(Received for publication, September 27, 1995; and in revised form, February 21, 1996)
From the
During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP. This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2. RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP. Thus, a direct linkage between promoter clearance and productive transcription is demonstrated. The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied. Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2. Because RpoB3449 demonstrates ``wild-type'' stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context. Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation. A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.
One of the potential regulatory steps in prokaryotic
transcription is promoter clearance, a transition step in transcription
initiation at which an RNA polymerase (RNAP) ()switches from
an initial transcribing stage to an elongation stage (for reviews see (1) and (2) ). During promoter clearance, RNAP usually
also makes two forms of nonproductive initiation products in a
promoter-dependent manner: (i) abortive
synthesis(3, 4, 5) and (ii) stuttering
synthesis(6) , although productive stuttering synthesis at some
promoters also has been
reported(7, 8, 9, 10) . It is
conceivable that productive initiation could be modulated by turning
the switch between these alternate pathways (nonproductive versus productive synthesis) during the promoter clearance step at these
promoters.
Nonproductive stuttering synthesis was reported only in a mutant promoter in the past few years(6) , whereas abortive synthesis is common at most promoters. Recently, evidence of biological significance for these nonproductive syntheses in gene expression is emerging. Nonproductive stuttering syntheses have been shown at the Escherichia coli native pyrBI and galP2 promoters(11, 12) , and the expression of these two promoters is sensitive to changes in UTP concentration(12, 13) . At these two promoters, RNAP makes an abundant amount of nonproductive stuttered products and a lesser amount of productive initiation products in vitro when the concentration of UTP is high and vice versa. An E. coli mutant RNAP that overproduces abortive initiation products and reduces productive initiation at the pyrBI promoter in vitro has reduced expression from the promoter in vivo(14) . CRP protein was shown to inhibit abortive initiation leading to an enhanced productive initiation at the malT promoter(15) . lac repressor was suggested to modify the initial transcribing complex so that promoter clearance was inhibited(16) . In addition, DNA sequences flanking the ``classical'' promoter region have been implicated to contribute to promoter function(17, 18) , suggesting that initially transcribed sequences of promoter may affect nonproductive syntheses and promoter clearance.
Our knowledge of the
parameters that affect RNAP on abortive and stuttering synthesis as
well as of the mechanisms underlying the switch between nonproductive
and productive initiations during promoter clearance is very limited.
One approach to study these questions is to analyze mutant RNAPs that
have altered function in the switching between the two alternate
pathways during promoter clearance and to determine how these mutant
RNAPs are perturbed in the process. For this purpose, the mutant RNAPs
that confer rifampicin resistance (Rif) were chosen first,
because the antibiotic rifampicin inhibits transcription by preventing
an initial transcribing complex from entering an elongation
mode(3, 19) . Presumably, rifampicin plugs the pathway
leading a nascent RNA out of the active center(20) ; thus the
rifampicin-binding sites of RNAP could potentially interact with a
nascent RNA. It is reasoned that some of the Rif
mutant
RNAPs, which have altered binding of rifampicin, are likely to be
altered in the interaction with nascent RNA leading to altered
nonproductive synthesis during promoter clearance. Most of the
Rif
mutations are clustered in the middle of the rpoB gene encoding the
subunit of RNAP(21, 22) ,
and their effects on transcription elongation,
termination/antitermination, and other functions have been
characterized(23, 24, 25, 26) .
Indeed, using this approach it was shown recently that some Rif
RNAPs that overproduced abortive initiation products have reduced
productive initiation and that abortive synthesis is sensitive to the K
of an initially transcribing complex
for the nucleotide UTP during transcription initiation(14) .
Some lethal mutant RNAPs that block the initiation-to-elongation
transition were described, but the mechanisms underlying the blockage
have not been elucidated clearly(27, 28) .
In this
study, the effects of previously described Rif mutant RNAPs
on the nonproductive stuttering synthesis at galP2 are studied
to uncover other parameters (in addition to be sensitive to changes in
UTP concentration) that might affect promoter clearance at this
promoter. It is found that a Rif
mutant RNAP, RpoB3449, has
diminished stuttering synthesis and enhanced productive initiation at
the galP2 promoter; thus it has altered the switch between the
two alternate pathways during promoter clearance. The effect of
RpoB3449 on the rate of productive initiation at galP2 is
analyzed, and the results indicate that promoter clearance is a
rate-limiting step for wild-type RNAP in the productive initiation of galP2. The mechanism by which RpoB3449 has altered promoter
clearance at galP2 is studied, and the results indicate that
the efficiency of incorporating CTP at the fifth position of galP2 transcript is critical in determining RNAP whether to engage in
nonproductive stuttering synthesis or in productive initiation.
Figure 1:
Transcription initiation from the gal and pyrBI promoters by wild-type and RpoB3449
RNAPs in vitro. RNA was labeled with
[-
P]UTP in initiation assays containing
either high (H) 0.2 mM or low (L) 0.02
mM UTP as described under ``Experimental
Procedures.'' The transcription products were separated by
electrophoresis on a 24% polyacrylamide, 7 M urea gel. The
stuttered products from the galP2 promoter (of which AU4 is
the shortest with the others increasing in length by an increment of
one UMP) (A) and from the pyrBI promoter (of which
AAU4 is the shortest with the others increasing in length by an
increment of one UMP) (B) are indicated. The other less
abundant smaller sized transcripts are aborted products either from the gal promoters as described previously (39) or from the pyrBI(11) and other promoters presented in pBHM332. C, the amounts of the stuttered products (the sum of AU4 to
AU30) produced from galP2 by the mutant RNAP (hatched
columns) and wild-type enzyme (solid columns) at
different UTP concentrations. D, the amounts of the stuttered
products (the sum of AAU4 and AAU30) produced from pyrBI by
the mutant RNAP (hatched columns) and wild-type enzyme (solid columns) at different UTP
concentrations.
Figure 2: Effects of different compositions of nucleotides on stuttering synthesis at galP2. A, transcription initiation at gal by wild-type and RpoB3449 RNAPs in vitro. The experiment was performed as described in Fig. 1, except that the compositions of NTPs were varied as indicated. The concentrations of UTP in odd numbered lanes were high (H) 0.2 mM and in even numbered lanes were low (L) 0.02 mM. The transcription products were separated by electrophoresis on a 24% polyacrylamide, 7 M urea gel as in Fig. 1. B, the amounts of the stuttered products (the sum of AU4 to AU30) produced from galP2 by the mutant RNAP (hatched columns) and wild-type enzyme (solid columns) at different conditions. Conditions for columns 1-8 are the same as that for lanes 1-8 (wild-type RNAP) or that for lanes 9-16 (RpoB3449) in A.
The effects of GTP and CTP on the stuttering synthesis by both RNAPs were studied (Fig. 2). For RpoB3449, omission of GTP from a complete transcription reaction leads to a large reduction in stuttering synthesis compared with that of wild-type RNAP (compare lanes 15 and 16 with lanes 7 and 8 in Fig. 2A); RpoB3449 behaves as if all four nucleotides were present (compare lanes 9 and 10 with lanes 1 and 2; see also Fig. 2B). However, when CTP was omitted from a complete transcription reaction, both the mutant and wild-type RNAPs made comparable amounts of stuttering synthesis products (compare lanes 13 and 14 with lanes 5 and 6 in Fig. 2A; see also Fig. 2B). These results indicate that the presence of CTP is responsible for the reduced stuttering synthesis by RpoB3449 at the galP2 promoter.
Because CTP is the next nucleotide to be incorporated after the three continuous uridine residues of the nascent RNA oligomer pppAUUU, it is hypothesized that RpoB3449 has increased its efficiency in incorporating CTP at the fifth position of the transcript. Therefore, this mutant RNAP accelerates the rate of polymerization of pppAUUUC, resulting in reduced stuttering synthesis and enhanced promoter clearance at galP2. If the proposed model is correct, one would predict that stuttering synthesis at galP2 by both wild-type and the mutant RNAPs will be sensitive to the concentration of CTP. Furthermore, at high enough concentrations of CTP, wild-type RNAP will have reduced stuttering synthesis, simulating the phenotype of RpoB3449. Conversely, when the concentration of CTP is low enough to reduce the efficiency of the mutant RNAP in incorporating the nucleotide, RpoB3449 should be able to make significant amounts of stuttered products.
To determine whether
the above predictions are true, transcription assays were performed at
a variety of CTP concentrations while keeping the other NTP
concentrations constant, and transcription products were analyzed. The
results are in complete agreement with the above hypothesis (Fig. 3). Overall, for both RNAPs there is an inverse
relationship between the amount of the stuttered products being made
and the concentration of CTP used in the assay (Fig. 3B). Furthermore, wild-type RNAP made few
stuttered products when the concentration of CTP was high (0.2
mM, lanes 1 and 2 in Fig. 3A) but an abundant amount of the stuttered
products when the concentrations of CTP were low (
0.02 mM, lanes 4-6). When the concentration of CTP was very low
(0.001 mM, lane 6), wild-type RNAP made almost the
maximum amount of the stuttered products, comparable with that made in
the absence of CTP (lane 7). This result indicates that at 1
µM CTP most of the initially transcribing complexes are
trapped in the nonproductive synthesis mode. On the other hand,
RpoB3449 made only very few of the stuttered products when the
concentrations of CTP were >0.005 mM (lanes
8-12) but a significant amount of the stuttered products
when the concentration of CTP was very low at 0.001 mM (lane 13). In the absence of CTP in the reaction, the
mutant RNAP made the maximum amount of the stuttered products (lane
14). The effect is CTP-specific, because the stuttering synthesis
at galP2 by wild-type RNAP was not sensitive to the
concentration of GTP (data not shown). These results indicate that the
rate of incorporation of CTP at the fifth position of the galP2 transcript is important in determining the efficiency of
stuttering synthesis and promoter clearance.
Figure 3: Effect of CTP concentration on stuttering synthesis at galP2. A, transcription initiation at gal by wild-type and RpoB3449 RNAPs in vitro as a function of CTP concentration. The experiment was performed as described in Fig. 1except that the concentration of UTP was kept at 0.2 mM in favor of stuttering synthesis, and CTP was varied as indicated: 1 (lanes 1 and 8); 0.2 (lanes 2 and 9); 0.08 (lanes 3 and 10); 0.02 (lanes 4 and 11); 0.005 (lanes 5 and 12); 0.001 (lanes 6 and 13); and 0 mM (lanes 7 and 14). The transcription products were separated by electrophoresis on a 24% polyacrylamide, 7 M urea gel as in Fig. 1. B, the amounts of the stuttered products (the sum of AU4 to AU30) produced from galP2 by the mutant RNAP (hatched columns) and wild-type enzyme (solid columns) at different CTP concentrations.
Figure 4: Effect of GTP concentration on stuttering synthesis at the new DNA template containing a C to G change at the fifth position of the galP2 sequence. The experiment was performed as described in the legend to Fig. 3except that the concentration of GTP was varied as indicated: 1 (lanes 1 and 6); 0.2 (lanes 2 and 7); 0.02 (lanes 3 and 8); 0.002 (lanes 4 and 9); and 0 mM (lanes 5 and 10). The transcription products were separated by electrophoresis on a 24% polyacrylamide, 7 M urea gel as in Fig. 1.
Because of its reduced stuttering synthesis, the mutant RNAP is likely to have an increased rate of promoter clearance leading to an increased productive initiation compared with wild-type RNAP. To test this hypothesis, the accumulation of both stuttered and the full-length galP2 transcripts (as the productive initiation products) by the two RNAPs was determined as a function of time in a single round transcription assay (i.e., in the presence of heparin) (Fig. 5A). In these assays only transcription from galP2 was measured because GalR repressor protein, which only inhibits transcription from galP1 in vitro(29) and has no effect on stuttering synthesis at galP2(12) , was included in the reactions. For wild-type RNAP, stuttered products appeared as early as 20 s, indicating that the time needed for RNAP to make oligomers of stuttered products is very short. Full-length transcript P2 appeared at 40 s, the time needed for both promoter clearance and the completion of the transcript elongation, and its amount was increased gradually as time increased. The accumulation of the full-length transcript was parallel with that of the stuttered products as a function of time, indicating that at an any given time only a fraction of the initially transcribing complexes has completed the promoter clearance at galP2 to enter transcription elongation and to finish a productive initiation cycle. Note that more full-length products were made at 15 min than at 6 min, indicating that even after 6 min there were still some wild-type RNAP molecules remaining at the promoter (Fig. 5C). Therefore, promoter clearance at galP2 is a rate-limiting step for productive initiation due to stuttering synthesis for wild-type RNAP.
Figure 5:
Kinetics studies of the production of galP2 products by wild-type and RpoB3449 RNAPs. The experiment
was performed as described in Fig. 1, UTP was kept at 0.2 mM in favor of stuttering synthesis, and the reaction was stopped at
indicated time. GalR protein was added during preincubation period to
prevent transcription from galP1; therefore only transcription
from galP2 is studied. The transcripts from the same
experiment were separated either by electrophoresis on an 8%
polyacrylamide, 7 M urea gel to analyze the galP2 full-length transcript (P2, 125 nucleotides) or by
electrophoresis on a 24% polyacrylamide, 7 M urea gel to
analyze the galP2 nonproductive initiation products. A, kinetics studies of the production of galP2 products in a single round transcription assay (heparin was added
at the beginning of reaction). B, kinetics studies of the
production of galP2 products in a multiple round transcription
assay (no heparin was presented in the assay). At later time course
RpoB3449 appeared making small amount of the galP1 full-length
products (
120 nucleotides), which migrated just below the galP2 full-length transcript, probably because some GalR
repressor was dissociated transiently from the operator sites to allow
reinitiating RNAP to initiate at galP1. C, the rates of
accumulation of the full-length galP2 transcripts by wild-type
and RpoB3449 RNAPs in a single round or multiple round transcription
assay. The full-length galP2 transcripts were scanned, and the
data were plotted as a function of time. Filled circle,
wild-type RNAP in a single round transcription assay; open
circle, wild-type RNAP in a multiple round transcription assay; filled triangle, RpoB3449 in a single round transcription
assay; open triangle, RpoB3449 in a multiple round
transcription assay.
However, in contrast to wild-type RNAP, the mutant RNAP made increasing amounts of the full-length transcript rapidly and lesser amount of the stuttered products (Fig. 5A). Furthermore, the accumulation of the full-length transcript approached the plateau within 3 min, when most RNAP molecules had finished a productive initiation cycle (Fig. 5C). These results indicate that at any given time, a larger fraction of the initially transcribing RpoB3449 complexes has completed promoter clearance, and consequently it takes less time for most of the mutant RNAP molecules to finish a productive initiation cycle compared with wild-type RNAP. Clearly, RpoB3449 has a faster rate of promoter clearance due to reduced stuttering synthesis.
The effect of the rate of promoter clearance on the productive initiation was further manifested in a multiple round transcription assay (i.e., in the absence of heparin). As shown above, during stuttering synthesis wild-type RNAP does not vacate the promoter, and it remains idling at the promoter for a long duration. Thus, it follows that promoter turnover must be also a rate-limiting step in productive initiation. One expected consequence of this event is a reduced ability to productively initiate in a multiple round transcription assay. In contrast, because RpoB3449 has enhanced the rate of promoter clearance or promoter turnover, there is a better chance for free mutant RNAP molecules to bind to vacated promoters leading to a relative large difference in productive initiation in a multiple round transcription assay compared with that in a single round transcription assay. To test this predication, a multiple round transcription assay was performed. The results are in complete agreement with the above hypothesis (Fig. 5B). For wild-type RNAP, the difference in the overall patterns of the production of galP2 products, in particular for the full-length transcript, was small between the multiple round transcription (Fig. 5B) and the single round transcription (Fig. 5A). Noticeable increase in the production of the full-length galP2 products only occurred after 6 min, and there was only about 40% increase in the production of the productive initiation products in a multiple round transcription compared with that in a single round transcription after 15 min (Fig. 5C). In contrast, for RpoB3449, there was a marked difference in the amount of the full-length galP2 transcript between the multiple round transcription (Fig. 5B) and the single round transcription (Fig. 5A). Significant increases in the production of the full-length galP2 products occurred at 3 min, and as time increased the difference in the production of the full-length transcript between the two assays becomes even larger, and there was an over 3-fold increase in the production of the productive initiation products in a multiple round transcription compared with that in a single round transcription after 15 min (Fig. 5C). At all time points, the mutant RNAP made more productive initiation products than wild-type RNAP, and the effects became most prominent as the time increases in the multiple round transcription assay (Fig. 5C). Clearly, the observed dramatic differences in the productive initiation of galP2 between wild-type and the mutant RNAPs at later time points reflect the amplified intrinsic differences in the rates of promoter clearance between the two RNAPs in a multiple round transcription assay.
Stuttering synthesis has been reported both in prokaryotic and eukaryotic organisms(33) . This work presents the first analysis of a mutant RNAP that has altered nonproductive stuttering synthesis during transcription initiation. The perturbation of stuttering synthesis at galP2 by RpoB3449 has provided a unique opportunity to study the effects of nonproductive synthesis on promoter clearance and to study the mechanism (other than UTP concentration) underlying the switch between nonproductive and productive initiation during promoter clearance.
Figure 6: A checkpoint model to explain the switch between two alternate pathways. Nonproductive stuttering synthesis versus productive initiation during promoter clearance at the galP2 promoter. For more detail, see text.
Furthermore, this study has demonstrated that the fifth position of
the galP2 initially transcribing sequence, rather than a
particular nucleotide at that position, is critical in determining
promoter clearance. This result indicates that this fifth position,
most likely because it lies immediately after the run of uridine
residues that are responsible for stuttering synthesis, imposes a high
energy barrier that an initially transcribing complex has to overcome.
However, it is not likely that in general the fifth position of the
initially transcribing sequence is critical in promoter clearance at
all promoters. Rather, it is the specific context at the galP2 sequence that makes the fifth position a critical one. It is known
that the stretch rUdA sequences before the fifth position of the galP2 sequence in an initially transcribing complex are
intrinsically unstable(14, 35) . When 5`-Br-UTP was
used in the place of UTP, the stuttering synthesis at galP2 was reduced dramatically (data not shown); presumably the more
stable 5`-Br-U
dA pairs prevented the short RNA oligomers from
dissociation from an initially transcribing complex. Therefore, the
competition between the rate of incorporation of an extra UTP, leading
to the release of a stuttered product, and the rate of incorporation of
CTP, leading to a stabilized initially transcribing complex at this
critical position of the galP2 sequence, will determine the
efficiency of stuttering synthesis and promoter clearance.
Why does
RNAP tend to make nonproductive initiation products during the
transition step between transcription initiation and elongation? It has
been speculated that in an initially transcribing complex, RNAP
establishes the RNA binding sites, which are postulated to be important
in maintaining the integrity of an elongation complex(36) . It
is conceivable that conversion from an initiation to an elongation
complex, accompanied by factor release is a stress-inducing
process. Such a stress state would likely sensitize RNAP to respond to
the availability of nucleotides for incorporation at putative critical
position(s) in the initially transcribed sequences at some promoters.
Because the mature RNA binding sites have not been established yet in
an initially transcribing complex, the association of oligomer to the
complex is likely to be weak and dissociation-prone, in particular, if
the initially transcribed sequences have a run of uridine residues. In
this regard, it is interesting to note that the termination efficiency
at intrinsic terminators also depends on the rate of RNAP incorporation
of a critical nucleotide that lies immediately after a run of uridine
residues at the end of terminator sites(37) . This similarity
between promoter clearance and termination suggests that there is a
common mechanism underlying the two processes. It would be interesting
to determine whether it is the particular nucleotide or the particular
position at those terminators that is important in determining
termination efficiency.
We are just beginning in understanding the
promoter clearance step in transcription initiation. This and other
work (14) indicate the importance of K of
an initially transcribing complex for critical nucleotide in promoter
clearance at some promoters. Other experiments show that at some other
promoters, promoter clearance is not sensitive to the changes in
nucleotide concentration but is stimulated by the transcript cleavage
factors GreA and GreB(38) . It is likely that the limiting
step(s) in promoter clearance at different promoters is different,
indicating that control of promoter clearance is a complex process and
appears to be promoter-specific (or context-dependent).