Changes in Conserved Region 3 of Escherichia coli sigma 70 Reduce Abortive Transcription and Enhance Promoter Escape*

Michael CashelDagger , Lilian M. Hsu§, and V. James HernandezDagger ||

From the  Department of Microbiology, Center for Microbial Pathogenesis, State University of New York at Buffalo School of Medicine, and Biomedical Sciences, Buffalo, New York 14214, Dagger  Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, and § Programs in Biochemistry, Mount Holyoke College, South Hadley, Massachusetts 01075

Received for publication, November 8, 2002, and in revised form, December 5, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutations within the Escherichia coli rpoD gene encoding amino acid substitutions in conserved region 3 of the sigma 70 subunit of E. coli RNA polymerase restore normal stress responsiveness to strains devoid of the stress alarmone, guanosine-3',5'-(bis)pyrophosphate (ppGpp). The presence of a mutant protein, either sigma 70(P504L) or sigma 70(S506F), suppresses the physiological defects in strains devoid of ppGpp. In vitro, when reconstituted into RNA polymerase holoenzyme, these sigma  mutants confer unique transcriptional properties, namely they reduce the probabilities of forming abortive RNAs. Here we investigated the behavior of these mutant enzymes during transcription of the highly abortive cellular promoter, gal P2. No differences between mutant and wild-type enzymes were observed prior to and including open complex formation. Remarkably, the mutant enzymes produced drastically reduced levels of gal P2 abortive RNAs and increased production of full-length gal P2 RNAs relative to the wild-type enzyme, leading to greatly reduced ratios of abortive to productive RNAs. These results are attributed mainly to a decreased formation of unproductive initial transcribing complexes with the mutant polymerases and increased rates of promoter escape. Altered transcription properties of these mutant polymerases arise from an alternative structure of the sigma 70 region 3.2 segment that permits efficient positioning of the nascent RNA into the RNA exit channel displacing sigma  and facilitating sigma  release.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The steps in transcription initiation performed by all DNA-dependent RNA polymerases are promoter binding, DNA strand melting, RNA chain initiation and nascent RNA chain formation, and finally escape from the promoter sequences. Failure to escape from the promoter results in the production of aborted RNA transcripts and reiterative cycling through the open complex (1-4). Promoter escape appears to occur concomitantly with the release of sigma 70 factor following the synthesis of a specific length of RNA and the conversion of the unstable initial transcribing complex to a stable elongating complex (5-8). Depending on the promoter, abortive RNA synthesis occurs to different degrees and can occur extensively enough that it becomes rate-limiting for the synthesis of productive RNAs (2, 9).

Important determinants of abortive initiation are contained along the region of melted DNA within the transcription complex encompassing the non-transcribed and the initial transcribed sequences (ITS).1 Changes in the composition of the ITS can alter the extent of productive RNA synthesis (10) and cause dramatic variation in the total number of abortive events and also alter abortive probabilities from position to position within the ITS (11). Base residue changes specifically in the non-template strand of the -10 promoter region, which weaken or strengthen open complex stability, can lead to decreased or increased abortive probabilities, respectively (12). Several examples of regulation at the level of promoter escape have been reported. The phage Phi 29 promoter A2c was repressed by the phage regulatory protein p4 through stabilization of RNA polymerase-promoter interactions to the extent that promoter escape is reduced, and abortive transcription is increased (13, 14). Alternatively, in phage P22, the Arc regulatory protein has been shown to activate transcription by increasing promoter escape through weakening the stability of open complexes (15). Upstream A-tracts, which activate promoter activity by improving complex formation between polymerase and promoter sequences (16, 17), can conversely inhibit the activity of a promoter presumably by stabilizing RNAP at the promoter to such a degree that it prevents efficient promoter escape (18). In vivo, RNA polymerase bound at a consensus promoter was shown to idle excessively along the ITS and escape the promoter poorly (19). Thus, enhancement of polymerase-promoter interactions can activate or repress promoter activity depending on whether the rate-limiting step of transcription initiation is associated with polymerase binding, open complex formation (20), or promoter escape (21).

The determinants of promoter escape do not reside exclusively with the promoter sequence but also involve components of the transcription machinery, especially the sigma  factor. Certain mutations in region 2.2 of sigma 70 were found to affect promoter escape (22, 23). We described previously (24, 25) the isolation of two mutant alleles of the gene encoding the sigma 70 transcription initiation factor, rpoD(P504L) and rpoD(S506F), and characterized the in vitro transcription behavior of these two mutant proteins, sigma 70(P504L) and sigma 70(S506F), reconstituted into RNA polymerase holoenzyme. On three bacteriophage promoters, T7 A1, T5 N25, and T5 N25antiDSR, the mutant enzymes transcribed to yield a reduced amount of abortive RNA chains compared with the wild-type polymerase without increasing the abundance of full-length RNA chains. These mutant enzymes behaved similarly at the lambda PR promoter (26), and it was determined that sigma 70(P504L) and sigma 70(S506F) RNA polymerases do not accumulate the so-called "moribund" complexes, the nonproductive complexes that yield only abortive products (27). Thus, a lower tendency to form moribund complexes by these mutant polymerases appears to bring about a lower production of abortive RNAs predominantly (26). These results do not fulfill the expectation that a lowered tendency to form non-productive complexes should increase the probability of forming productive complexes, consequently resulting in increased productive transcription.

To resolve this discrepancy, we extended the investigation of these mutant polymerases to other well characterized highly abortive promoters. As reported below, we found that on the E. coli gal P2 promoter, the mutant polymerases drastically reduced abortive transcription while greatly stimulating productive RNA synthesis when compared with the wild-type enzyme. Subsequently, we determined the various transcription initiation constants to pinpoint the basis of this altered transcription property.

    EXPERIMENTAL PROCEDURES
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Proteins-- The cloning of rpoD+, rpoD(P504L), and rpoD(S506F) and the overexpression and purification of sigma 70, sigma 70(P504L), and sigma 70(S506F) proteins have been described (25). Core RNA polymerase was purchased from EpiCentre Technologies (Madison, WI). Holoenzyme was reconstituted by mixing sigma 70 with core RNA polymerase at a ratio of 5:1 and incubating at room temperature for 15 min immediately prior to use in the transcription reactions.

Transcription-- The minicircle DNA template bearing the gal P1 and P2 promoters was prepared as described (28). Abortive initiation assays to measure the lag times for tau -plot analysis (29) were performed in potassium chloride buffer (40 mM Tris-HCl, pH 7.9; 10 mM magnesium chloride; 100 mM potassium chloride; 1 mM dithiothreitol; 100 µg/ml acetylated bovine serum albumin) with 2 nM of template DNA per reaction and different concentrations of RNA polymerase holoenzyme added to initiate transcription. The gal P2-specific initiating dinucleotide ApU was present at 500 µM plus 10 µM [alpha -32P]UTP (specific activity of 15 cpm per fmol). Abortive initiation products were resolved on a 7 M urea, 20% polyacrylamide gel (acrylamide:N,N'-methylenebisacrylamide = 59:1) (30) to verify formation of the appropriate abortive products. Extensive quantification at different polymerase concentrations was determined as a percentage of the total radioactivity incorporated following ascending paper chromatography as described (30). The radioactivity on paper chromatograms was quantitated with the radioanalytic imaging system from Ambis (San Diego, CA). The average time for open complex formation, tau obs, corresponds to the lag prior to the final steady-state rate of product formation and was determined by least square fit of the data to the theoretical exponential equation derived by Hawley and McClure (31). Apparent binding and isomerization constants (KB(app) and kf(app)) were solved by least square fit of the data to the classical derivation of McClure (29). Curve fitting was accomplished with the SigmaPlot program (Jandel Scientific, San Rafael, CA).

Steady-state transcription reactions were performed in potassium glutamate buffer (20 mM Tris acetate, pH 7.8; 10 mM magnesium acetate; 100 mM potassium glutamate; 10 mM beta -mercaptoethanol) with 10, 20, 50, or 100 µM [gamma -32P]ATP (specific activity of 50 cpm per fmol), GTP, CTP, and unvarying UTP (at 10 µM) for 10 min at 37 °C in a reaction volume of 20 µl. Reactions were initiated by mixing 50 nM RNA polymerase holoenzyme together with 2 nM template plus nucleoside triphosphates all prewarmed to 37 °C for 2 min.

Single-round transcription kinetics (Fig. 7) were performed by mixing RNAP (20 nM) with template (2 nM) followed by preincubation for 5 min prior to the simultaneous addition of heparin (25 µg/ml) with 100 µM ATP, GTP, CTP, and 10 µM [alpha -32P]UTP (specific activity of 50 cpm per fmol) in a reaction volume of 100 µl, and 10-µl aliquots were removed at subsequent times. Reactions were stopped, precipitated, resuspended, and treated as described previously (25). Abortive RNAs were analyzed by electrophoresis of 1/4 of each sample on a 7 M urea, 25% polyacrylamide gel (acrylamide:N,N'-methylenebisacrylamide = 12.5:1). Electrophoresis and buffer conditions were as described before (25). Full-length RNAs were separated on a 7 M urea, 6% polyacrylamide gel (19:1) using a conventional Tris borate-EDTA buffer (TBE) system. Radioactivity was quantified using a Storm PhosphorImager and ImageQuant program version 4.2 from Amersham Biosciences.

Determination of Open Complex Stability-- An excess of RNA polymerase holoenzyme (100 nM) was mixed with template (2 nM) in potassium chloride buffer (above) in a volume of 100 µl and incubated at 37 °C for 10 min to allow the formation of open complexes. Heparin or poly[d(AT)] was then added to RNA polymerase-template mix to a final concentration of 25 µg/ml. At subsequent times aliquots of 10 µl were removed and mixed with an equal volume of a pre-warmed chase mixture (400 µM ATP, GTP, CTP; 10 µM [alpha -32P]UTP (200 cpm per fmol); 25 µg/ml sodium poly[d(AT)] or sodium heparin). Transcription reactions were carried out for an additional 5 min at 37 °C and then stopped by mixing with 5 µl of 70% formamide, 100 mM EDTA, 1× TBE, 0.025% bromphenol blue, and 0.025% xylene cyanol. Full-length RNAs were analyzed and quantified as described above. An initial or zero time point was taken prior to the addition of heparin or poly[d(AT)].

Gre Factor Purification-- GreA and GreB were purified from the Escherichia coli strain JM109 bearing the GreA and GreB overexpression plasmids, pDNL278 and pGF296, respectively, as described (32).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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sigma 70 Mutations-- Missense alleles rpoD(P504L) and rpoD(S506F) contain single amino acid substitutions of leucine for proline and phenylalanine for serine at residues 504 and 506, respectively, in subregion 3.2 of sigma 70 (Fig. 1); subregion assignment within region 3 is based on the structural domains identified in the Thermus thermophilus RNAP holoenzyme crystal structure of Vassylyev et al. (41) rather than the original assignment based on sequence alignment (44). Proline 504 and serine 506 are 2 of only 10 residues in region 3 that are invariant among the primary sigma  factors (Fig. 1).


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Fig. 1.   Mutational changes in region 3.1. Four protein regions and subdivisions highly conserved among the major sigma  factors are represented on a linear map of the 613-amino acid E. coli sigma 70 (44). Region 3 lies between conserved regions 2 and 4 involved in the recognition and binding of the -10 and -35 promoter elements, respectively. The amino acids of region 3 are shown above the sigma 70 map; residues of identity among all major sigma  factors are denoted by large boldface letters. Missense substitutions at amino acid positions 504 and 506 are indicated.

In Vitro Transcription System-- The gal P1 and P2 promoters have been extensively characterized in vitro with respect to RNA polymerase binding, rate of open complex formation, abortive RNA synthesis, and promoter escape (28, 30, 33). The activity of gal P1 is limited by promoter binding (33), whereas that of gal P2 is intrinsically limited at the step of promoter escape (28). The gal P1 promoter lacks an identifiable -35 promoter element; however, polymerase binding is aided by an extended -10 promoter element (34). The gal P2 promoter has both the -35 and -10 elements with a non-optimal spacing of 16 bp in addition to an extended -10 promoter element (35) (Fig. 2). Following binding at gal P2 there is extensive "idling" or abortive cycling of polymerase along the initial transcribed sequences prior to promoter escape. The probability that an RNA chain initiated at P2 is aborted rather than extended is estimated to be greater than 95%. For reasons that are unclear, abortive yields are especially high at specific sites, i.e. at positions +2, +3, and +6, within the initial transcribed sequence of gal P2 (28) (Fig. 3). In addition, at high UTP concentrations polymerase tends to "stutter" at the run of three U residues encoded in the initial sequence ATTTC of gal P2 after initiating at the normal A residue start site (36). Stuttering refers to the nonproductive mode of reiterative incorporation of UMP producing poly(U) RNA chains that are released at a variety of lengths and can grow to hundreds of nucleotide residues (37). At low UTP concentrations, this stuttering does not occur. This substrate dependence is one feature that distinguishes stuttering from the normal abortive RNA synthesis; the latter occurs readily at low and high nucleotide concentrations (11).


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Fig. 2.   Promoter region of mini-gal P1 and P2. The RNA synthesis start site of gal P2 is located 5 bp upstream of the gal P1 start site. The Pribnow boxes for gal P1 and P2 overlap and are indicated by boxes labeled -10, the shaded box is for gal P1. The overlines indicate extended -10 sequences, TG, for both gal P1 and P2. The -35 promoter element for gal P2 is labeled and indicated by the box. The positions within the initial transcribed sequences of gal P2 where the majority of "natural" abortive transcriptions occur are labeled, denoted by large boldface letters and asterisks. Transcripts initiating at gal P2 terminate on the mini-gal template at the rpoC terminator 125 bp downstream and those from gal P1 terminate 120 bp downstream as indicated (28).


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Fig. 3.   Complete RNA synthesis reactions on gal P1 and P2. A, the production of naturally abortive RNAs and full-length terminated RNAs arising from the mini-gal template was visualized. Transcription was performed as described under "Experimental Procedures" with increasing concentrations of ATP, GTP, and CTP as indicated with UTP held constant at 10 µM. Abortive RNA species were separated on a 25% polyacrylamide gel and full-length RNAs on a 6% polyacrylamide gel (see "Experimental Procedures"). RNA was labeled with [gamma -32P]ATP. Length of RNAs are labeled as follows: 2, dimer; 3, trimer; 4, tetramer; etc. (P1)120 = gal P1 and (P2)125 = gal P2 transcripts. B, PhosphorImager counts of the radioactive abortive and full-length RNAs at different nucleoside triphosphate concentrations determined from the gel of A. Wild-type RNAP, filled circles; sigma 70(P504L) RNAP, open triangles; and sigma 70(S506F) RNAP, open circles. C, the ITS of gal P1 and P2. The relative degree of reduction in the abundance of abortive transcripts produced by sigma 70(P504L) RNAP at different positions along the ITS of gal P2 is indicated by the length of downward arrows.

The recombination-excision system of Choy and Adhya (28) was used to generate a supercoiled minicircle template containing only the gal P1 and P2 promoters upstream of a single Rho-independent terminator. This "mini-gal" template specifies the transcription of short abortive RNAs as well as a 120- and 125-nucleotide RNA originating from gal P1 and P2, respectively, without contaminating RNAs arising from endogenous promoters, a limitation usually encountered when using regular supercoiled plasmids as templates.

Synthesis of Abortive and Full-length RNAs-- Multiple round in vitro transcription reactions were performed in the presence of all four NTPs to determine the continuous production of abortive and productive gal RNAs (11). We used increasing concentrations (10-100 µM) of ATP, GTP, and CTP and a fixed concentration of UTP (10 µM); the UTP concentration was kept low to prevent polymerase stuttering (see above). Because both gal P1 and P2 initiate with an A residue, the transcripts were all 5'-labeled at a constant specific activity with [gamma -32P]ATP. End labeling of RNA transcripts has the advantage of allowing direct stoichiometric comparison of radioactivity incorporated into different RNA species regardless of size. The wild-type RNA polymerase transcribes gal P1/P2 to yield an abortive RNA ladder up to a length of 7 nucleotides and the terminated P1 and P2 RNA of 120 and 125 nucleotides, respectively (Fig. 3A). The abortive RNAs arise predominantly from gal P2 because very little difference in abortive products was observed from a mini-circle template bearing a gal P1 knock-out mutation (28). With the wild-type enzyme, the level of abortive and productive RNA synthesis increased concomitantly with increasing NTP concentration, suggesting that the abortive pattern did not result from NTP substrate limitation. Rather, a higher concentration of NTP supported a higher frequency of initiation, with a fraction of the initiated chains becoming productive RNA and the remainder abortive.

Compared with the wild-type enzyme, mutant polymerases accumulated many fewer abortive RNA chains and more full-length RNA chains from both gal promoters (Fig. 3A). The reduction of abortive RNA chains appears to be differentially affected by the mutant polymerases. The dimer RNA is reduced about 40%, and the trimer and hexamer RNAs are reduced by 4- and 10-fold, respectively (Fig. 3B). Relative reduction of abortive RNA by the mutant RNA polymerases at all positions along the initial transcribed sequence of gal P2 is indicated with downward arrows in Fig. 3C.

The mutant RNA polymerases also gave rise to increased accumulation of both gal P1 and P2 transcripts. The abundance of full-length RNA chains produced by mutant enzymes is higher by about 2-fold for gal P1, 4-fold for gal P2, and 3-fold overall (Fig. 3B). Several interesting comparisons can be made on the changes in productive synthesis. On a number of promoters tested previously, the sigma 70 mutant polymerases showed reduced abortive transcription but no increase of productive synthesis (25, 26); thus, increased productive synthesis, which can be equated with an enhanced ability at promoter escape, by the mutant enzymes appears to be a promoter-dependent phenomenon. We further noticed that at low NTP concentrations with the wild-type enzyme, P1 was the predominant full-length transcript, but at higher NTP concentrations, P1 and P2 RNA achieved parity. This observation can be explained by the excessive idling of wild-type polymerase at the P2 promoter relative to P1 (28), and the extent of idling was reduced when NTP concentration was raised. For the mutant enzymes, however, P2 was the more abundant productive RNA at all NTP concentrations. Thus, the sigma 70 mutations seemed to have brought about two effects on gal P2 transcription, overcoming an NTP concentration-dependent ability at escape and increasing the intrinsic rate of promoter escape.

To compare further the abortive and productive synthesis by the wild-type and mutant enzymes, we plotted the abortive and productive yields (see Ref. 11; i.e. the mole fraction of abortive and full-length RNA per total product) and abortive probabilities at each position along the initial transcribed sequence of gal P2 (Fig. 4). This revealed aspects of mutant enzyme behavior not obvious from the data in Fig. 3. The abortive yield (Fig. 4A) of dimer RNA is actually greater for the mutant polymerases, that of trimer and hexamer RNA is significantly reduced, and other abortive RNAs are represented by about equal yields. Full-length gal P2 RNA is increased from 1% of total RNA for the wild-type enzyme to ~6 and 5% for P504L and S506F sigma 70 containing holoenzyme, respectively (Fig. 4A). Abortive probabilities were determined at each position by correcting the percentage of each abortive RNA chain by the fraction of polymerases that actually advanced to that position. This revealed that the wild-type and mutant enzymes had very similar overall probabilities of producing abortive RNAs of 5 nucleotides or shorter. Most pronounced differences occurred at +6 and +7 where the probabilities of forming the 6- and 7-mer abortive transcripts were reduced 50-70% for the mutant enzymes (Fig. 4B). Perhaps not coincidentally, these positions correspond to the maximum length of RNA synthesized prior to escape and thus likely demarcate the positions where the initial transcribing complex makes the transition to an elongating complex. Taken together, the above analysis allows us to conclude that the mutant polymerases are more facile at promoter escape, thus producing fewer abortive RNA and more full-length RNA.


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Fig. 4.   Abortive yields and probabilities on gal P2. A, the percent of total incorporated counts represented by each length of RNA species, called the abortive yield, is presented (11). For this analysis RNA products transcribed with 50 µM [gamma -32P]ATP, GTP, CTP, and 10 µM UTP were quantified. This was the highest concentration of nucleoside triphosphates for which dimer, trimer, and unincorporated radioactive ATP were sufficiently well resolved so that the signal to noise ratio for accurate quantitation of these small RNA chains was tolerable. Wild-type RNAP, black bar; sigma 70(P504L) RNAP, lightly shaded bar; sigma 70(S506F) RNAP, darkly shaded bar. B, the abortive probabilities were calculated by Equation 1 (see Ref. 11),
P<SUB>i</SUB>=<FENCE><FR><NU>X<SUB>i</SUB></NU><DE>100%–<LIM><OP>∑</OP><LL>2</LL><UL>i−1</UL></LIM> X<SUB>i</SUB></DE></FR></FENCE> × 100% (Eq. 1)
where Pi is the abortive probability that the transcript will be aborted from the initial transcribed complex at the ith position. Xi is the abortive yield of the ith RNA species (in percent). Histogram bars are the same as in A. Total PhosphorImager counts were 2.02 × 106 for wild-type enzyme, 1.43 × 106 for P504L enzyme, and 0.98 × 106 for the S506F enzyme.

Open Complex Formation at gal P2-- To gain insight into the differential transcriptional outcome during RNA synthesis from gal P2 by wild-type and sigma 70 mutant enzymes, we determined and compared the various transcription initiation constants for these enzymes. First, we evaluated whether the sigma 70 mutants could affect the general process of promoter binding or open complex formation by determining the apparent equilibrium binding constant, KB, and the forward rate constant, kf, of open complex formation at the gal P2 promoter. This was done by the steady-state abortive initiation assay using a subset of nucleoside triphosphates followed by the derivation of tau  plots (29, 30). Because the binding of polymerase at gal P1 and P2 is mutually exclusive, we specifically determined the apparent KB and kf values (KB(app) and kf(app)) only for P2 by using the initiating dinucleotide ApU and [alpha -32P]UTP. These limited substrates permit the synthesis of only the trimer (pApUpU) and tetramer (pApUpUpU) RNAs, the production of which was verified by PAGE (Fig. 5A). By monitoring the length of time required for the appearance of RNA following the addition of enzyme to template and substrates, the average lag time (tau obs) for polymerase binding and open complex formation was measured at five different enzyme concentrations to determine KB and kf values for the wild-type and mutant enzymes. At the low polymerase concentrations the steady-state rate of trimer and tetramer RNA production was lower for the P504L mutant than the wild-type enzyme (Fig. 5B). However, tau obs was approximately the same at all RNA polymerase concentrations (Fig. 5C). Similarly for the S506F mutant, limited measurements at low and high polymerase concentrations revealed approximately the same tau obs value (data not shown). From tau obs, we obtained the values for KB(app) and kf(app) according to Goodrich and McClure (30); these values are 10.5 ± 1.2 × 106 M-1 and 9.76 ± 0.4 × 10-2 s-1 for both the wild-type and the mutant enzymes. Thus, the mutant polymerases appear to bind promoter and form open complexes normally on gal P2.


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Fig. 5.   Determination of open complex formation constants on gal P2. A, kinetics of accumulation of gal P2-specific ApUpU and ApUpUpU abortive products. Shown are sample time points from 0.5 to 10 min of reactions initiated by addition of 100 nM of either wild-type or sigma 70(P504L) bearing RNA polymerase holoenzyme to 2 nM mini-gal template in the presence of ApU and [alpha -32P]UTP. Radioactive reaction components were fractionated on a polyacrylamide gel system that separates short RNAs based on charge rather than size ("Experimental Procedures") (45). B, kinetics of percent radioactivity incorporated using 2 nM mini-gal template and 100 nM or 10 nM RNAP. Percent incorporation into trimer and tetramer RNA was determined following paper chromatography as described under "Experimental Procedures." C, observed lag times (tau obs) were plotted as a function of the reciprocal of RNAP concentration. tau obs were determined at five RNAP concentrations from kinetics of percent radioactive incorporation determined as described under "Experimental Procedures." Wild-type RNAP, filled circles; sigma 70(P504L) RNAP, open circles.

Stability of RNA Polymerase-Promoter Open Complexes-- A correlation between lower open complex stability and increased rates of productive RNA synthesis was originally noted for mutant lac promoter variants (9). In its simplest interpretation, this correlation suggests the notion that the formation of a stable open complex and promoter escape are antagonistic processes. We next examined the stability of pre-formed open complexes of wild type, and mutant sigma 70 polymerases on gal P1 and P2 were monitored in the presence of a nonspecific competitor, heparin or poly[d(AT)]. Open complexes were formed in the absence of NTP using a 50-fold molar excess of RNA polymerase to template to maximize promoter occupancy. Heparin was added next, and the promoter occupancy of gal P1 or P2 was assayed at subsequent times by adding high concentrations of NTP and monitoring the production of full-length RNA. The wild-type enzyme formed very stable open complexes on the gal promoters as found previously (33). After 2 h, only about 5-20% of the wild-type open complexes had dissociated from either gal P1 or P2 (Fig. 6). By contrast, the mutant polymerases exhibited a 50% loss of the gal P1 and gal P2 open complexes at ~80 and 40 min, respectively, after the addition of heparin (Fig. 6). The shorter open complex half-life with the mutant polymerases is not caused by the competitive nature of heparin (38); we compared the effect of heparin and poly[d(AT)] as nonspecific competitors and found that they gave comparable results (data not shown). The open complex half-life for the mutant polymerases corresponds to an k-f(app), value of 2.1 and 0.4 × 10-4 s-1, respectively, for gal P1 and gal P2.


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Fig. 6.   Stability of open complexes formed on gal promoters. A 100-fold molar excess of RNA polymerase was incubated with mini-gal template for 10 min at 37 °C followed by addition of either heparin or poly[d(AT)] to a final concentration of 25 µg/ml. At subsequent times aliquots were removed and chased with nucleoside triphosphates to monitor transcription activities at gal P1 and P2 (see "Experimental Procedures"). Activities are expressed as a fraction of the activity prior to addition of competitor and represent the fraction of open complexes remaining at the indicated times. Panels labeled P1 and P2 indicate promoter occupancy at gal P1 and gal P2, respectively. RNAP, filled circles; sigma 70(P504L) RNAP, open triangles; sigma 70(S506F) RNAP, open circles. Zero time point values for PhosphorImager counts of gal P1 and P2 transcripts in the presence of heparin for wild-type enzyme were 2.40 × 105 and 2.72 × 105; for P504L enzyme, 2.71 × 105 and 2.74 × 105; and for S506F enzyme, 2.72 × 105 and 2.98 × 105, respectively.

Comparing the Kinetics of Promoter Escape and Abortive Transcription-- So far, we have shown that the mutant polymerases are not compromised at open complex formation at gal P2, but the open complexes formed are less stable than that of the wild-type enzyme. When NTP is present, the mutant polymerases are also more facile at promoter escape. To confirm that the sigma 70 mutations have altered the rate of escape, we assayed the lag time for the appearance of each RNA species under single-round conditions. The lag time was determined for the 3-mer, 6-mer, gal P1, and gal P2 RNAs by quantifying these RNA species following the preincubation of an excess of polymerase to mini-gal templates followed by heparin and all four nucleoside triphosphates (see "Experimental Procedures"). As shown in Fig. 7A (right-hand column), the lag time for the appearance of the 3-mer and 6-mer RNAs is about 10-15 s for both the mutant and wild-type polymerases. This value closely matches the rate of open complex formation determined by tau -plot analysis at this polymerase concentration (see Fig. 5B; 100 nM RNAP). Similarly, the lag time for the appearance of the gal P1 transcript, also 10-15 s, is virtually identical for all enzymes. A major difference, however, is observed for the time required for the appearance of the gal P2 transcript; the wild-type polymerase requires about 45 s, while both mutants require 10-15 s. Under these conditions only about 10-15 s are required to achieve the first initial transcribing complex (ITC); thus, unlike the wild-type enzyme, no detectable idling of mutant polymerases occurs at gal P2. These results indicate that the mutant enzymes differ from the wild-type enzyme in that they appear to escape the gal P2 promoter without idling.


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Fig. 7.   Kinetics of RNA synthesis from gal promoters. Kinetics of the accumulation of hexamer (6-mer), trimer (3-mer), full-length gal P1 (P1), and gal P2 (P2) transcripts were measured in the presence of 50 µM ATP, GTP, CTP, and 10 µM [alpha -32P]UTP. Transcription conditions, resolution, and quantitation of RNA species were as in Fig. 4, except that heparin (50 µg/ml) was added simultaneously with nucleoside triphosphates. Wild-type RNAP, filled circles; sigma 70(P504L) RNAP, open triangles; sigma 70(S506F) RNAP, open circles. A shows the kinetics of RNA accumulation; the left-hand column represents a full time scale of 15 min; the right-hand column represents a time scale of 2.5 min. B shows the ratio of hexamer RNA to gal P2 full-length RNA (6-mer/gal P2) and trimer to gal P2 full-length RNA as a function of time after substrate addition.

Furthermore, because formation of the 6-mer RNA occurs with no detectable further lag, and the 6th and 7th positions in the initial transcribed sequence appear to demarcate the transition point of the ITC to the elongation phase, the slow step preventing the wild-type polymerase from escape may be the escape reaction itself that most likely occurs just after position +6. One straightforward interpretation of these results is that the mutant sigma 70 proteins are rapidly released from the ITC compared with the wild-type enzyme at a critical RNA length following 6 nucleotides, thus allowing rapid promoter escape.

Formation of Moribund Complexes by gal P2-- Previously, the lambda PR promoter was shown to have a high tendency to form the moribund abortive complexes with wild-type polymerase (27), but that tendency was lowered with the sigma 70 P504L and S506F mutants (26). On the gal promoters, the wild-type polymerase showed a similar high propensity to form moribund complexes; this is evidenced by the continuous accumulation of abortive RNA transcript which increased linearly for 15 min of transcription while the full-length transcripts accumulated linearly for less than 4 min and then began to plateau (Fig. 7A, left-hand column). As was observed before (26, 27), this differential pattern of RNA accumulation is indicative of the existence of two populations of initial transcribing complexes: one productive and the other moribund. In this single-cycle transcription, the productive complexes are rapidly depleted following promoter escape, but the long lived moribund complexes are trapped at abortive cycling to produce the short abortive RNAs continuously. Interestingly, the moribund fraction of gal P2 complexes formed with mutant polymerases is drastically reduced. This can be seen when comparing the rate of abortive RNA synthesis of mutant enzymes with the wild-type enzymes (Fig. 7A, 3-mer and 6-mer); however, this comparison assumes that the chain initiation and abortive cycling rates of the wild-type and mutant moribund complexes are the same.

The existence of moribund complexes is even more clearly demonstrated when one plots the ratio of abortive to productive RNAs over the time course of the transcription reaction (Fig. 7B). For the wild-type enzyme, the initial ratio of 6-mer and 3-mer abortive RNAs to productive transcripts is 8 and 15, respectively. These levels stay fairly constant between 3 and 5 min after the start of the reaction when the abortive to productive ratio begins to rise sharply, reaching a value of 20 and 45, respectively. The initial constant abortive to productive ratio is the result of parallel increases of abortive and productive RNA at early times; the later sharp rise results when productive synthesis has ceased and the abortive synthesis by the moribund complexes continues. For the sigma 70 mutant holoenzymes, the initial ratio of abortive to full-length RNAs is close to unity at the beginning of the reaction and increases only modestly, up to 8-fold at the most, at later times. These patterns of abortive versus productive synthesis indicate that the mutant polymerases form a much lower percentage of moribund complexes at gal P2, in agreement with the observation made with the lambda PR promoter (26).

Effect of Gre Factor Addition on Abortive Transcription-- Earlier, GreA and GreB transcript cleavage factors were shown to stimulate promoter escape in an escape-limited promoter (32). Recently, these factors were found to relieve the initiation arrest of moribund complexes (39). The above results suggesting that the highly abortive nature of gal P2 on wild-type RNA polymerase can be attributed in part to its high tendency to form moribund complexes prompted us to test the sensitivity of gal P2 abortive transcription to the Gre factors. As shown in Fig. 8, the presence of GreA and GreB factors dramatically reduced the formation of the 6-mer and 7-mer abortive RNAs but showed almost no effect on shorter (<6-mer) abortive RNAs. At the same time, a 3-5-fold increase in full-length gal transcripts is evident. This is consistent with the observed effects of Gre factors on the moribund complexes at the lambda PR promoter (39). Thus, the Gre factors also stimulate the conversion of gal moribund complexes into productive complexes. Addition of excess Gre factors to the mutant RNAP holoenzymes had no observable effect on gal P2 transcription apart from those previously attributed to the presence of the mutant sigma  factors per se (data not shown).


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Fig. 8.   Gre factor sensitivity of gal P2 promoter transcription. Purified GreA and GreB were added at an increasing stoichiometry to RNA polymerase (Gre/RNAP) as indicated starting at a ratio of 1:1 stoichiometry and raising the Gre factor stoichiometry as high as 10:1. Abortive and productive full-length RNAs were visualized as described in the legend to Fig. 3. Length of RNA species is indicated. Transcription was performed with 50 µM ATP, GTP, CTP, and 10 µM UTP. RNA was labeled with [gamma -32P]ATP, as described under "Experimental Procedures."


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Differential Behavior of sigma  Mutants on Different Promoters-- In this report, we investigated the transcription properties of RNA polymerase holoenzyme containing either sigma 70(P504L) or sigma 70(S506F) on the highly abortive cellular promoter gal P2 and found that the mutant polymerases reduced abortive RNA synthesis and increased the abundance of full-length RNA. By examining the various rate constants associated with transcription initiation, we showed that the mutant enzymes are equally capable as the wild-type enzyme in binding and melting the promoter DNA to form open complexes. However, the open complexes with the mutant enzymes are less stable, exhibiting a much lower open complex half-life than the wild-type enzyme. This difference alone between the mutant and wild-type enzymes can account for the diminished level of abortive RNA synthesis by the mutant enzymes, basically reflecting a lower amount of "trapped" open complexes at the promoter. In this regard, gal P2 transcription by the mutant enzymes resembles lambda PR transcription in that the amount of open complex formed is less than that with the wild-type enzyme (26). This explanation may also account for the reduced abortive transcription from T7 A1, T5 N25, and T5 N25antiDSR promoters by the mutant enzymes, although in that study, the stability of the open complexes was not examined (25).

More interestingly, the sigma 70 region 3.2 mutant polymerases showed differential effect with regard to increasing promoter escape. These mutant enzymes did not stimulate promoter escape from T7 A1, T5 N25, T5 N25antiDSR, nor lambda  PR but greatly enhanced productive synthesis from gal P2. On gal P2, the enhanced productive synthesis appears to have resulted from two factors as follows: one, a greatly diminished fraction of moribund complexes (Fig. 7A, top frames) and therefore a greatly elevated fraction of productive complexes that carries out full-length RNA synthesis; two, the mutant enzymes display an increased rate of promoter escape (i.e. a shorter t1/2 of productive synthesis of ~2 min) compared with that of the wild-type enzyme (t1/2 of ~5 min; see Fig. 7A, bottom frame). The increase in productive synthesis was not observed with lambda PR, although the mutant enzymes were shown to form a lower fraction of moribund complexes at this promoter (26). What might be the cause of this discrepancy? Is the stimulation of promoter escape by the mutant enzyme simply a promoter-dependent phenomenon? This would be likely if the promoters are very different in sequence, but this does not appear to be the case. Is it possible that the number of abortive complexes can be lowered without necessarily increasing the number of productive complexes? Another possible difference between our previous and present results could be the source of RNA polymerase; however, our previous and current results were obtained using identical batches of commercial sigma -free core RNAP (obtained from Epicentre Technologies, Madison, WI) for holoenzyme reconstitution (see "Experimental Procedures" and Ref. 25). An important factor that likely accounts for the above discrepancy is the supercoiling status of the promoter. Here, gal P2 transcription was performed with supercoiled minicircle templates, whereas those of lambda PR, T7 A1, T5 N25, and T5 N25antiDSR were all performed with linear templates. That supercoiling of the template can lead to a greatly increased level of productive synthesis is currently under investigation and will be reported elsewhere. We summarize the initiation constants measured for gal P1 and gal P2 in Table I. We further use a summary diagram to illustrate the steps of transcription initiation where the sigma 70 mutations have brought about different effects for the wild-type versus the mutant enzymes (Fig. 9).

                              
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Table I
Summary of initiation constants of gal P2/P1 with wild-type and mutant RNA polymerases


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Fig. 9.   Kinetic scheme of wild-type and mutant enzyme behavior on gal P2 promoter. The symbols represent the following: R, wild-type RNAP holoenzyme; R', mutant sigma 70 RNAP holoenzyme; P, promoter; RPc, closed promoter complex; RPo, open promoter complex; ITC, initial transcribing complex; ITCp, productive ITC; ITCu, unproductive ITC; EC, elongation complex. KB, equilibrium binding constant; kf, forward rate of open complex formation; k-f, rate of decay of open complex. The thickness of the arrow is meant to indicate the different extent of a reaction.

Based on the current findings, we propose that the P504L and S506F mutations lead to a rapid release of sigma 70 from the initial transcribing complexes to allow RNA synthesis to proceed to longer lengths. This idea is consistent with the original proposal of Hansen and McClure (5) that the presence of sigma 70 sterically hinders the elongation of the nascent RNA beyond a specific length and that the release of the sigma  factor is a physical prerequisite for continued RNA synthesis. Specifically, the idea of a physical competition between sigma 70 region 3 and the nascent RNA in the "RNA exit channel" is verified by analysis of the x-ray crystallographic structures of prokaryotic RNA polymerase holoenzyme recently reported (Refs. 40 and 41 and see below). In these two studies, it is predicted in the former and observed in the latter that sigma 70 region 3.2 occupies the entire length of the RNA exit channel and is predicted to cause steric clash with the advancing nascent RNA transcript during initial steps of RNA polymerization, providing the physical basis for abortive transcription.

Finally, consistent with the observation that moribund complexes are sensitive to the presence of the Gre transcription factors and fail to accumulate in their presence (39), we tested and observed a reduction in abortive RNA synthesis at the gal P2 promoter concomitant with a stimulation of full-length productive RNAs in the presence of Gre factors (Fig. 8). In their study, Shimamoto and colleagues (39) concluded that Gre factors cause a direct conversion of moribund complexes into productive complexes by opening a pathway of inter-convertibility between these two types of initial transcribing complexes. We propose that the sigma  mutant proteins also fail to accumulate moribund complexes because they form mutually exclusive productive complexes (Fig. 7B). The high propensity to form productive complexes is due to structural alterations in the holoenzyme in which the region 3.2 mutant sigma  factors do not sterically hinder the positioning of the nascent RNA in the RNA exit channel as tenaciously as wild-type sigma 70. Furthermore, we observed no additive effect of excess Gre factor on mutant RNAP behavior likely indicating that mutant sigma  factors mimic the presence of the Gre factors, namely allowing the interconvertibility of moribund into productive initial transcribing complexes.

Phenotypic Suppression of the ppGpp0 Condition-- The two sigma 70 mutants, P504L and S506F, characterized in this study were originally isolated as phenotypic suppressors of strains devoid of the transcriptional effector ppGpp. Strains deleted for the genes encoding the two known ppGpp synthetases in E. coli, the relA and spoT genes, lose the ability to accumulate the intracellular stress messenger ppGpp and as a consequence display pleiotropic defects in growth and stress adaptation (42). A newly discovered role of ppGpp in the regulation of sigma  factor competition (43) sheds new light on the behavior of these sigma  mutants with respect to their ability to suppress defects associated with the intracellular loss of ppGpp. It was originally reported that a striking characteristics of these two sigma  mutants was their lower affinity for core RNA polymerase leading to a lower abundance of sigma 70 holoenzyme in the cell (24). In light of the newly understood role of ppGpp with respect to lowering sigma 70 core affinity, we can now interpret the lowered ability of these sigma  mutants to associate with core RNAP as mimicking the normal effect of ppGpp in vivo. Furthermore, it seems that effects leading to increased alternative sigma  competition are fundamental to the global effect of ppGpp.

Structural Basis and Mechanism of Mutant sigma 70 Behavior-- In the crystal structure of the open complex (40), sigma  domains 2 and 4 are located on the surface of the holoenzyme interacting with base residues of the -10 and -35 promoter elements, respectively. The predicted path of the sigma  region 3 peptide that links domains 2 and 4 (referred to as the "Linker Domain") first descends into the enzyme crevice toward the active site and then makes a "hairpin turn" away from the active site to lay down the remainder of acidic region 3.2 into the path of the basic RNA exit channel. The presence of region 3.2 in the RNA exit channel is conjectured to pose steric clash between the advancing nascent RNA and the sigma 70 protein, causing abortive release of the RNA (40, 41).

Residues 504 and 506 of sigma 70 are located in the beginning stretch of region 3.2 according to the structural domain assignments of Vassylyev et al. (41) and may directly alter the path of the sigma  peptide backbone in the linker domain. Overall the sigma  subunit appears to be composed entirely of alpha -helices connected by either turns or loops (41), and the two sigma 70 mutations we examined are located, by structural analogy, at the end of the 11th alpha -helix in the T. thermophilus holoenzyme structure (41). By using the SOPMA secondary structure prediction algorithm, both mutations are predicted to extend the alpha -helical potential of the 11th helix beyond proline 504 to position 510 where the next proline residue occurs. This would extend the 11th alpha -helix through the N-terminal stem of the hairpin sequence ending just prior to the turn in the hairpin turn structure as discussed above and would re-direct the sigma 70 peptide backbone away from the catalytic center and away from the RNA exit channel. By disrupting the normal helical and hairpin turn pattern, P504L and S506F mutations lead to a situation where sigma  3.2 is now likely positioned loosely in the RNA exit channel. Consequently, sigma  3.2 becomes less of an impediment to the advancing RNA, lowering abortive initiation; at the same time, it might be more easily displaced from the RNA exit channel, facilitating sigma  release and resulting in promoter escape. Consistent with this notion is the observation that mutant sigma 70 proteins preferentially reduce abortive probabilities at RNA lengths of 6 and 7 and not 2-5 nucleotides (Fig. 4B). Thus, the abortive probabilities are reduced specifically at a length when sigma  release is likely occurring (Fig. 4). In addition, the supposition that the P504L and S506F mutations in sigma 70 alter and disrupt the normal path of region 3.2 with respect to its normal disposition within the RNA exit channel is supported by the physical defects displayed by these mutant sigma 70s. The loss or "loosening" of the interaction between the highly negatively charged region 3.2 with the highly positively charged RNA exit channel (40, 41) would be expected to reduce sigma 70-core affinity which in fact is what has been observed (24).

    ACKNOWLEDGEMENTS

We thank Dr. Sankar Adhya for originally suggesting our investigation of the gal promoters and Drs. Agamemnon Carpousis and Maria-Laura Avantaggiati for particularly insightful experimental suggestions. We also thank Drs. Hyon Choy and Susan Garges for kindly providing strains. We are especially indebted to Drs. Sergei Borokhov, Daniel Gentry, and Daniel Vinella for helpful discussions and suggestions during the course of this work. We are also obliged to the "Friday Seminar" group for their continuing interest and critical scientific review of results during the development of this project.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM57189 (to V. J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. E-mail: vjh@buffalo.edu.

Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M211430200

    ABBREVIATIONS

The abbreviations used are: ITS, initial transcribed sequences; RNAP, RNA polymerase; ITC, initial transcribing complex.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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