(Received for publication, May 8, 1995; and in revised form, July 13, 1995)
From the
Transcription initiation at the 54-dependent glnAp2 promoter was studied to follow the state of polymerase
as RNA synthesis begins.
54 polymerase begins transcription in
abortive cycling mode, i.e. after the first bond is made,
approximately 75% of the time the short RNA is aborted and synthesis
must be restarted. Polymerase is capable of abortive initiation until
it reaches a position beyond +3 and before +7, at which stage
polymerase is released from its promoter contacts and an elongation
complex is formed. Initial elongation is accompanied by two
transcription bubbles, one moving with the polymerase and the other
remaining at the transcription start site. The
54-associated
polymerase shows an earlier and more efficient transition out of
abortive initiation mode than prior studies of
70-associated
polymerase.
54 is an alternative bacterial transcription factor that
directs transcription of specific subsets of genes (reviewed by
Magasanik(1989), Kustu et al.(1989), and Merrick(1993)).
54 is the only sigma factor that is not a member of the
70
family of proteins, as defined by sequence similarity (Merrick and
Gibbins, 1985; Lonetto et al., 1992). Regulation of
54-dependent promoters differs from regulation of
70-dependent promoters (Gralla, 1991; Collado-Vides et
al., 1991) despite the fact that both sigma factors bind the same
core RNA polymerase. All known
54-dependent promoters are
controlled by activator proteins rather than by repressors
(Collado-Vides et al., 1991). The activators are generally
enhancer-binding proteins (Reitzer and Magasanik, 1986), which work
from locations that are too remote to activate transcription in
70-dependent systems.
Certain steps in the transcription cycle
at 54 promoters differ from analogous steps at
70
promoters.
54 can bind certain promoters without being part of
holoenzyme (Buck and Cannon, 1992), whereas
70 cannot (Dombroski et al., 1992). The two holoenzymes recognize different
sequence elements (see review by Merrick, 1993). In addition the
54 promoter elements are located in different positions near
-12 and -24 (Morett and Buck, 1989). ATP hydrolysis is
required to form open complexes at the
54 promoters (Popham et al., 1989; Weiss et al., 1991) but not at
70
promoters.
Recently we identified another difference; 54 can
remain bound to a promoter after RNA polymerase begins transcription
elongation (Tintut etal., 1995). This is in contrast
to the well established
70 transcription cycle, in which
70
is released when an elongation complex is formed. The observation has
led us to attempt to investigate the transition from open promoter
complex to elongation complex for
54 holoenzyme. The process is
well known for
70 promoters (Hansen and McClure 1980; Carpousis
and Gralla, 1985; Krummel and Chamberlin, 1989). At those promoters RNA
synthesis begins with an abortive cycling phase, in which
promoter-bound holoenzyme synthesizes short RNAs (Carpousis and Gralla,
1980). Subsequently, a longer RNA of length 10 or 11 is made, and at
this point polymerase is released from the promoter (Carpousis and
Gralla, 1985; Krummel and Chamberlin, 1989).
70 is released both
from the polymerase and from the DNA, and the complex becomes fully
committed to elongation.
In this paper we investigate the transition
of 54 holoenzyme from open complex to elongation complex at the Escherichia coli glnAp2 promoter. Popham et al(1989)
showed that open complexes formed at an analogous promoter are capable
of synthesizing short, probably abortive, RNAs. We confirm this finding
in a different context and go on to characterize the transition from
abortive to productive transcription. The results indicate that several
important aspects of this transition differ from those observed in
prior studies of
70 systems.
For DNase I digestion, 2 µl of
DNase I (0.45 µg/ml, including 45 mM MgCl and
22.5 mM CaCl
) was added for 30 s at 37 °C,
followed by an addition of 2 µl of 0.5 M EDTA to stop the
reaction. For KMnO
footprinting, 4 µl of 92.5
mM KMnO
was added for 1 min at 37 °C, and the
reactions were quenched by adding 6 µl of
-mercaptoethanol.
The modified DNA was isolated by extracting with phenol and passed
through a 1-ml syringe packed with G50-80 (Sigma) equilibrated with
water. The samples were subjected to primer extensions using P-labeled primers (Sasse-Dwight and Gralla, 1991). The DNA
products of the Taq reaction was isolated on 6% denatured
polyacrylamide gel.
The initial goal was to monitor the progress of 54
holoenzyme as it transcribed from the glnAp2 promoter. In
order to do that, the polymerase was stalled at different positions
along the template as it attempted to move downstream during
transcription initiation. The stalling was accomplished by omitting a
subset of the required nucleotides. The expected positions of stalling
are shown in Table 1along with the nucleotide combinations used.
Three assays were used to characterize these stalled complexes: DNase I
footprinting to locate the polymerase, abortive initiation to assay
abortive transcripts, and permanganate probing to locate melted
regions.
Plasmid pLR1, which contains the 54-dependent
promoter glnAp2 with upstream NtrC enhancer sites, was used
(Reitzer and Magasanik, 1986). Primer extension footprinting methods,
which allow probing of both DNA strands of the same sample, were used
(see Gralla(1985)). This method also allows the use of supercoiled DNA,
which may be an important parameter in this system.
The footprint pattern of the top strand of the glnAp2 promoter is shown in Fig. 1A. Lane
1 shows the protection pattern of a closed complex formed from
54 and core polymerase. The closed complex footprint covers from
-34 to -2 (compare lane1versus the lane2 control pattern). This encompasses
both the -12 and -24 promoter recognition elements but not
the transcribed region, in agreement with prior experiments (Popham et al., 1989). When phosphorylated NtrC and ATP were also
present, the open complex formed extends into the transcribed region to
position +23 (lane3), covering at total of
approximately 57 bases.
Figure 1: DNase I footprints of the glnAp2 promoter. A, top strand; B, bottom strand. Lane1, closed complex (cc); lane2, control; lane3, open complex (oc); lane4, polymerase stalled at +2 position; lane5, +7 position; lane6, +18 position; lane7, >+18 position. The arrow indicates the +1 start site and the direction of transcription. Dideoxy sequencing reactions are shown on the left.
Next, the holoenzyme was allowed to
transcribe to different positions by adding different combinations of
nucleotides (see Table 1). In order to prevent binding by new
polymerases from solution, free proteins were titrated with a
competitor promoter DNA. The competitor contains the 54-dependent
tightly binding R. meliloti nifH promoter (Buck and Cannon,
1992) carried on a 160-base pair fragment. A
100-fold excess of
this competitor was added after open complexes were formed with glnAp2 DNA, but before the addition of nucleotides, which
caused the polymerase to begin RNA synthesis. The lack of protection in
control lane2, where competitor was mixed with glnAp2 before addition of proteins, confirms that a sufficient
amount of the competitor is present to titrate excess proteins.
In the first experiment dinucleotide primer UpA and CTP were added to the open complex to allow the polymerase to form a single bond, creating the product UpApC, where ``A'' is the initiating nucleotide (see ``Transcription'' below) (Popham et al., 1989). This corresponds to incorporating the nucleotide at position +2 in the natural mRNA. Under these conditions the result shows no change in the DNase protection pattern compared to the open complex (lane4versuslane3). Not surprisingly, the polymerase can make the first bond of the mRNA without moving along the DNA.
By contrast, the polymerase did change its interaction with DNA when GTP was added to allow transcription as far as the +7 position (see Table 1). This is demonstrated by the shortened protection seen in lane5. The polymerase now fully protects the DNA only from -2 to +23 with additional partial protection as far as -11 upstream and perhaps to +27 downstream. The overall protection pattern corresponds to less than a 40-base pair region, compared to open and first bond-making complexes, both of which protect approximately 57 base pairs. The comparison indicates that when the polymerase transcribes to the +7 position, it is released from its upstream promoter contacts. The important promoter element near -24 is no longer protected, and the protection near the -12 element is quite weak.
Next, the polymerase is allowed to transcribe to position +18 (see Table 1). The primary protection (lane6) now extends from +3 to +23 and is not as complete as in other cases. In addition there is partial protection from +23 to +39. Thus, the stalled polymerase covers a total of 36 bases, approximately the same extent as seen at the +7 position, but the protection is weaker. Finally, when all four nucleotides are present no protection is seen (lane7), as expected for a polymerase that has moved to downstream positions.
As also observed on the top strand, a drastic change in the footprint occurs when polymerase is allowed to move to position +7 (lane5). The protection is strong from -13 to +22. The result is similar to that from the top strand in that polymerase no longer protects the critical -24 promoter element. A transition indicative of loss of promoter interaction has occurred on both strands. The footprints shorten a little more when transcription is halted at +18 (lane6), at which positions from -3 to +31 are partially protected.
In addition, lanes
5, 6, and 7, corresponding to stalling at
+7, at +18, and steady-state transcription, show partial
protection of the region upstream from the start site (lesser intensity
in these lanes compared to the control lane2). This
is a reflection of the weak DNase protection due to not being
released from this region, as shown by Tintut et al.(1995).
A pictorial summary of the DNase I footprinting results is presented in Fig. 7. The main point at this stage is that a transition releasing polymerase from the promoter elements has occurred before polymerase reaches position +7.
Figure 7: Summary of data. Solidbar, strong protection; stripedbar, weak protection; openoval, melted region.
The production of UpApC was
used as a measure of abortive initiation. This product is made using
dinucleotide primer UpA and P-labeled CTP as the only
source of nucleotides (Fig. 2). When all required abortive
transcription components were present, an abortive initiation product
was seen (lane7 with ATP). The specificity of this
reaction was confirmed by showing its dependence on various required
components. No signal is seen with the omission of: NtrC (lane1), carbamyl phosphate to phosphorylate NtrC (lane2),
54 (lane3), core polymerase (lane4), ATP (lane5), and UpA (lane6). Parallel experiments (data not
shown) to produce long transcripts under these same conditions
confirmed the specificity of glnA transcription.
Figure 2:
Abortive initiation. The component that is
left out from the transcriptions is as follows: lane1, NtrC; lane2, CBP; lane3, 54; lane4, core polymerase; lane5, ATP; lane6, UpA. In lane7 all the required transcription components are
present.
Next we characterized the amount of abortive initiation that occurs when polymerase is permitted to move to various positions (see Table 1) as described in the DNase footprinting experiments. The goal is to learn at what position the polymerase ceases to make large amounts of abortive initiation products. Open complexes were formed, and a subset of nucleotides containing radioactive CTP (see Table 1) were added. The reactions were allowed to proceed for 10 min to achieve the steady state conditions. Reaction products were loaded directly onto an acrylamide gel, separated by electrophoresis, and detected by autoradiography.
In the presence of dinucleotide UpA and labeled CTP, the polymerase produced a large amount of abortive product UpApC (see band indicated by the arrow in Fig. 3A, lane 1). When the polymerase was allowed to move as far as position +7, a much smaller amount of abortive product was made (much lighter band in the same position of lane2, quantified below). In addition small amounts of slightly longer products were made, as expected; we have not definitively characterized the size of these longer products. The data indicate that few abortive products are made when polymerase is allowed to move as far as position +7.
Figure 3: Abortive initiation at certain stalled positions. A, lane1, +2 position; lane2, +7 position; lane3, +18 position; lane 4, >+18 position. B, lane5, +2 position; lane6, +3 position; lane7, +18 position. The arrow indicates the position of the abortive product UpApC.
When additional nucleotides were present (see Table 1) to promote further downstream movement of the polymerase, the amount of abortive product was only slightly lessened (lane3; band in same position denoted by arrow). Two major longer products were also produced under these conditions. Calibration using short RNAs (data not shown) suggests that these two products correspond to RNAs of the expected length, 19, and also of length 22; the 22-mer probably occurs from the mis-incorporating the omitted UTP, followed by stalling at the next position of UTP where two UTPs must be incorporated. This has been seen previously (Carpousis and Gralla, 1985). Finally, when all four nucleotides were present, the amount of abortive product was essentially unchanged (lane4).
This qualitative analysis indicates that a transition from a frequent abortive initiation state to a lower abortive state has occurred between position +2 and +7 (lane1versuslane2). Note that the +7 position is the same point at which DNase footprinting revealed a physical change in the state of the transcription complex.
One further combination of nucleotides was used to narrow the position at which the transition occurs. By the use of UpA, CTP, and 3-methyl-GTP the polymerase could be stalled at position +3. The result (Fig. 3B, lane 6) indicates that the polymerase stalled at +3 still produces large amounts of abortive products (quantified below). That is, the amount is comparable to that seen when transcription is stalled at +2 (lane5) but much more than that seen when transcription is stalled later (lane7). Thus the transition out of frequent abortive mode seems to occur after the polymerase reaches +3 but before it reaches +7.
For each of several samples where the polymerase stalled at positions +2, +3, +7, +18, and >+18, the amount of abortive products and productive transcripts were quantified using a PhosphorImager. From the quantitative data, the relative molar amount was determined by normalizing to the number of radiolabeled CTPs incorporated (Table 2). Note that the longer products incorporate more than one radioactive cytidine, and thus their molar amount is over-represented on the autoradiographs. The molar excess of abortive RNA UpApC (compared to productive RNA, 18 + 19 nucleotides long, produced in a parallel experiment) was determined. The data (Table 2) showed that during the 10-min abortive initiation period, a 50-fold excess of abortive product UpApC was made from polymerase stalled at the +2 and +3 positions.
The relative amount of abortive products associated with stalling at each position is plotted in Fig. 4to display the transition from frequent abortive phase to more stable elongation complex. When transcription is artificially stalled at position +2, the amount of abortive product UpApC made is approximately 15-fold higher than the amount of abortive product made at position +7. This huge overproduction of UpApC is maintained when polymerase is artificially stalled at position +3. Apparently, a transition out of abortive mode has occurred at position +7, as the polymerase aborts transcription rarely after reaching this position. This much lower production of UpApC is maintained under conditions in which polymerase can reach position +18 and, indeed, under conditions of free transcription. The quantitative analysis confirms that a transition away from abortive mode has occurred between positions +3 and +7.
Figure 4: The total of all abortive RNAs produced (see Table 2) is plotted against the position at which transcription is stalled.
A key parameter in the synthesis of productive transcripts is the probability of aborting synthesis at a particular position (Carpousis, 1983; Carpousis and Gralla, 1985). The probability can be defined as the amount of RNA shorter than a certain length divided by the sum of all RNA products made (abortive plus longer). For example, the data showed that UpApC constitutes approximately 75% of all RNAs made under various conditions (Table 2). That is, in several experiments where the polymerase was stalled at position +7 or +18, the probability of aborting synthesis at +2 was calculated to be 0.75. Thus, approximately three out of four times, polymerase aborts synthesis after forming UpApC and must restart; the remaining time, it goes forward to make productive transcript. This predicts that only a small (roughly 2-fold) excess of abortive product will be seen during productive transcription, as is observed. Note that huge amounts of abortive products are only produced when polymerase is artificially stalled at a position that is not far enough to be associated with the transition to elongation mode. Significant amounts of abortive transcripts have been seen previously under productive transcription conditions, but this is probably a consequence of the use of low concentrations of CTP, GTP, and UTP (Popham et al., 1989).
In order to confirm that this abortive phase is not restricted to dinucleotide-primed synthesis, we repeated selected experiments using the normal initiating nucleotide ATP. Abortive transcripts could not be detected directly because the presence of the triphosphate end caused them to migrate within a region dominated by radioactive products from the unincorporated labeled CTP. Thus after transcription the samples were subjected to calf intestine alkaline phosphatase to cleave off the 5`-phosphate groups (Jacob et al., 1994) before loading onto gels. Under these conditions abortive product can be seen. Lane1 of Fig. 5shows that a large amount of abortive product is made when ATP and labeled CTP are used to form pppApC. When the same experiment is done in the presence of additional GTP and UTP to allow elongation, the result shown in lane2 is obtained. The much reduced amount of abortive product under these conditions is similar to the result obtained with dinucleotide-primed synthesis. This confirms that abortive initiation can occur using the natural initiating nucleotide ATP.
Figure 5: Abortive initiation using the initiating nucleotide ATP. Lane1, +2 position; lane2, >+18 position.
In order to follow the single-stranded regions in this
transition, the exposed single-stranded DNA was probed with
KMnO. KMnO
reacts selectively with
single-stranded thymines and has been used previously to detect the
melted transcription bubble at this promoter (Sasse-Dwight and Gralla,
1988; 1990). In these experiments the bottom strand of the glnAp2 promoter was probed. As a control, open complexes,
formed as described above, yield a strong permanganate signal (Fig. 6, lane 2) corresponding to bottom strand
thymines at positions -9 and +1. As expected, this
reactivity was absent in closed complexes (Fig. 6, lane
1).
Figure 6:
KMnO footprinting of the glnAp2 promoter. Lane1, closed complex (CC); lane2, open complex (OC); lane3, polymerasestalled at +2
position; lane4, +7 position; lane5, +18 position; lane6,
>+18 position. The bottom strand was probed. Arrows indicate the positions of thymines.
Next, samples were probed with permanganate using the same conditions described above to move the polymerase to various positions along the template. No change in pattern was observed when complexes forming product UpApC were probed (Fig. 6, compare lane3 with the open complex signal of lane2). Recall that at this stage the polymerase has not moved (as assayed using DNase footprinting; Fig. 1) and is in abortive mode (as assayed by abortive initiation; Fig. 3A).
By contrast, when the polymerase is stalled at position +7, new permanganate hypersites extended to positions +7 and +9 (Fig. 6, lane4). Recall that under these conditions the polymerase has been largely released from abortive mode (see above). When polymerase is stalled further downstream in the +18 to +21 region, additional permanganate-sensitive sites are seen corresponding to thymines within this region (lane5). As expected, no hypersites are seen in the transcribed region when polymerase transcribes in the presence of all nucleotides (lane6). The results provide evidence that a melted bubble moves downstream with the elongating polymerase. The differences in intensities of permanganate-sensitive bands is probably due to different environments surrounding thymines at different positions.
Note that under all conditions the transcription start site remains
open (Fig. 6, bracket indicating bands in lanes
2-6). This is in agreement with prior experiments, not
involving abortive initiation, analogous to those in lanes 2,5, and 6
but probing the top strand with permanganate (Tintut et al.,
1995). The result supports the view that, as polymerase is released
from the promoter, the original open complex bubble splits into two
bubbles; one moves with the elongating polymerase and the other remains
behind with 54.
These experiments describe the transition from abortive
initiation mode to elongation mode for 54 holoenzyme at the glnAp2 promoter (see summary in Fig. 7). The holoenzyme
makes the first mRNA bond without moving from the promoter. After
making this short RNA product, the data show that there is a 75% chance
that the synthesis will be aborted and started anew. Thus, even when
all elongation substrates are present, this abortive product
accumulates in modest excess over long RNA. However, longer abortive
products do not accumulate in significant amount under these
conditions, leading to relatively efficient productive transcription.
The polymerase is capable of abortive initiation when RNA synthesis reaches position +3 but loses this property prior to position +7 (Fig. 4). At this stage, footprinting shows that the polymerase is released from the contacts that hold it to the promoter region (see summary in Fig. 7). This leads to a reduction in the size of the protected region by approximately 20 base pairs. Permanganate probing shows that the transcription bubble has moved forward to cover the position that is transcribed. Thus the polymerase appears to have reached elongation mode prior to +7 position of the glnA gene. Probing of complexes stalled further downstream also show elongation complexes with characteristic of shortened footprints and transcription bubbles covering the point of synthesis.
This
pathway may be compared to the analogous pathway used by the common
70 form of RNA polymerase holoenzyme. The comparison reveals both
similarities and differences. The main similarity is that both pathways
require the polymerase to pass through an abortive initiation mode
prior to being committed to transcription elongation (Carpousis and
Gralla, 1985). The molecular basis for this requirement is not known,
but it was speculated previously to be related to the
primer-independent nature of mRNA initiation (Carpousis and Gralla,
1985). The lack of primers, common to RNA polymerases but not DNA
polymerases, may cause difficulty in initiating RNA synthesis. The
abortive mode can be thought of as a phase in which a primer is created
with some difficulty. For example, cycling to produce abortive RNAs may
occur because these short RNAs are associated with the ``loose
product site'' and are not translocated to the ``tight
product site'' (see Chamberlin(1992-1993)) until a stable
ternary complex is formed (Mustaev et al., 1994).
Alternatively, the need to break strong contacts between polymerase and
promoter may also retard the transition to elongation phase.
There
are several differences in this pathway compared to prior studies,
which used the 70 form of polymerase. One important difference is
that the
54-dependent glnAp2 promoter involves a
relatively efficient transition out of abortive cycling mode. The data
show that approximately 25% of the glnAp2 RNA 5` ends that are
produced will end up in productive transcripts. In the two
70
cases studied, the probabilities are much lower: 2% at lacUV5
(Carpousis and Gralla, 1980; Carpousis, 1983) and less than 8% at T7A1
promoters (Krummel and Chamberlin, 1989). (
)This difference
is largely a consequence of what happens after the first bond of the
mRNA is formed. Although the
54 polymerase is capable of abortive
initiation up to position +7, it aborts primarily after forming
the first bond. That is, the release of abortive products at glnAp2 during transcription is observed primarily from
polymerases that have formed a single bond. By contrast, in prior
studies at
70 promoters, longer RNA products are also aborted.
A second difference is that polymerase is released from the contacts
that hold it to the promoter sooner in the 54 case studied here
than in the
70 cases studied previously (Carpousis and Gralla,
1985; Krummel and Chamberlin, 1989). As discussed above, at glnAp2 this occurs after formation of the second bond but
prior to formation of the sixth bond. In the prior cases elongation
mode is not attained until the RNA is approximately twice as long, and
abortive cycling occurs at several positions. One possible cause of
these differences might be a lower affinity of core polymerase for
54 (see Lesley et al., 1991), allowing polymerase to
dissociate more readily; the two sigma factors use different
determinants to bind polymerase (Tintut et al., 1994; Tintut
and Gralla, 1995).
Another difference is that the initial
transcription bubble is split into two parts only during initiation by
54 holoenzyme. That is, as RNA synthesis begins the initial
bubble separates into two bubbles; one moves downstream with the
polymerase and the other remains transiently over the start site. We
show elsewhere that this is because
54 remains promoter-bound
during initial transcription due to its stronger affinity for DNA
(Tintut et al., 1995).
The mechanism of 54-dependent
transcription has been described as a hybrid between prokaryotic and
eukaryotic mechanisms (Gralla, 1991; North et al., 1993). If
this analogy extends to the pathway studied here, then one might expect
eukaryotic mRNA synthesis to go through an analogous, quite brief,
abortive to elongation transition (see Jacob et al.(1994), and
references therein). Indirect experiments (
)support this
possibility, but more direct experiments will be required to test this
further.
54 is also used more commonly in other bacteria, and it
will be useful to learn if there are promoter- or species-specific
differences in the mechanism of transcription initiation by
54
holoenzyme.