex
can be chased by prolonged incubation with NTP (data
not shown). In other words, at a randomly chosen sequence site, artificial weakening of the hybrid caused
TEC to enter into an apparently discontinuous arrestprone phase of elongation.
Cell
38
Figure 6. Model of TEC
Schematic representations of a productive and backtracked TEC
are shown. Mg 21 denotes the active site. DBS symbolizes the frontend DNA-binding site responsible for processivity. RBS or RNAbinding site was denoted the RNA “ tight binding site” (TBS) in previous models. The heteroduplex area is equivalent to RNA “ loose
binding site” (LBS) in previous models (Nudler et al., 1994, 1995).
Discussion
The principal results of this work are the conclusive
proof of the existence of an 8–9 bp RNA–DNA hybrid
in TEC and the demonstration of its functional role in
keeping the complex in register with the transcribed
sequence. It should be noted that the structure of the
hybrid may be different from a true helix because of
interaction with protein in TEC, e.g., it may be bent or
partially unwound. Our results agree with the early data
of Hanna and Meares (1983), who detected an RNA–DNA
hybrid in ternary initiation complexes on the promoter.
Because of the central position of the hybrid in TEC,
the uncertainty about its very existence was the major
stumbling block to our understanding of RNAP structure
and function. Resolution of the hybrid debate thus provides the basis for more definitive model building. We
wish to propose a model of TEC (Figure 6) that explains
elongation, pausing, and termination in structure-functional terms. In presenting the model, the role of the
RNA–DNA hybrid is discussed below in the context of
the following three groups of data: (i) irregularities of
TEC footprints and other evidence suggesting internal
plasticity of RNAP (inchworming); (ii) instances of TEC
moving backward, occasionally causing arrest of elongation; and (iii) the mechanism of processivity that, as
we showed previously (Nudler et al., 1996), does not
involve the hybrid.
RNAP Is Not an Inchworm
The central notion of the discontinuous (a.k.a. inchworm)
model of elongation was that advancing RNAP undergoes conformational transitions that are not synchronous with single-step nucleotide additions, i.e., TEC
moves nonmonotonically (Chamberlin, 1995). Mechanistically, this was envisioned as contraction and expansion of advancing RNAP. Originally, it was proposed
that inchworming is intrinsic to elongation, i.e., it constitutes the very mechanism of TEC advancement (Krummel and Chamberlin, 1992; Chamberlin, 1995). Subsequently, we showed that TEC advances mostly
monotonically, with the exception of incidental situations when specific sites in DNA induce irregularities of
ExoIII footprints (Nudler et al., 1994, 1995).
Recently, Komissarova and Kashlev (1997b) demonstrated reversible loss of catalytic activity in TEC at the
inchworming sites. In such complexes, RNAP translocated backward with time after halting, as shown by fast
DNA footprinting. Antisense oligonucleotides hybridized
upstream of RNAP restored the initial position of the
enzyme. On this basis, transient backtracking of RNAP
was suggested as an alternative to inchworming. Similar
suggestion has been made for yeast pol II by Reeder
and Hawley (1996).
Here, we directly demonstrate that TEC backtracks
as the result of weakness of the 39-proximal RNA–DNA
hybrid. The RNAP molecule that has backtracked appears to be in the same overall conformation as that in
the productive elongation-competent complex, including the constant distance between the active site and the
front boundary of ExoIII protection (the CzF parameter).
Moreover, in the backtracked complex, the RNA–DNA
hybrid appears to have shifted to an upstream region.
This conclusion is based on the observation of an RNA–
DNA cross-link reemerging in the upstream region in a
backtracked complex. Because of technical constraints,
the reemerging cross-link could be demonstrated only
for one site. The notion of hybrid shifting, however, is
supported by our preliminary result that nucleotide substitutions in the upstream region affect backtracking in
the direction opposite to the same substitutions in the
39-proximal region.
A principal conclusion from these studies is that elongation-competent productive TEC is always monotonic,
while apparently discontinuous advancement reflects
a reversible side pathway rather than a succession of
elongation intermediates (Figure 6). The irregular DNA
and RNA footprints that have been observed by us and
others (Krummel and Chamberlin, 1992; Nudler et al.,
1994, 1995; Wang et al., 1995; Zaychikov et al., 1995; Gu
et al., 1996; Samkurashvili and Luse, 1996) will have to be
reinterpreted as reflections of mixed populations of TEC
alternating between the productive and backtracked
states. Similarly, reversible unwinding–rewinding and
hybrid shifting may explain variable values of the length
of the hybrid and partial protection from chemical degradation reported earlier (Kainz and Roberts, 1992; Zaychikov et al., 1995).
It should be noted that repudiation of the inchworming
mechanism of elongation does not invalidate the notion
of RNAP plasticity. There is compelling evidence that
during the early steps of RNA chain synthesis, when
the RNA–DNA hybrid is first formed, the active center
domain “ stretches” to accommodate the nascent RNA
chain of 8–9 nt. This evidence was obtained with RNA
primers secured at the 59 face of the active center
The RNA–DNA Hybrid during Transcription
39
through a cross-link (Mustaev et al., 1993) or the Rifanchor (Mustaev et al., 1994), which excludes any possibility of RNA sliding through the protein. In view of this
evidence, our present results only demonstrate that the
stretched-out conformation of the active center, once
established during initiation, is monotonically preserved
throughout elongation and is probably maintained during backtracking. However, after transcription terminates and TEC dissociates, the active center must return
to its original “ compressed” state.
The RNA–DNA Hybrid and the Active Site:
The Zip-Lock Analogy
In our model, during backtracking the hybrid unwinds
in the 39 proximal region and shifts upstream. We propose that channels that guide DNA and RNA chains
through RNAP (Darst et al., 1991; Polyakov et al., 1995)
are shaped to accommodate the RNA–DNA heteroduplex just upstream of the active site. In effect, the
active site is “ glued” to the heteroduplex like a ziplock is to the locked section of the zipper. When the
heteroduplex unwinds and shifts upstream, it pulls back
the active site together with the whole RNAP.
What triggers and then determines the distance of
backtracking? Backtracking signals are A–U rich. However, the stability of the 39-terminal heteroduplex is not
everything to it, since there are instances of A-tracks in
the template that do not induce backtracking (E. N.,
unpublished data). Sequences behind and ahead of the
heteroduplex area were reported to affect elongation
arrest (Krummel and Chamberlin, 1992; Wiest et al.,
1992; Nudler et al., 1994, 1995). Since backtracking
seems to involve associated rewinding of the hybrid
upstream, as well as unwinding–rewinding of DNA duplexes ahead and behind of a transcription bubble, the
specific parameters of the event must depend on the
overall sequence context. Thus, in some cases backtracking may be short and reversible (as in TEC80), or
it may go too far and never come back (as in TEC27).
Backtracking may be resolved through internal RNA
cleavage and resumption of elongation from the newly
generated 39 terminus. It is easy to imagine that mismatched ribonucleotides that destabilize the 39 proximal
hybrid would facilitate backtracking. Thus, in addition
to keeping the active site in register with the template,
our model proposes a second principal role for the hybrid, namely sensing mismatches in RNA and then
launching corrective action (Erie et al., 1993; Jeon and
Agarwal, 1996).
Segregation of Processivity and Fidelity Functions
In combination with our previous work (Nudler et al.,
1996), these results demonstrate that separate sets of
interactions are responsible for the two principal biochemical features of TEC, i.e., processivity and fidelity.
The operational distinction between the two types of
interactions is that the former serves to counter dissociation of TEC, whereas the latter serves to counter its
wanton sliding along DNA.
Thus, in our hypothesis processivity is achieved by
the front-end DNA-binding site (DBS) that locks around
the double-helical DNA ahead of the bubble (Nudler et
al., 1996). The locking is induced allosterically by RNA
when it enters the RNA-binding site (RBS) behind the
heteroduplex. Interactions in DBS and RBS prevent TEC
dissociation (Nudler et al., 1996) but permit threading
of DNA and RNA through RNAP, i.e., lateral translocation
of TEC.
Fidelity, in our hypothesis, is achieved by heteroduplex interactions upstream of the active site, as this
work shows. While these interactions are weak and insufficient for stably holding the three components of
TEC together, they serve the principal role of maintaining
the register of TEC. When these interactions are disrupted, TEC slides back and forth even though it does
not dissociate.
Of course, the two types of interactions may crossinfluence each other. Since backtracking must involve
threading of RNA and DNA through RBS and DBS, respectively, it is not inconceivable that “ friction” in these
sites may affect backtracking. Conversely, weak protein–nucleic acid interactions in the heteroduplex area
are capable of holding the ternary complex together,
even when the strong processivity interactions in DBS
and RBS are destroyed. Such a complex is nonprocessive and highly unstable (Nudler et al., 1996).
The notion of separation of processivity and fidelity
functions has been discussed by Guajardo and Sousa
(1997), who attribute stability of TEC to structural devices (thumbs, locks, etc.) and explain positioning of
RNAP on the template through local free energy minima.
In their model, the key role in keeping RNAP in register
is played by the incoming substrate. Our views are in
general agreement with theirs, except that we give the
RNA–DNA hybrid the primary role in setting the freeenergy equillibrium that keeps TEC in register.
Implications for Pausing and Termination
From the proposed model of TEC, simple mechanisms
of pausing and termination follow. Backtracking of
RNAP induced by an unstable RNA–DNA hybrid is a
likely cause of a pause. Suppression of the elongation
rate by inosine in eukaryotic (Matsuzaki et al., 1994) and
prokaryotic (E. N., unpublished data) systems supports
this notion. Since the processivity interactions in the
backtracked complex are intact, such a pause would
not lead to termination. Thus, there is no mechanistic
difference between a paused and an arrested complex,
except that the latter cannot restart because of a particularly unfavorable sequence context. The factors that
accelerate elongation, such as bacteriophage antitermination factors N and Q (Yang and Roberts, 1989; Mason
et al., 1992; Das, 1993), E. coli NusG (Burova et al., 1995),
or eukaryotic elongin (Aso et al., 1995), may act through
suppressing RNAP backtracking.
Termination must involve disruption of the processivity interactions. This is likely to occur by an allosteric
mechanism triggered by detachment of RNA from RBS,
e.g., through the formation of a hairpin. The resulting
complex would be held together only by weak fidelity
interactions in the heteroduplex area. We speculate that
such a complex is a principal intermediate in the pathway leading to termination.
In conclusion, it should be emphasized that our model
Cell
40
combines elements from both of the initial conflicting
views on TEC (see Introduction). The hybrid is clearly
there, in agreement with the classical model (Yager and
von Hippel, 1986, 1991), but its primary role is not holding
together the components of TEC. This function is
achieved through protein–nucleic acid interactions outside of the hybrid area, in accord with the revisionist
view (Chamberlin, 1995).
Experimental Procedures
The T7 A1 promoter-containing template (226 bp) was obtained by
polymerase chain reaction (PCR) from Template 2 (Nudler et al.,
1995). Its transcribed sequence is ATCGAGAGGG ACACGGCGAA
TAGCCATCCC AATCGACACC GGGGTCCGGG ATCTGGATCT GGA
TCGCTAA TAACATTTTT ATTTGGATCC CCGGGTACCG AGCTCGA
ATT CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC
TGGCG.
His-tagged RNAP was purified and immobilized on Ni-NTA-agarose as described (Nudler et al., 1996). The preparation of a start-up
ternary complex with a 12 nt 32P-labeled transcript and the walking
reaction were performed as described (Nudler et al., 1996).
The cross-linking reagent U• is an alkylating derivative of UTP
containing 3-[39-(N,N-bis-2-iodoethyl)amino-4’-formylphenyl]propionate moiety attached to the 5 position of the pyrimidine ring through
an aminoallyl spacer. The complete synthesis of U• will be published elsewhere. Upon exposure of U• to NaBH4, the aromatic bis(2iodoethyl)amino group of U• is converted to a reactive aziridinium
intermediate shown in Figure 1A. The reaction proceeds through
reduction of the aldehyde group of U• , which in turn activates the
2-iodoethylamino group (Gall et al., 1979).
In a typical cross-linking reaction, TEC stalled at appropriate position was washed 3 times with the buffer containing 20 mM HEPES
(pH 7.25), 100 mM KCl, 2 mM MnCl 2 . The U• probe was added to
20 ml of reaction mixture to the final concentration of 40 mM. The
complex was incubated for 30 min at 208C followed by the walking
reaction in the buffer (20 mM HEPES [pH 7.25], 100 mM KCl, 10 mM
MgCl2 ). To activate the cross-link, freshly prepared solution of NaBH4
(4 mg/ml, Aldrich) was added to 0.4 mg/ml for 20 min at 208C. The
reaction was stopped by adding 2 vol of the loading mix (10 mM
EDTA, 12 M Urea and BPB), heated at 808C for 1 min, and the crosslinked species were resolved on a 12% PAGE (19:1 acrylamide:bisacrylamide, 7M urea, 0.53 TBE).
Incorporation of substrate analogs during the walking reaction
(Nudler et al., 1996) was performed at 208C for 10 min. The analog
concentrations were as follows: inosine triphosphate (I; Boehringer
Manheim), 20 mM; 5-bromouridine-59-triphosphate (brU; Sigma), 10
mM; 5-iodocytidine-59-triphosphate (iC; Sigma), 20 mM; sU (synthesized from 4-thio-UMP [Sigma] as described [Hoard and Ott, 1965]),
40 mM.
GreB cleavage and ExoIII footprinting were performed as described previously (Nudler et al., 1994), except that the time of incubation with ExoIII was 2–3 min.
In experiments with Fe2 1-mediated transcript cleavage, TEC was
washed 6 times with the buffer containing 20 mM HEPES (pH 7.25),
50 mM KCl, followed by addition of 40 mM (NH4)2Fe(SO4)2• 6H2 O
(Aldrich) and 10 mM DTT for 20 min at 208C. The reaction was
stopped and cleavage products resolved on a 12% or 6% PAGE as
described above. It should be noted that Fe2 1 cleavage of RNA
is a low yield reaction that occurs concomitantly with nonspecific
degradation of RNAP protein. As a consequence, the specific RNA
cleavage reaction rapidly reaches a plateau and can be used only
in qualitative assays.
Acknowledgments
We are grateful to E. Avetissova for His-tagged RNAP, S. Borukhov
for GreB protein, M. Kozlov for advice on cross-linking techniques,
and N. Komissarova and M. Kashlev, who were the first to suggest
RNAP backtracking and shared their ideas. This work was supported
by NIH grant GM49242.
Received December 13, 1996; revised February 18, 1997.
References
Altman, C.R., Solow-Cordero, D.E., and Chamberlin, M.J. (1994).
RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme RNA complex. Proc. Natl.
Acad. Sci. USA 91, 3784–3788.
Aso, T., Lane, W.S., Conaway, J.W., and Conaway, R.C. (1995).
Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II. Science 269, 1439–1443.
Borukhov, S., Sagitov, V., and Goldfarb, A. (1993). Transcript cleavage factors from E. coli. Cell 72, 459–466.
Burova, E., Hung, S.C., Sagitov, V., Stitt, B.L., and Gottesman, M.E.
(1995). Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro. J. Bacteriol. 177, 1388–1392.
Chamberlin, M.J. (1995). New models for the mechanism of transcription elongation and its regulation. Harvey Lect. 88, 1–21.
Chan, C.L., and Landick, R. (1994). New perspectives on RNA chain
elongation and termination by E. coli RNA polymerase. In Transcription: Mechanisms and Regulation, R.C. Conaway and J.W. Conaway,
eds. (New York: Raven Press, Ltd.), pp. 297–321.
Darst, S.A., Edwards, A.M., Kubalek, E.W., and Kornberg, R.D.
(1991). Three-dimensional structure of yeast RNA polymerase II at
16 Å resolution. Cell 66, 121–128.
Das, A. (1993). Control of transcription termination by RNA-binding
proteins. Annu. Rev. Biochem. 62, 893–930.
Erie, D.A., Hajiseyedjavadi, O., Young, M.C., and von Hippel, P.H.
(1993). Multiple RNA polymerase conformations and GreA: control
of the fidelity of transcription. Science 262, 867–873.
Farnham, P.J., and Platt, T. (1980). A model for transcription termination suggested by studies on the trp attenuator in vitro using base
analogs. Cell 20, 739–748.
Gall, A.A., Kurbatov, V.A., Mustaev, A.A., and Shishkin, G.V. (1979).
Reagents for directed modification of biopolymers. Aromatic
2-chlortoaethylamines with controlled reactivity. Izv. Sib. Otd. Acad.
Nauk SSSR Ser. Khim. Nauk 4, 99–104.
Gamper, H.B., and Hearst, J.E. (1982). A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes. Cell 29, 81–90.
Gu, W., and Reines, D. (1995). Identification of a decay in transcription potential that results in elongation factor dependence of RNA
polymerase II. J. Biol. Chem. 270, 11238–11244.
Gu, W., Wind, M., and Reines, D. (1996). Increased accommodation
of nascent RNA in a product site on RNA polymerase II during arrest.
Proc. Natl. Acad. Sci. USA 93, 6935–6940.
Guajardo, R., and Sousa, R. (1997). A model for the mechanism of
polymerase translocation. J. Mol. Biol. 265, 8–19.
Hanna, M.M., and Meares, C.F. (1983). Topography of transcription
path: the leading end of nascent RNA through the Escherichia coli
transcription complex. Proc. Natl. Acad. Sci. USA 80, 4238–4242.
Hoard, D.E., and Ott, D.G. (1965). Conversion of mono- and oligodeoxyribonucleotides to 59-triphosphates. J. Am. Chem. Soc. 87,
1785–1789.
Izban, M.G., and Luse, D.S. (1992). The RNA polymerase II ternary
complex cleaves the nascent transcript in 39-59 direction in the presence of elongation factor SII. Genes Dev. 6, 1342–1356.
Izban, M.G., and Luse, D.S. (1993). The increment of SII-facilitated
transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes. J. Biol.
Chem. 268, 12874–12885.
Jeon, C., and Agarwal, K. (1996). Fidelity of RNA polymerase II
transcription controlled by elongations factor TFIIS. Proc. Natl.
Acad. Sci. USA 93, 13677–13682.
Johnson, T.L., and Chamberlin, M.J. (1994). Complexes of yeast
RNA polymerase II and RNA are substrates for TFIIS-induced RNA
cleavage. Cell 77, 217–224.
Kainz, M., and Roberts, J. (1992). Structure of transcription elongation complex in vivo. Science 255, 838–841.
Kashlev, M., Martin, E., Polyakov, A., Severinov, K., Nikiforov, V., and
The RNA–DNA Hybrid during Transcription
41
Goldfarb, A. (1993). Histidine-tagged RNA polymerase: dissection of
the transcription cycle using immobilized enzyme. Gene 130, 9–14.
Kerppola, T.K., and Kane, C.M. (1991). RNA polymerase: regulation
of transcript elongation and termination. FASEB J. 5, 2833–2842.
Komisarrova, N., and Kashlev, M. (1997a). Arrest of transcription:
E. coli RNA polymerase translocates backward leaving the 39 end
of the RNA intact and extruded. Proc. Natl. Acad. Sci. USA 94, in
press.
Komisarrova, N., and Kashlev, M. (1997b). RNA polymerase switches
between inactivated and activated states translocating back and
forth along the DNA and the RNA. J. Biol. Chem. 272, in press.
Krummel, B., and Chamberlin, M.J. (1992). Structural analysis of
ternary complexes of Escherichia coli RNA polymerase. Individual
complexes halted along different transcription units have distinct
and unexpected biochemical properties. J. Mol. Biol. 225, 221–234.
elongation and termination. In Escherichia coli and Salmonella typhimurium, 2nd Ed., F.C. Neidhardt, ed. (Washington, D.C.: American
Society for Microbiology), pp. 822–848.
Rudd, M.D., Izban, M.G., and Luse, D.S. (1994). The active site of
RNA polymerase II participates in transcript cleavage within arrested
ternary complexes. Proc. Natl. Acad. Sci. USA 91, 8057–8061.
Samkurashvili, I., and Luse, D.S. (1996). Translocation and transcriptional arrest during transcript elongation by RNA polymerase II. J.
Biol. Chem. 271, 23495–23505.
Sweetser, D., Nonet, M., and Young, R.A. (1987). Prokaryotic and
eukaryotic RNA polymerases have homologous core subunits. Proc.
Natl. Acad. Sci. USA 84, 1192–1196.
Wang, D., Meier, T.I., Chan, C.L., Feng, G., Lee, D.N., and Landick,
R. (1995). Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex.
Cell 81, 341–350.
Markovtsov, V., Mustaev, A., and Goldfarb, A. (1996). Protein–RNA
interactions in the active center of transcription elongation complex.
Proc. Natl. Acad. Sci. USA 93, 3221–3226.
Wiest, D.K., Wang, D., and Hawley, D.K. (1992). Mechanistic studies
of transcriptional arrest at the adenovirus majour late attenuation
site. J. Biol. Chem. 267, 7733–7744.
Mason, S.W., Li, J., and Greenblatt, J. (1992). Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage l. J. Biol. Chem.
267, 19418–19426.
Yager, T.D., and von Hippel, P.H. (1987). Transcript elongation and
termination in Escherichia coli. In Escherichia coli and Salmonella
typhimurium, F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik,
M. Schaechter, and H.E. Umbarger, eds. (Washington, D.C.: American Society for Microbiology), pp. 1241–1275.
Matsuzaki, H., Kassavetis, G.A., and Geiduschek, E.P. (1994). Analysis of RNA chain elongation and termination by Saccharomyces
cerevisiae RNA polymerase III. J. Mol. Biol. 235, 1173–1192.
Mustaev, A., Kashlev, M., Zaychikov, E., Grachev, M., and Goldfarb,
A. (1993). Active center rearrangement in RNA polymerase initiation
complex. J. Biol. Chem. 268, 19185–19187.
Mustaev, A., Zaychikov, E., Severinov, K., Kashlev, M., Polyakov,
A., Nikiforov, V., and Goldfarb, A. (1994). Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide
compounds. Proc. Natl. Acad. Sci. USA 91, 12036–12040.
Nudler, E., Goldfarb, A., and Kashlev, M. (1994). Discontinuous
mechanism of transcription elongation. Science 265, 793–796.
Nudler, E., Kashlev, M., Nikiforov, V., and Goldfarb, A. (1995). Coupling between transcription termination and RNA polymerase inchworming. Cell 81, 351–357.
Nudler, E., Avetissova, E., Markovtsov, V., and Goldfarb, A. (1996).
Transcription processivity: RNA polymerase–DNA interactions holding together the elongation complex. Science 273, 211–217.
Orlova, M., Newlands, J., Das, A., Goldfarb, A., and Borukhov, S.
(1995). Intrinsic transcript cleavage activity of RNA polymerase.
Proc. Natl. Acad. Sci. USA 92, 4596–4600.
Platt, T. (1997). RNA structure in transcription elongation, termination, and antitermination. In RNA Structure and Function, R. Simons
and M. Grunberg-Manago, eds. (Cold Spring Harbor, New York:
Cold Spring Harbor Laboratory Press), in press.
Polyakov, A., Severinova, E., and Darst, S.A. (1995). Three-dimensional structure of E. coli core RNA polymerase: promoter binding
and elongation conformations of the enzyme. Cell 83, 365–373.
Puhler, G., Leffers, H., Gropp, F., Palm, P., Klenk, H.P., Lottspeich, F.,
Garrett, R.A., and Zillig, W. (1989). Archaebacterial DNA-dependent
RNA polymerases testify to the evolution of the eukaryotic nuclear
genome. Proc. Natl. Acad. Sci. USA 86, 4569–4573.
Reeder, T.C., and Hawley, D.K. (1996). Promoter proximal sequences modulate RNA polymerase II elongation by a novel mechanism. Cell 87, 767–777.
Reines, D. (1994). Transcript elongation and gene regulation in eukaryotes. In Transcription Mechanisms and Regulation, R.C. Conaway and J.W. Conaway, eds. (New York: Raven Press, Ltd.), pp.
263–278.
Rice, G.A., Kane, C.M., and Chamberlin, M.J. (1991). Footprinting
analysis of mammalian RNA polymerase II along its transcript: an
alternative view of transcription elongation. Proc. Natl. Acad. Sci.
USA 88, 4245–4249.
Richardson, J.P., and Greenblatt, J. (1996). Control of RNA chain
Yager, T.D., and von Hippel, P.H. (1991). A thermodynamic analysis
of RNA transcript elongation and termination in E. coli. Biochemistry
30, 1097–1118.
Yang, X., and Roberts, J.W. (1989). Gene Q antitermination protein
of Escherichia coli phages 82 and l suppress pausing by RNA
polymerase at a r-dependent terminator and other sites. Proc. Natl.
Acad. Sci. USA 86, 5301–5305.
Zaychikov, E., Denissova, L., and Heumann, H. (1995). Translocation
of the Escherichia coli transcription complex observed in the registers 11 to 20; “ jumping” of RNA polymerase and asymmetric expansion and contraction of the “ transcription bubble.” Proc. Natl. Acad.
Sci. USA 92, 1739–1743.
Zaychikov, E., Martin, E., Denissova, L., Kozlov, M., Markovtsov, V.,
Kashlev, M., Heumann, H., Nikiforov, V., Goldfarb, A., and Mustaev,
A. (1996). Mapping of catalytic residues in the RNA polymerase
active center. Science 273, 107–109.