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INTRODUCTION |
Abortive transcription by Escherichia coli RNA
polymerase is characterized by reiterative synthesis and release of
short transcripts of variable length in a
template-dependent manner (1-5). This nonproductive
process occurs at most promoters, even in the presence of high
concentrations of all four substrates (6). During this process RNA
polymerase does not usually dissociate from the template (7). The RNA
cleavage factors GreA and GreB have been found to reduce the production
of abortive transcripts while concomitantly increasing that of
full-length transcripts at some weak promoters (8-10). Therefore, it
is conceivable that there could be a regulatory switch operating at
this initiation step that affects the efficiency of productive
elongation.
The
70 subunit is believed to be retained during
abortive transcription, and the establishment of an elongation complex
is correlated with release of
subunit from the holoenzyme (11-13), which suggests a possible involvement of this subunit in abortive synthesis. Two spontaneously generated missense alleles have been isolated, rpoD(P504L) and rpoD(S506F), whose
mutations alter region 3 of the
70 subunit and have been
shown to compensate for the lack of ppGpp-dependent functions in a ppGpp0 strain (14). It is known that region
3 can be cross-linked to the catalytic center of the enzyme (15) and
becomes inaccessible from outside upon holoenzyme formation (16).
Holoenzymes with these mutant
subunits also exhibit reduced
abortive initiation at several phage promoters (17). Thus their study
should shed light on the mechanism of abortive initiation and the
involvement of region 3 of
70 in this process.
Recently Kubori and Shimamoto (18) demonstrated the existence of
nonproductive complexes yielding only abortive products at modified
PR and lacUV5 promoters. These
nonproductive complexes were termed "moribund complexes," since
they are incapable of productive elongation and gradually become fully
inactive. Consistent with the inactivation, these promoters yield
significantly fewer than one full-length transcript per promoter in a
single-round transcription (19). In the present study, we investigated
the reduced output of abortive transcripts at a modified
PR promoter (
PRAL)
caused by the
70 mutations P504L and S506F. The faster
dissociation and concomitant lesser accumulation of moribund complexes
were found to be the major causes of the low levels of abortive
synthesis by these two mutant enzymes.
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EXPERIMENTAL PROCEDURES |
Materials--
Nucleoside triphosphates were obtained from
Yamasa (Tokyo), and all radioisotopes were from NEN Life Science
Products (Bethesda, MD). All other chemicals were of analytical grade.
Restriction enzymes were purchased from Takara and Toyobo (Tokyo,
Japan). Taq DNA polymerase was purchased from Boehringer
Mannheim.
All the linear DNA templates were prepared from parent plasmids by the
polymerase chain reaction amplification method. The construction of
these plasmids has been described elsewhere (19). The templates
carrying the
PR promoter have 32 (
PRAL32) or 73 bp1
(
PRAL73) A-less initial transcribed
sequences, with total lengths of 190 and 230 bp, respectively. The
transcription start site is situated 85 bp away from the upstream end
of each DNA fragment. The original
PR
promoter partially overlaps the divergent
PRM promoter in its upstream region. We inactivated
PRM by changing each of the bases
7A,
11T,
and
12A into C (the positions are numbered with respect to the start
site of PRM) to produce the modified promoter
PRAL. A 160-bp fragment harboring the T7A1 promoter (T7A1 DNA) was prepared as described previously (19). The
run-off transcript produced on this template was 74 nucleotides in
length. All the polymerase chain reaction products were purified on 8%
polyacrylamide gels and eluted. Immobilized template DNA was prepared
as described previously (16, 18, 19).
The wild-type holoenzyme and core enzyme were purified according to
Gonzalez et al. (20) by using Biorex 70 (Bio-Rad) instead of
phosphocellulose. Mutant
factors were purified from overexpressing strains (17) by the following modified procedure. After the ammonium
sulfate fractionation, the samples were subjected successively to
phenyl Toyopearl M, DEAE-Toyopearl M (TOSO; Tokyo, Japan) and phenyl-Superose HR (Amersham Pharmacia Biotech) column chromatography. The purity of the mutant
factors was judged using a 7.5%
polyacrylamide gels stained by the reverse staining method (21).
Holoenzyme was reconstituted by mixing core enzyme with a 1.5 molar
excess of mutant
subunit. We confirmed that this amount of mutant
was optimal (data not shown) and that a further increase in the amount did not significantly change the activities (17).
In Vitro Transcription Assay--
All the transcription assays
were carried out as described previously (18, 19) in T buffer (50 mM Tris-HCl, pH 7.9, 100 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml partially hydrolyzed casein) at 37 °C for 20 min. The
concentration of the
-32P-initiating nucleotide (GTP or
ATP) was 5 µM, whereas the other nucleoside triphosphates
were 0.1 mM. Unless otherwise indicated, 40 µg/ml heparin
was added simultaneously with the substrates. For pulse-labeling
experiments in the presence of 100 mM NaCl, the RNA
polymerase-promoter binary complexes were formed in T buffer. NaCl was
then added in both the unlabeled and labeled substrate solutions.
The dissociation of the binary complexes was measured by trapping the
dissociated enzyme with heparin (100 µg/ml) or with a 40-fold molar
excess of T7A1 promoter-containing DNA (described in the text).
Gel Shift Assay--
Binary complexes were formed in T buffer
containing 12% glycerol at 37 °C for 10 min. Samples were then
loaded onto a 4% polyacrylamide gel containing 6.75 mM
Tris acetate, pH 8.0, and 1 mM EDTA. Electrophoresis was
carried out for 1.5 h at a constant voltage of 150 V at room temperature in a circulating buffer system.
Permanganate Footprinting--
Potassium permanganate
footprinting was performed according to Suh et al. (22) with
the following modifications. After the permanganate reaction, the
samples were passed through Sephadex G-25 micro spin columns, treated
with phenol/chloroform/isoamyl alcohol, and then precipitated with
ethanol in the presence of sodium acetate and glycogen. DNA was cleaved
by incubating the samples in 10% piperidine at 90 °C for 30 min,
and the products were resolved on an 8% sequencing gel.
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RESULTS |
(a) Affinities of the Mutant Enzymes for the
PR
Promoter and the Lifetime of Their Binary Complexes--
We examined
the effects of the mutations in region 3 of
70 on the
stability of the binary complex of RNA polymerase with the
PRAL promoter. First, a gel shift assay was
employed in the absence of heparin to measure the apparent dissociation
constants of complexes of the promoter with the wild-type and the two
mutant holoenzymes. Essentially the same binding isotherm was observed
for all three enzymes, so the mutations do not change the equilibrium
constant for formation of the binary complex. The observed isotherm was sigmoidal, with a half-saturation around 20 nM (data not
shown). This sigmoidal behavior is likely to be an artifact of the gel shift assay, because a single-round transcription assay using various
concentrations of the wild-type enzyme yielded the usual hyperbola,
with a half-saturation at 14 ± 3 nM (data not
shown).
Heparin traps free RNA polymerase and therefore can be used to measure
the rate of dissociation of binary complexes (7). We compared the
heparin sensitivity of the binary complexes formed by the three
enzymes. Complexes were formed by incubating the
PRAL32 DNA with a nearly saturating
concentration of each enzyme at 37 °C for 10 min, which was
sufficient for the binding to reach equilibrium. Incubation was
continued for another 10 min in the presence of 100 µg/ml heparin,
and the gel shift assay was then performed. Only 7 and 13% of the
binary complexes formed by the S506F and P504L enzymes, respectively,
resisted heparin, whereas 60% survived in the wild-type case (data not
shown). This suggests that a smaller fraction of the mutant enzymes
form heparin-resistant complexes such as the open complex and/or that
reversion from open to closed complex is more rapid.
Next we studied the dissociation kinetics of the binary complexes of
wild-type and S506F enzymes with the
PRAL32
promoter. In these experiments, binary complexes were first formed with
PRAL32 at 37 °C, then further incubated
for times ranging from 15 s to 20 min with either 100 µg/ml
heparin or a 40-fold molar excess of T7A1 promoter as competitor.
Finally, nucleotide substrates were added. The amounts of competitors
used were enough to prevent the rebinding of the enzyme to
PRAL32. We compared the dissociation kinetics
using two different competitors because it has been reported that
heparin is not a simple competitor for some promoters (23). The assay
measures the fraction of open complex that has survived in the presence
of a competitor for each period of time. We monitored the formation of
both the full-length (32-mer) and abortive (9-mer) products.
Figs. 1, a and b
show the decay profiles for the two enzymes. The profile for the
wild-type enzyme was almost monophasic, whereas that for the S506F
mutant was biphasic and characterized by an early rapid decay. The
kinetic parameters obtained are listed in Table
I, proving that there is more than one
type of binary complex of the S506F enzyme and that the major type is
short-lived. Thus the back reaction into the free components is faster
for the mutant enzyme, although the overall equilibrium constant of binary complex formation was not significantly different from wild
type, as shown above by the gel shift assay in the absence of
competitors.

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Fig. 1.
Dissociation of the binary complexes at the
PRAL32 promoter. The wild-type
(Wt) holoenzyme (a) or S506F holoenzyme
(b) were preincubated with PRAL32
for 10 min at 37 °C. They were then incubated with either 100 µg/ml heparin or a 40-fold excess of T7A1 DNA for the indicated
times. Substrates were added next to initiate transcription reactions
of 20 min. The amounts of transcripts were plotted against the time of
incubation with the competitors. ATP was excluded from the substrate
mixture so that a 32-mer transcript was the full-length product. The
filled circles and squares show the amounts of
32-mer and 9-mer transcripts, respectively, when T7A1 DNA was used as
competitor. Open circles and squares show the
amounts of 32-mer and 9-mer, respectively, when heparin was used as
competitor. The concentrations of enzyme and DNA were 35 and 12 nM, respectively, except that the concentration of template
DNA was 5 nM when T7A1 DNA was used as competitor.
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Table I
The values of the dissociation rate constants (k) for binary complexes
of RNA polymerase carrying wild-type or S506F at the
PRAL32 promoter
Dissociation rate constants (k) were obtained by fitting the
data to single exponential or double exponential expressions of the
forms y = A exp( k1t)
and y = A exp( k1t) + B
exp( k2t) for the wild-type and S506F
enzymes, respectively. In the case of wild-type enzyme, the decay is
monophasic, so it is designated by one rate constant.
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Both heparin and T7A1 DNA exerted the same effect, within experimental
error, as shown in Table I. Therefore in our system heparin acts as a
simple competitor of the binary complexes by binding to free RNA
polymerase. In the following sections all the interpretations of
experiments using heparin assume this simple competition model.
(b) Promoter Melting by the Mutant Enzymes--
Permanganate
preferentially modifies thymine residues in single-stranded or
distorted regions of DNA in open promoter complexes (24). In the
presence of a saturating concentration of any of the enzymes, the
thymine residues at
13,
10,
7,
4,
3, and +2 of the
nontemplate strand and
8 and
9 of the template strand exhibited
strongly increased permanganate sensitivity (Fig.
2). These residues were much more
sensitive in the wild-type than in the mutant complexes, with S506F
showing the least sensitivity. But the relative sensitivities of the
residues in a given complex remained the same. These results suggest
that smaller fractions of the binary complexes are in the "open"
form in the case of the mutant enzymes, especially S506F. These
mutations in region 3 of
70, therefore, shift the
equilibrium from the open toward the closed promoter state. This result
is consistent with the presence of a large fraction of short-lived
complexes among the binary complexes formed by the S506F mutant enzyme
(Fig. 1); the major fraction of the mutant binary complexes is
presumably in a closed state.

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Fig. 2.
KMnO4 footprinting of the binary
complexes with PRAL promoter.
Footprinting experiments were done with linearized
PRAL73 template labeled at the 5' end of
either the nontemplate (a) or the template (b)
strand. A sequence ladder was used to identify the
permanganate-sensitive bases (lanes not shown). Control lanes
(C) show the pattern in the absence of enzyme. Enzyme and
DNA concentrations were 35 and 12 nM, respectively.
Wt, wild-type; T, thymine.
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(c) Productive and Abortive Transcriptions by the Mutant
Enzymes--
The A-less template (
PRAL32)
was designed so that by excluding ATP during transcription, a 32-mer
paused product would be formed. Both the full-length (32-mer) and the
shorter abortive products of this template can easily be quantified on
the same polyacrylamide gel (18). 5-mer and 9-mer transcripts were the major products observed along with the 32-mer using this template (Fig.
3a). These short 5- and 9-mers
were indeed abortive products, as shown in section "d."

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Fig. 3.
Transcription of RAL32
template for 20 min. a, transcripts synthesized by wild-type
(Wt), P504L, and S506F holoenzymes in the presence (+) and
absence ( ) of heparin. The transcripts were labeled with
[ -32P]GTP. The enzyme and DNA concentrations were 35 and 12 nM, respectively. b, amounts of 32-mer
(filled) and 5-mer (open) products in the
presence (+) and absence ( ) of heparin are plotted in different
scales. c, amounts of 5-mer products relative to those of
the 32-mer full-length products.
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The amounts of products formed in the presence or absence of heparin
are quantified in Fig. 3, b and c. At a nearly
saturating concentration of the enzyme (~90% bound), both mutants
produced less RNA than the wild-type enzyme (panel b). The
amounts of abortive products (4-9-mer) were particularly reduced by
the mutations, as is evident from the ratios of 5-mer to 32-mer
transcripts (panel c). The 5-mer and 32-mer syntheses are,
therefore, differentially inhibited by the mutations, suggesting the
involvement of more than one pathway in the syntheses of these two
products.
(d) Time Courses of Accumulation of Full-length and Abortive RNA
Products of
PRAL32--
Next we examined the time
course of accumulation of the transcripts generated by both free and
immobilized
PRAL32 templates in the presence
of heparin. Templates that had been immobilized on avidin-acrylic beads
were used to separate the released abortive products from those
retained in the transcription complexes. After transcription with
immobilized templates and brief centrifugation, the supernatant
contained only released products. Fig.
4a shows that most of the
products shorter than 12 nucleotides were released and, thus, indeed
products of abortive transcription. This was true not only for S506F
but also for the other two enzymes (data not shown). Panels
b and c are plots of the time courses of accumulation of the two major products (5-mer and 32-mer) using the free template with the wild-type and S506F enzymes, respectively. The production of
the full-length 32-mer transcript had a time lag of about 48 s for
the wild-type and P504L enzymes and about 18 s for S506F enzyme,
respectively (panel c). 32-mer synthesis by all the enzymes reached a plateau at around 10 min (data for P504L are not shown). In
the case of the wild-type enzyme, the time course of 5-mer production
had an initial burst phase and a slow phase continuing beyond 20 min
(panel b). The latter slow accumulation has been interpreted
as evidence for the formation of moribund complexes, which are defined
as complexes capable of persistent production only of abortive
transcripts. However, the slow accumulation phase was not observed or
barely detectable with the mutants, and both 5-mer and 32-mer
production reached plateaus within 10 min (panel b).

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Fig. 4.
Time-courses of transcription of the
PRAL32 template. a, the
immobilized PRAL32 template was transcribed
by S506F for the indicated times, and reactions were then stopped with
25 mM EDTA. The reaction mixture was centrifuged, half the
supernatant (1/2 Sup) was examined separately, and the rest
was remixed with the pellet (1/2 Sup + Pellet). The
triangles denote the time points as 15 and 30 s and 1, 3, 5, 10, 15, and 20 min. The enzyme and DNA concentrations were the
same as in Fig. 3. b, the time courses of 32-mer ( ) and
5-mer ( ) synthesis by wild-type (Wt), and the time
courses of 32-mer ( ) and 5-mer ( ) syntheses by S506F holoenzyme
determined by experiments using nonimmobilized templates under the same
conditions. c, the first 5 min of the time course of 32-mer
synthesis by wild-type ( ) and S506F ( ) enzymes, respectively. In
panels b and c, the amount of each transcript was
normalized to the amount of 32-mer formed at 20 min. Experiments were
done in the presence of heparin. In a previous study, the 9-mer was the
transcript found to be most characteristic of the moribund complex, and
it continued to be produced up to 20 min (18), but in the present study
the persistent production of 5-mer was more pronounced. Although the
reasons for this difference are unknown, it was associated with the use
of different brands of radioisotope and/or with a change in the
template (190-bp template instead of 1130 bp).
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(e) Pulse-labeling Assay to Detect Moribund Complex--
The
analysis of time course data could be obscured if cleavage of
transcripts were taking place. So we have used the pulse-labeling assay
to confirm the difference between the temporal behaviors of abortive
and full-length transcription. In this assay, transcription was first
initiated with cold substrates, and then the
-32P-labeled initiating nucleotide was added at various
time points, followed by transcription for a further 5 min. This assay
can detect the initiation of abortive products even after the synthesis of full-length transcripts has ceased. Such a persistent initiation of
short transcripts is characteristic of moribund complexes at the
PRAL32 promoter (18).
The time course of synthesis of abortive products by the wild-type
enzyme at the T7A1 promoter did not show any slow phase (data not
shown). Therefore, moribund complexes (if any) do not accumulate at
this promoter. To confirm the reliability of the pulse label assay, we
used the T7A1 system as a control. As expected, incorporation of
[
-32P]ATP was rapid, and neither full-length nor
abortive transcripts were labeled after 3 min (Fig.
5a).

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Fig. 5.
Pulse-labeling experiments in the presence of
40 µg/ml heparin. Transcriptions were carried out in the
presence of 35 nM wild-type (Wt) enzyme and 12 nM T7A1 (a) and PRAL32
(b) templates. Panel c is the same experiment
with S506F enzyme on the PRAL32 template. See
the text for details.
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With the
PRAL promoter, that the
persistent initiation of 9-mer and 5-mer products after 32-mer
initiation had ceased was observed in the case of the wild-type enzyme
(Fig. 5b) but was almost undetectable (Fig. 5c)
for S506F enzyme. Since most binary complexes of the mutant enzyme
dissociate within 1 min and the released enzyme is trapped by heparin,
the absence of persistent initiation might be an artifact caused by
heparin, which was added with the substrates to prevent enzyme
turnover. To avoid this potential artifact, we repeated the
pulse-labeling experiments in the absence of heparin by using an
alternative method to block turnover.
PRAL32
template was kept in 3-fold excess over enzyme, and the elongation
complex was stalled at +32 by excluding ATP. In a control experiment,
to confirm the absence of turnover in this protocol, we added a
template harboring a 73-bp A-less leader sequence (AL73 template) plus
labeled substrates to the pre-incubated mixture of
PRAL32 template, enzyme, and unlabeled
substrates. We did not detect the 73-mer transcript, confirming that
enzyme turnover had been blocked. The elimination of heparin did not significantly affect the persistent synthesis of abortive product by
the wild-type enzyme (data not shown).
As shown in Fig. 6a, in the
absence of heparin, S506F enzyme showed the persistent initiation of
abortive transcripts. The ratios of abortive to full-length products
increased with time for mutant enzyme (Fig. 6b) as for the
wild-type (Fig. 5b and 6d). Although the S506F
enzyme produced much smaller amounts of 5-mer RNA than the wild-type,
the accumulation of 9-mer was more comparable with that for wild-type
enzyme. Therefore S506F enzyme does form moribund complexes at the
PR promoter, but the complexes are readily
trapped by heparin after their rapid dissociation. The moribund
complexes of the mutant are equilibrated with the free DNA and
protein more rapidly than their wild-type counterparts, strongly
suggesting the existence of binary complexes in moribund conformations.

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Fig. 6.
Pulse-labeling experiments in the absence of
heparin or in the presence of additional salt. a,
PRAL32 template (105 nM) was
transcribed S506F enzyme (35 nM) in the absence of heparin.
See details in the text. b, the ratios of the amounts of
5-mer ( ) and 9-mer( ) to that of 32-mer product shown in
panel a are plotted against the time of addition of
[ -32P]GTP. c, a similar assay using 12 nM DNA and 35 nM wild-type (Wt)
enzyme in the presence of 40 µg/ml heparin. 100 mM NaCl
was present during the labeling period. d, the ratios of the
amounts of 5-mer ( , 5+) and 9-mer ( , 9+) to
that of the full-length (32-mer) product shown in panel c
are plotted. The broken lines are the same ratios of 5-mer
( , 5-) and 9-mer ( , 9-) without added NaCl
derived from Fig. 5b as controls.
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If the interpretation of the pulse-labeling results mentioned above is
correct, any conditions preferentially destabilizing wild-type moribund
complexes should mimic the effect of the S506F mutation when combined
with heparin. We performed the same assay with the wild-type enzyme in
the presence (only during the pulse period) of 100 mM extra
NaCl, so that the resulting total salt concentration was 100 mM KCl and 100 mM NaCl. We chose this condition because it maintained the level of full-length transcription and the
length distribution of abortive products from this promoter, which were
altered at 200 mM KCl or by higher concentrations of NaCl
(data not shown). As shown in Fig. 6c, the persistent
production of abortive products was significantly reduced in this
condition, and the ratios of abortive to productive transcripts
remained constant after 3 min (Fig. 6d). Therefore moribund
complexes are salt-sensitive, indicating that ionic interactions play
an important role in their stabilization.
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DISCUSSION |
Contribution of Region 3 of
70 to Transcription
Initiation--
To understand the reasons for the reduced output of
abortive transcripts by the
70 mutants P504L and S506F
(17), we examined the nature of the DNA-holoenzyme binary complexes,
the abortive transcription process, and the accumulation of moribund
complexes at the
PRAL promoter. In the open
complex at the natural
PR promoter, about 14 bp of duplex DNA were melted in the
10 region (22). Recently, it has
been shown that a
70 fragment comprising amino acids
374-448, corresponding to a nearly complete region 2, can bind to the
single-stranded form of the
10 region, specifically the nontemplate
strand, with high affinity, and thereby stabilizes the melted duplex in
this region (25). Here we present evidence that mutations in region 3 strongly affect the process of promoter melting or the stability of the
open complex (Fig. 2) without changing the overall equilibrium constant
of the binary complex formation. The predominant fraction of binary complexes of the mutants consists of a closed complex(es), which rapidly equilibrates with the free promoter and free holoenzyme.
Open and closed binary complexes are, each, populations containing
several different species (26, 27). The rapid equilibrium of moribund
complex with free mutant enzyme further suggests the existence of
binary complexes with moribund conformation. Such binary complexes
should be a component of the abortive cycle of moribund complexes,
because the cycle involves interconversion between ternary and binary
complexes. An accurate assignment of species to the two phases observed
in the decay curve of the mutant (Fig. 1b) is impossible. We
conclude only that the mutation increases the rate of equilibration of
open complex and moribund complex with their free components because
both k1 and k2 for the
mutant enzyme are larger than k1 of the
wild-type (Table I).
The lag time for productive transcription is reduced by the S506F
mutation (Fig. 4c). A similar acceleration by this mutation has also been observed at the galP2
promoter.2 Therefore, a
mutation in region 3 can accelerate the step(s) in the reaction pathway
leading to productive transcription yet can simultaneously reduce the
fraction of the open form in binary complexes and the overall yield of
full-length product.
Mechanism of Reduction in Abortive Synthesis from the
PRAL Promoter--
At the
PRAL promoter, abortive transcripts are
continuously produced for more than 20 min, whereas synthesis of
full-length 32-mer ceases in 5 min (Ref. 18; also in Fig.
5b). Due to this long duration of abortive cycling compared
with productive RNA synthesis, it is reasonable to suggest that a
significant fraction of the abortive products generated from this
promoter are made by moribund or nonproductive binary complexes. Here
we report that mutations in the
subunit or the addition of high
salt not only reduce abortive transcription but also increase the rate of equilibration of moribund complexes with free enzyme. A rapid exchange of moribund complexes with free enzyme facilitates their conversion into other complexes, including open complexes, the inactivated ternary complexes (19), and a complex with heparin (when it
is added). These processes reduce the accumulation of moribund
complexes and therefore reduce the output of abortive products. This
appears to be the key reason for the observed decrease in abortive
transcription by the
mutants at this promoter. The existence of
several alternative fates of mutant moribund complexes can also explain
the absence of any concomitant increase in output of full-length
products and the effects of heparin on full-length and abortive
products. Recently, using DNA footprinting, it has been shown that
moribund complexes are promoter-bound species (19). Therefore, it is
understandable that alteration of the RNA polymerase-promoter
interaction by the mutations in the
subunit (Figs. 1 and 2) should
also influence the nature of the moribund complexes.
As already mentioned, the reduction of abortive synthesis by a
decreased accumulation of moribund complexes did not cause a
concomitant increase in productive transcription. Similarly the
presence of high salt, which selectively destabilized the moribund
complexes, did not affect productive transcription (Fig. 6c). This lack of correlation suggests that the abortive
cycling associated with moribund complexes is not part of the
sequential multistep initiation process leading to productive
transcription. On the contrary, some moribund complexes convert into
catalytically inactive complexes at this promoter (19). Although
moribund complexes are very likely to be the major source of abortive
products at this promoter, there is no evidence to prove or disprove
the possibility that some abortive products arise from the productive pathway.
Since very few, if any, moribund complexes appear to accumulate at the
T7A1 promoter, it seems that their formation depends on the promoter
sequence. Evidently some promoters, such as
PR, have a much greater tendency to form
moribund complexes. It will be of great interest to evaluate how common
is the formation of such complexes at promoters in general.
Furthermore, structural characterization of moribund complexes will be
essential for a full understanding of transcription initiation.
We are grateful to Dr. Richard
Hayward and Dr. Jun-Ichi Tomizawa for critically reading the manuscript
and for stimulating discussions. We would like to acknowledge Dr. Akira
Ishihama for discussions and his CREST project for supporting
R.S.