©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance (*)

(Received for publication, September 27, 1995; and in revised form, February 21, 1996)

Ding Jun Jin (§)

From the Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

One of the potential regulatory steps in prokaryotic transcription is promoter clearance, a transition step in transcription initiation at which an RNA polymerase (RNAP) (^1)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^r) 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^r 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^r mutations are clustered in the middle of the rpoB gene encoding the beta 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^r 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^r 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^r 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.


EXPERIMENTAL PROCEDURES

Materials

Nucleotides were from Boehringer Mannheim; salts of glutamate were from Fluka; and P-labeled nucleotides were from Amersham Corp. or ICN. Plasmids were isolated using Qiagen column according to the manufacturer's manual followed by phenol extraction, and DNA fragments were purified by electroelution after isolation from a 1.5% agarose gel followed by phenol extraction. The 348-bp EcoRI-BamHI fragment of pSA509 containing the gal promoter-regulatory region and the very strong Rho-independent transcription terminator of the rpoC gene of E. coli was used as the DNA template for in vitro transcription(29) . Plasmid pBHM332 (a kind gift from Dr. Charles Turnbough, Jr., University of Alabama at Birmingham), which was constructed by replacing the 322-bp PvuII fragment of pUC19 with the 758-bp PvuII fragment containing the pyrBI promoter region(30) , was also used as the DNA template for in vitro transcription. In pBHM332, the transcriptional direction of pyrBI is opposite that of bla. RNAPs were purified from E. coli K12 MG1655 background as described(31) . Both the wild-type and mutant RNAP, RpoB3449, were highly pure (>95%) and had comparable activities in synthesizing the RNAI transcripts of pBR322. The gal repressor (GalR protein, a kind gift of Dr. Yan Ning Zhou) was purified as described(32) .

Localized Mutagenesis, Cloning, and DNA Sequencing

The C residue at the fifth position of the galP2 coding sequence in the pSA509 plasmid was replaced with G residue as follows. First, two DNA fragments (A and B), which share 33-nucleotide sequences around the starting site (+1) of galP2 coding region, were each amplified by polymerase chain reaction using pSA509 as a DNA template. The sequences of the two primers used for amplifying DNA fragments A (300 bp) were: 5`-TTCTAGACCTTCCCGTTTCGCTCAAGTTAG (DJJ121), which covers the sequence upstream of the EcoRI site in pSA509, and 5`-TAGGCTTATGGTATCAAATAACCATAGCATAAC (DJJ122), which covers the complementary sequences to the coding sequences from +19 to -14 position and contains a G to C change at the +5 position. The sequences of the two primers used for amplifying DNA fragments B (200 bp) were: 5`-GTTATGCTATGGTTATTTGATACCATAAGCCTA (DJJ123), which covers the coding sequences from -14 to +19 position and contains a C to G change at the +5 position, and 5`-TCATAGAGTCTTGCAGACAAACTGCGCAAC (DJJ124), which covers the sequence downstream of the BamHI site in pSA509. The amplified fragments A and B were isolated from a 1.5% agarose gel and purified by electroelution followed by phenol extraction. The purified DNA fragments were then denatured and annealed at the overlapping sequences followed by Taq polymerase extension in a limited polymerase chain reaction (four cycles) to generate enough double strand DNA to be used as DNA template. Another 20 cycles of polymerase chain reaction were continued after adding two primers (DJJ121 and DJJ124) into the above reactions to amplify the fragment C (500 bp), which contains EcoRI and BamHI sites at either end of the fragment and a mutational change at the +5 position of galP2 coding sequence. The amplified DNA fragment C was digested with EcoRI and BamHI restriction enzymes followed by purification of the resulting 350-bp EcoRI-BamHI fragment as described above. This EcoRI-BamHI fragment containing the mutation was ligated with the purified large EcoRI-BamHI-digested fragment of pSA509 resulting in pDJJ51, followed by transformation. Plasmid pDJJ51 DNA was purified from the Amp^r transformants, and the C to G change at the +5 position of galP2 sequence was confirmed by automated DNA sequencing carried out on a model 373A DNA sequencer (Applied Biosystems Inc.) using the Taq polymerase dye terminator sequencing protocol (Applied Biosystems Inc.) according to the manufacturer's manual.

In Vitro Transcription Assays

In vitro transcription assays were essentially as described(12) . A complete transcription reaction mixture containing 20 mM Tris-glutamate (pH 8.0), 100 mM potassium glutamate, 10 mM magnesium glutamate, 5% glycerol, acetylated bovine serum albumin (100 µg/ml), DNA at 5 nM, and RNA polymerase at 20 nM for wild-type and 24 nM for RpoB3449, was preincubated for geq10 min at 37 °C. When indicated, GalR protein (87 nM as dimer) was also included during the preincubation period. Unless otherwise mentioned, NTP concentrations were 0.2 mM for ATP, GTP, and UTP and 0.02 mM for CTP. The reaction was started by the addition of NTPs including P-labeled nucleotide as indicated and stopped after 10 min or at the indicated times by the addition of a 0.2 volume of 0.2 M EDTA in 40% glycerol with dyes and put on ice. For single round transcription assays, heparin (final concentration, 100 µg/ml) was present in the NTP mixture. After heating for 3 min in boiling water, samples were loaded directly onto a polyacrylamide gel containing 7 M urea, and transcripts were visualized by autoradiography. The nonproductive initiation products were analyzed on a 24% gel, and the productive initiation products were analyzed on a 8% gel. The transcription products were quantified with an AMBIS Imaging System(TM) (San Diego, CA), and the data were corrected for background.


RESULTS

RpoB3449 Has Reduced Stuttering Synthesis during Transcription Initiation at galP2

RNAP makes nonproductive stuttering synthesis products at the galP2 promoter, and the stuttering synthesis is sensitive to the concentration of UTP(12) . As shown in Fig. 1A, at high UTP (0.2 mM), wild-type RNAP makes a large amount of stuttered products, whereas at low UTP (0.02 mM), the synthesis of stuttered product is reduced. To study the effects of previously described Rif^r mutant RNAPs on stuttering synthesis from the galP2 promoter, transcription initiation assays were performed. Most Rif^r mutant RNAPs behaved like wild-type RNAP in stuttering synthesis (data not shown). However, one mutant RNAP, RpoB3449, which has a deletion of an alanine at the 532 position of the beta subunit of RNAP, has greatly reduced nonproductive synthesis at galP2 compared with wild-type RNAP (Fig. 1, A and C). Apparently, the effect of the mutant RNAP on stuttering synthesis is promoter-specific, because RpoB3449 behaves like wild-type RNAP at the pyrBI promoter: both RNAPs make a large amount of stuttering synthesis products at 0.2 mM UTP (high) and a lesser amount at 0.02 mM UTP (low) (Fig. 1, B and D). These results indicate that the mutant RNAP is capable of engaging in stuttering synthesis but has an altered function at the galP2 promoter (when comparable amounts of the proteins were used).


Figure 1: Transcription initiation from the gal and pyrBI promoters by wild-type and RpoB3449 RNAPs in vitro. RNA was labeled with [alpha-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.



The Efficiency of an RNAP in Incorporating CTP at the Fifth Position of the galP2 Nascent RNA Determines the Extent of Stuttering Synthesis

To determine the mechanism by which RpoB3449 has altered stuttering synthesis at galP2, the effects of different conditions on this nonproductive initiation at galP2 were compared between wild-type and the mutant RNAPs (Fig. 2). It is known that wild-type RNAP stutters at galP2 when only the two nucleotides ATP and UTP are present (12) . Under this condition, RpoB3449 is capable of stuttering synthesis at galP2 (Fig. 2A, lanes 11 and 12), just like wild-type RNAP (Fig. 2A, lanes 3 and 4; see also Fig. 2B). Because the patterns of stuttering syntheses by the two RNAPs are similar and sensitive to the concentration of UTP, it rules out the possibility that RpoB3449 had an altered apparent K(m) for UTP leading to reduced stuttering synthesis. Because RpoB3449 has diminished stuttering synthesis in the presence of four nucleotides, whereas it is proficient in stuttering synthesis in the presence of only ATP and UTP, it suggests that the presence of either GTP or CTP affects the ability of the mutant RNAP to make stuttering synthesis products at the galP2 promoter.


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 (geq0.2 mM, lanes 1 and 2 in Fig. 3A) but an abundant amount of the stuttered products when the concentrations of CTP were low (leq0.02 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.



The Fifth Position of the galP2 Transcript, Which Lies Immediately after the Stretch of Uridine Residues, Is Critical for Stuttering Synthesis

It is possible that it is not CTP per se but rather the fifth position of the galP2 sequence (e.g., the base that follows the run of uridine residues) that is critical in determining the efficiency of stuttering synthesis by RNAP. If this was the case, one would predict that by changing CTP to another nucleotide at that position, stuttering synthesis at galP2 would become sensitive to the concentration of the new nucleotide. To determine whether in stuttering synthesis at galP2 the effect is CTP- or position-specific, the C at the fifth position was replaced with G by localized mutagenesis, and the effect of the concentration of GTP on stuttering synthesis was analyzed. The results showed that with the new DNA template, stuttering synthesis by both RNAPs became sensitive to the changes in the concentration of GTP and the amount of stuttered products made was an inverse function of the concentration of GTP (Fig. 4). These results have demonstrated that the effect is position-specific and not CTP-specific, indicating that the fifth position of the galP2 sequence is a critical point in determining stuttering synthesis and promoter clearance at galP2. Furthermore, in contrast to the stuttering synthesis with the wild-type galP2 sequence (Fig. 3), the patterns of stuttering synthesis between the wild-type and the mutant RNAPs are basically the same with the new DNA template containing a C to G change at the fifth position of the galP2 sequence (Fig. 4). The wild-type phenotype of stuttering synthesis by the mutant RNAP with the new DNA template further argues for the notion that the mutant RNAP has an enhanced efficiency in incorporating CTP at the fifth position of the wild-type galP2 sequence, leading to its reduced stuttering synthesis at the promoter. At the present, the possibility that this mutant RNAP affects the K(m) for CTP or other nucleoside triphosphates during transcription at other contexts cannot be excluded.


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.



Stuttering Synthesis Delays Promoter Clearance and Reduces the Rate of Productive Initiation of an RNAP

-Because an initially transcribing RNAP either completes promoter clearance to enter elongation mode or engages in nonproductive stuttering synthesis at galP2, one would predict that promoter clearance is a rate-limiting step for productive initiation when stuttering synthesis occurs. The alteration of RpoB3449 in stuttering synthesis at galP2 provides a unique opportunity to study the effects of the nonproductive initiation on promoter clearance and productive initiation.

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.


DISCUSSION

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.

Promoter Clearance Is a Rate-limiting Step in the Productive Initiation Due to Stuttering Synthesis

During stuttering synthesis, RNAP is able to engage in multiple cycles of initiation without dissociation from the promoter. Furthermore, for each new cycle of initiation, only a fraction of RNAP molecules is able to incorporate the CTP at the fifth position of the galP2 transcript. Only then an initially transcribing complex will be able to complete the promoter clearance step to enter the elongation mode and finish a productive initiation cycle. This is consistent with the observation that the accumulation of the nonproductive initiation products is parallel with that of the productive initiation products in the presence of heparin over a long period of time. Such a prolonged delay in promoter clearance will reduce the rate of the production of the productive initiation products accompanied by an exaggerated accumulation of nonproductive initiation products (both abortive and stuttered products). The mutant RNAP has diminished nonproductive synthesis; therefore it ``by-passes'' the rate-limiting step leading to a faster rate in productive initiation at galP2 compared with wild-type RNAP. Therefore, the observed dramatic differences between wild-type and the mutant RNAPs in the synthesis of the galP2 products (Fig. 5) demonstrates a direct link between nonproductive stuttering synthesis and productive initiation during promoter clearance. This is an analogy to another case study showing a direct link between abortive initiation and productive initiation during promoter clearance(14) . These results demonstrate that the strength of a promoter can be regulated beyond the steps of RNAP binding to a promoter and isomerization, two parameters that have been studied extensively in determine the strength of a classical promoter (34) .

A Checkpoint Model for the Switching between Nonproductive Stuttering Synthesis and Promoter Clearance

By analyzing the mechanism by which the mutant RNAP has altered stuttering synthesis at galP2, a ``checkpoint'' model has evolved for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance (Fig. 6). According to this model, the rate of incorporation of CTP at the fifth position of galP2 transcript serves as a checkpoint in determining the fate of an initially transcribing complex containing the pppAUUU oligomer. Thus, if the effective concentration of CTP is limiting relative to UTP, the probability of an initially transcribing complex to produce pppAUUUC will be low, leading to enhanced stuttering synthesis. Conversely, when the effective concentration of CTP is abundant, the probability of an initially transcribing complex to produce pppAUUUC will be high, leading to reduced stuttering synthesis and enhanced promoter clearance. In essence, the competition between the rate of incorporating an extra uridine and the rate of incorporating the cytidine to the nascent RNAP oligomer pppAUUU will determine the outcome of an initially transcribing complex into either of the alternate pathways during transcription initiation. RpoB3449 is in a sense a checkpoint mutant of RNAP that has altered the switch between alternate pathways in transcription initiation of galP2, because it has increased efficiency in incorporating CTP at the +5 position of galP2 transcript compared with wild-type RNAP. The fact that RpoB3449 demonstrates wild-type stuttering synthesis at the mutant galP2 promoter, which contains the G residue at the +5 position, indicates that the alteration of the mutant RNAP is probably in binding CTP.


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 rUbulletdA 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-UbulletdA 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(m) 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).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Molecular Biology, NCI, NIH, Bldg. 37, Rm. 2E14, 9000 Rockville Pike, Bethesda, MD 20892-4255. Tel.: 301-496-3209; Fax: 301-402-1344; DJJIN{at}helix.nih.gov.

(^1)
The abbreviations used are: RNAP, RNA polymerase; Rif^r, rifampicin resistance; bp, base pair(s).


ACKNOWLEDGEMENTS

I am grateful to Drs. Sue Gargess, Jeff Roberts, Lucia Rothman-Denes, and Yan Ning Zhou for comments on the manuscript, and I thank Dr. Yan Ning Zhou for sequencing the mutated galP2 DNA template.


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