The Efficiency of Promoter Clearance Distinguishes T7 Class I1 and Class I11 Promoters*

Promoter strength has been defined as the relative production of transcripts from a promoter. For T7 transcription it has frequently been observed that T7 class I11 promoters are qualitatively stronger than T7 class I1 promoters. In previous work it was observed that the maximum rates of initiation of three class I11 and three class I1 promoters show no class distinctions R. A., A. 2640-2649). This suggests that the effi- ciency of the conversion of the polymerase initiation complex to a stable transcription complex contributes to the overall strength of T7 promoters.

The efficiency of promoter clearance is then determined by measuring the relative production of small transcription products in comparison with the production of run-off transcripts. These measurements clearly distinguish the T7 class 111 promoters from the T7 class I1 promoters. It is found that 68-75% of all initiations at the T7 class I11 promoters 46~5,410, and 413 produce a run-off transcript, while only 16-36% of the initiations at the T7 class I1 promoters + l . l B , 41.3, and 43.8 produce a run-off transcript. Clearly, promoter clearance contributes to the difference in promoter strengths of the T7 class I1 and 111 promoters.
Transcription of the bacteriophage T7 genome is unidirectional from left to right across the T7 chromosome; however, complete transcription of T7 requires two different RNA polymerases, Escherichia coli RNA polymerase and T7 RNA polymerase. The early genes or class I genes are transcribed by E. coli RNA polymerase, while the middle and late genes or class I1 and I11 genes are transcribed by T7 RNA polymerase. Although promoters for T7 RNA polymerase consist of a highly conserved sequence of 23 continuous bases (1)(2)(3), it is known that transcription from T7 promoters both in vivo * This work was supported by National Institutes of Health FIRST Grant AI-24905. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Tel.: 404-894-4037; Fax: 404-894-7452. and in uitro is sequence dependent (4)(5)(6)(7)(8)(9)(10)(11). In vitro, the relative strength of T7 class I1 and class I11 promoters depends not only upon promoter sequence but also upon reaction conditions, specifically ionic strength, temperature, and the superhelicity of the DNA template: factors that are known to affect helix stability (5, [11][12][13][14][15][16][17][18][19][20][21]. In uiuo, T7 transcription from the class I1 promoters, which differ in sequence from the consensus T7 promoter, is weaker than transcription from the absolutely conserved class I11 promoters (6, 7,22). The nature of the transcriptional differences between T7 class I1 and I11 promoters has been addressed, and different studies have provided different answers. With T7 restriction fragments McAllister and Carter (5) showed that class I1 promoters are preferentially inhibited by conditions that stabilize the DNA helix. They also noted that the establishment of stable RNA-polymerase complexes required the synthesis of longer RNA transcripts at class I1 promoters (5, 20). On cloned T7 promoters Smeekens and Romano (11) found no differences in the binding strength of T7 RNA polymerase to the class 11 and class I11 promoters 41.1B and 413. Similarly, Burgess and co-workers' comparison (23) of T7 RNA polymerase footprints on a class I11 promoter and a class II/III hybrid promoter showed that there was no difference in promoter binding in the absence of GTP, but that in the presence of the initiating nucleotide the binding constant for the class I11 promoter was almost 3 times that of the hybrid promoter. All of this suggested that the efficiency of initiation is reflected in the stability of the initiated polymerase-promoter complex, and that the overall process of initiation might differentiate strong T7 class I11 promoters from the weaker T7 class I1 promoters.
We previously showed that initiation at T7 promoters can be assayed by following the rate of appearance of small initiation products. These studies showed that the in uitro initiation activities of the T7 class I1 promoters $l.lB, 41.3, and 43.8, and the T7 class I11 promoters 46.5, 410, and 413 only mimic the in viuo observation that T7 transcription from class I1 promoters is weaker than transcription from class I11 promoters when the T7 class I1 and I11 promoters are assayed on linear templates (24). On these linear templates, the class I1 promoters generally required a higher promoter concentration to produce half of the maximum rate of initiation ([P]vmax/2 values) than the class I11 promoters. Although the [P]v,,,,J2 measurements differentiated the T7 class I1 and I11 promoters, curiously there was no class difference observed in the maximum rates of initiation (V,,, values) of the six promoters. Based on V, , , , , , only the T7 410 promoter is significantly more active than any of the other five promoters.
This similarity in the maximum rates of initiation at the three T7 class I1 promoters, @l.lB, 41.3, and 43.8, and the two T7 class I11 promoters, 46.5 and 413, seems to contradict the many observations that T7 transcription from its class I11 promoters is qualitatively much stronger than transcrip-tion from its class I1 promoters. What is the basis for this apparent contradiction? Since promoter strength has been defined as the relative production of transcripts from a promoter (25), promoter strength is the net combination of the rate of promoter bindinglopening, the rate of formation of the initial internucleotide bonds, and the ratelefficiency of conversion to a stable transcription complex. [P]v,Jz and Vmax values for the production of small initiation products are measures of the efficiency of the early stages of initiation and reflect both the characteristics of promoter bindinglopening and the rate of formation of the initial internucleotide bonds (24).
[P]v,,,2 and Vmax values for the production of small initiation product do not measure the efficiency of the late stages of initiation and do not reflect the ratelefficiency of conversion t o a stable transcription complex (24 suggests that the ratelefficiency of conversion to a stable transcription complex is a significant factor in the determination of the overall strength of these promoters. Relative promoter strengths and efficiencies of conversion to a stable transcription complex are measured for the three T 7 class I1 promoters, dl.lB, 41.3, and 43.8, and the three T7 class I11 promoters, 46.5, $10, and 413. These measurements are made on the same templates previously used to measure [P]vm,~2 and V,,, values (24) to allow direct comparison of the two studies. It is found that the relative strengths of the class I11 promoters are generally stronger than the class I1 promoters and that the class I11 promoters are much more efficient than the class I1 promoters at converting from the initiation complex to a stable transcription complex. This suggests that the promoter clearance step is a significant factor in differentiating the T7 class I1 and 111 promoters.

Materials
Enzymes-Restriction endonucleases and Klenow fragment of DNA polymerase I were purchased from New England Biolabs and Boehringer Mannheim Biochemicals.
Nucleotide~-[a-~~P]GTP and [cY-~'P]ATP (800 Ci/mmol) were purchased from Du Pont-New England Nuclear. Unlabeled, high purity dNTPs and NTPs were purchased from Pharmacia LKB Biotechnology Inc., and the purity of the unlabeled NTPs was confirmed by thin layer chromatography on PEI-cellulose F (EM Science) (26,27). No contaminants were detected in the unlabeled NTPs.

T7 RNA Polymerase
T7 RNA polymerase was prepared from E. coli HMS12/pGP1-1/ pGP1-5 (29). This strain was a gracious gift from Drs. Stanley Tabor and Charles Richardson (Harvard Medical School). The T7 RNA polymerase was purified as previously described (30), and the activity of the purified enzyme was assayed under standard conditions (31). The T7 RNA polymerase had a specific activity of 483,000 units/mg (1 unit of activity is defined as the amount of enzyme necessary to incorporate 1 nmol of AMP into RNA in 1 h at 37 "C).
Preparation of Template DNA All plasmids were prepared by standard procedures and were confirmed by restriction mapping (32). The purified, supercoiledplasmids were then cut with an appropriate restriction enzyme to produce linear transcription templates (Table I). For pHI1.3 and pSR135, the overhanging ends created by linearization with AjlIII were repaired with E. coli large fragment DNA polymerase I and the 4 dNTPs.
The restricted DNA was analyzed on a 1% agarose gel to check for complete cleavage, and afterward, the DNA was purified by extraction with phenol and chloroform and precipitation with ethanol. The repaired pHI1.3 andpSR135 templates were purified over a Schleicher & Schuell Elutip-d column to remove unincorporated dNTPs, and the purified DNA was precipitated. The precipitated DNA was redissolved in 10 mM Tris-HC1, pH 7.8, 1 mM EDTA, and DNA concentrations were determined by measuring the absorbance of the solutions at 260 nm. In general, the reactions minus T7 RNA polymerase were preincubated at 37 "C for 5 min to warm the mixture. The reactions were then started by the addition of T7 RNA polymerase. At 3, 6, and 9 min, 7.0-pl samples were withdrawn from the reaction and were stopped by addition of an equal volume of stop buffer (90% formamide, 50 mM EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol).

Measurement of Promoter Strength
The samples were heated in a boiling water bath for 30 s and cooled on ice. Samples of 7 p1 were loaded onto a 5% polyacrylamide denaturing gel, and the samples were electrophoresed under standard conditions (32). After electrophoresis the gels were soaked in 10% methanol, 10% acetic acid to remove the urea and were subsequently dried. The products were visualized by autoradiography using Kodak XAR-5 x-ray film. The bands corresponding to the run-off transcripts were cut out of the gel, and the bands were quantitated by liquid scintillation.

Measurement of the Efficiency of Run-off Transcription
The efficiency of producing a run-off transcript was determined by measuring the ratio of small transcription products to run-off transcripts produced in a T7 transcription reaction. Reaction conditions were identical to the reactions described in the previous section except for the inclusion of only one template at 20 nM, the use of 20-25 pCi of [a-"PIGTP to label the RNA, and the withdrawal of samples at 3, 9, and 27 min. The specific activity of the [a-32P]GTP in the reaction mixtures was determined for each individual experiment and ranged from 2100 to 2600 cpm/pmol.
Half of each sample was loaded onto a 19% polyacrylamide (acrylamide to bisacrylamide ratio = 9 to 1) denaturing gel to analyze the small transcription products, and the remainder of each sample was loaded onto a 5% polyacrylamide denaturing gel (32) to analyze the run-off transcripts. The bands corresponding to the run-off transcripts and the bands corresponding to transcripts that were 3-8 nucleotides long were cut out of the gels. These bands were then quantitated by liquid scintillation analysis.

RESULTS
Relative Strengths of T7 Promoters-It has been previously noted that the experiment-to-experiment variability of T7 RNA polymerase assays makes it difficult to accurately determine the absolute activity of T7 RNA polymerase (24,33). These same experimental variations also make it difficult to measure the absolute strengths of T7 promoters; consequently, the strengths of the T7 class I1 promoters 4l.lB, 41.3, and 43.8 and the T7 class I11 promoters 46.5 and 413 were determined in a comparative assay in relation to the strength of the T7 class I11 promoter 410. Measurements were made by comparing the synthesis of run-off transcripts in a transcription reaction containing an excess of T7 RNA polymerase, a linear transcription template bearing the 410 promoter, and a second linear template bearing the promoter to be assayed. This comparative, run-off assay minimizes the problems associated in determining absolute promoter strength with an enzyme whose apparent activity can vary from day to day. Furthermore, the assay produces specific RNA transcripts by run-off synthesis. Fortunately, this eliminates the requirement for an efficient transcriptional terminator, since the known T7 terminator, T4, is not sufficiently active (34) for this assay. Finally, the assay minimizes the effects of template-dependent pausing and premature termination on the measurement of promoter strength by exploiting the sequence-independent processivity of the stable T7 transcription complex (19).
To prepare the run-off templates for the assay, plasmids containing the T7 promoters +l.lB, 41.3,43.8,46.5,413, and 410 were linearized by cleavage with restriction enzymes (Table I). These plasmids were used to allow direct comparison of the relative promoter strengths obtained in this study to the previous measurements of promoter V, , , and [P]vmdz (24). The restriction enzymes for linearizing the plasmid templates were chosen with three criteria in mind. First, the restriction enzyme must have a unique cleavage site on the plasmid to eliminate the generation of promoter-less DNA fragments that might compete for T7 RNA polymerase. Second, restriction of the plasmid must result in flush ends to avoid the possible competitive binding of T7 RNA polymerase to staggered ends (35). Third, where possible, the plasmids should be cut so that the T7 promoter is placed 200-500 bases upstream of the end of the DNA. This was not possible with the plasmids pSR135 and pHI1.3; consequently, pHI1.3 and pSR135 were cleaved with AflIII, and the overhanging ends created by cleavage with AflIII were repaired with E. coli large fragment DNA polymerase I and the 4 dNTPs.
Typically, excess T7 RNA polymerase (200 nM) was added t o a prewarmed (37 "C) transcription reaction containing 20 nM pHIlO/EcoRV and 20 nM of a second transcription template. Samples taken at 3, 6, and 9 min were electrophoresed on a 5% acrylamide denaturing gel to separate the transcripts. The products were visualized by autoradiography; the bands corresponding to the run-off transcripts were cut out of the gel, and the amount of each transcript was determined from scintillation analysis after correcting for the adenylate content of the transcript. Since it was observed that the accumulation of transcripts was linear with respect to time (data not shown), the rate of synthesis of each transcript was determined from the slope of a least squares analysis of the data. The relative strength of a promoter was then defined to be the ratio of the rate of transcript synthesis from the promoter/template being assayed to the rate of transcript synthesis from the pHIlO/EcoRV template. The concentrations of nucleoside 5"triphosphates used for the measurements of promoter strength are the same concentrations that were used for the previously reported initiation studies (24) (Table 11).
Additional experiments demonstrated that the relative strength of 43.8 was insensitive to the concentration of T7 RNA polymerase, to changes in the length of incubation, and to changes in the order of addition of the nucleotides and RNA polymerase (Table 11). Conversely, the relative strength of 43.8 was sensitive to high total template concentration in the assay. If the concentration of @lO/EcoRV plus the concentration of 43.8/SmaI was comparable with the concentration of T7 RNA polymerase then the apparent strength of 63.8 decreased; however, if T7 RNA polymerase was present in excess the apparent strength of 43.8 was constant and insensitive to promoter concentration (Table  11) Efficiencies of Run-off Transcription-The large differences in relative promoter strength (Table 111) contrast with the smaller differences previously observed in the maximum rates of initiation (24). This dichotomy suggests that the efficiency

TABLE I1
The strength of 63. 8    of conversion from an initiation complex to a stable transcription complex is a significant factor in the determination of the overall strength of T7 promoters. The efficiency for producing a run-off transcript (the efficiency of promoter clearance) was determined by measuring the percentage of total initiations that produce a run-off transcript during T7 transcription. The reaction conditions used for determination of the efficiencies of promoter clearance were identical to the assay conditions used for determination of relative promoter strengths except for the inclusion of only one transcription template (20 nM) in the efficiency assays. This allows direct comparison of the relative promoter strengths and the efficiencies of run-off transcription.

relative to 610: controls for the measurement of promoter strength ([PI, [RNAPI, [GTP], order of addition) [PI, the concentration of each promoter in the assay
Generally, excess T7 RNA polymerase (200 nM) was added to a prewarmed (37 'C) transcription reaction containing 800 pM GTP, 400 pM ATP, 400 pM UTP, 400 p M CTP, 20-25 pCi of [cY-~'P]GTP, and 20 nM of a transcription template. [cY-~'P] GTP was used in this assay so that trinucleotides produced by promoter dependent initiation of T7 RNA polymerase would be labeled. The RNA was analyzed by gel electrophoresis (Fig. 2), and the bands corresponding to run-off transcripts or to small initiation products 3-8 nucleotides in length were cut out of the gel. The quantity of each product was then determined by scintillation analysis after correcting for the guanylate content of the individual products.
Although small initiation products longer than 8 nucleotides are visible on the autoradiographs (Fig. 2), these products are significantly less abundant (data not shown) than The l a h s in the bottom panel show the small initiation products from 3 to 8 nucleotides in length that are produced from the transcription templates 46.5/SspI, @13/AfZIII, and &lO/SspI during run-off transcription after 3-, 9- the products 3-8 nucleotides long. The exclusion of these longer initiation products from the calculation of the efficiency of run-off transcription does not appreciably affect the results of the assay.
The proportions of small transcription products and runoff transcripts were measured for transcription from the T7 promoters on the templates dlO/SspI, +6.5/SspI, 413/AfcIII, $l.l/EcoRV, 41.3/AflIII, and 43.8/SmuI. These results are listed in Table IV as the percentage of initiation events that result in a run-off transcript and as the number of small initiation products per transcript. The data clearly distinguish the T7 class I11 promoters 46.5, 410, and 413, from the T7 class I1 promoters, dl.lB, 41.3, and 43.8, showing that the

1V
The distribution of T7 transcription products and the efficiency of initiation The rows labeled 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, and 8-mer tabulate the picomoles of RNA that are 3, 4, 5, 6, 7, and 8 nucleotides. Total oligonucleotides = The total picomoles of RNA 3-8 nucleotides in length. Run-off, the total picomoles of run-off transcripts produced in the sample. Total prods., total oligonucleotide + run-off (in pmol). The data tabulated on the first nine rows of the table are the results obtained from the 27-min sample of a single typical experiment. The % Trans. and Unpr. init. rows list the average of at least four separate measurements. % Trans., the percent of initiation events that result in a run-off transcript (run-off/total prods). Unpr. init., the number of small initiation products produced per complete run-off transcript (total oligonucleotide/run-off). The reactions contained 20 nM promoter and 200 nM T7 RNA polymerase.

TABLE V
T7promoter sequences Asterisks mark the bases that differ from the consensus sequence for a T7 promoter. +1 is the position in the promoter corresponding to the first base in the transcript that would be synthesized from the promoter. The sequences shown are the strands that correspond to the sequence that would appear in the RNA. The class of the promoters is designated by the Roman numerals following the promoter names. In addition, it was observed that the percentage of initiation events that result in a run-off transcript is constant over time (data not shown) and that the distribution of small initiation products 3-8 nucleotides long differs from promoter to promoter ( Fig. 2 and Table IV). For 41.1B, the amounts of small initiation products synthesized generally decrease as the size of the products increase; however, the pentamer, GGAGA, and octamer, GGAGAACC, break this trend and are more abundant than the tetramer and heptamer, respectively. The two remaining T7 class I1 promoters, $3.8 and 41.3, only produce significant amounts of trimers, tetramers, pentamers, and hexamers, but these promoters produce different major products. The most abundant initiation products produced by 93.8 are the trimer, GGG, and the tetramer, GGGA, while the most abundant products produced by 41.3 are the tetramer, GGGA, and the pentamer, GGGAC.
Different distributions of initiation products are not only observed for the nonidentical T7 class I1 promoters, but are also observed for the identical T7 class I11 promoters (Tables IV and V and Fig. 2). For (610, significant amounts of all initiation products from trimer to octamer are observed, and their abundance generally decreases with increasing product length. For 46.5 and 413, all initiation products from trimer t o octamer are observed, but for 46.5 the hexamer, GGGAGA, predominates, while trimer, tetramer, pentamer, and heptamer are the major initiation products observed for 413.

DISCUSSION
Transcription by T7 RNA polymerase has been studied extensively, but although much is known about the enzyme, the mechanistic details of T7 transcription have not been fully characterized. The studies of transcription by T7 RNA polymerase suggest that the mechanism for the initiation of transcription by T7 RNA polymerase (Fig. 3) might be similar to the mechanism proposed for E. coli RNA polymerase (36-38). Coleman and co-workers (39, 40) have studied the association of T7 RNA polymerase with its promoter in the absence of ribonucleoside triphosphates. They found no evidence of a closed complex (RP,), but did identify an open complex (RP,) on native polyacrylamide gels. This suggests that either the closed complex is difficult to observe or the recognition and binding of a promoter by T7 RNA polymerase involve opening of the DNA double helix. Addition of ribonucleoside triphosphates to the binary polymerase-promoter complex allows for the initiation of RNA synthesis. Footprinting indicates that the polymerization of the first few ribonu-cleotides stabilizes the polymerase promoter complex and suggests that initiation produces a complex (RPJ that is different from the open complex (8,23,41). Finally, both McAllister et al. (5,20) and Coleman et ul. (19) have shown that T7 RNA polymerase forms a stable transcription complex (RPt) after synthesis of an oligoribonucleotide of approximately 8-12 nucleotides.
Comparison of the +1 to +8 sequences of the T7 promoters (Table V) (1) with the distributions of initiation products (Table IV) shows that addition of a pyrimidine to an oligoribonucleotide less than 8 nucleotides in length frequently increases the relative frequency of abortive initiation either before the incorporation of the pyrimidine (46.5 and 413) or after the incorporation of the pyrimidine (41.1B and 41.3). Increased abortive initiation has been previously observed when UMP is incorporated into a transcript before the 8th and 12th nucleotides (19,20), but the observations that the preponderance of abortive initiation products produced by a T7 promoter are 8 nucleotides or less in length and that only 4 of the 17 natural T7 promoters incorporate UMP in the first 8 nucleotides of their transcripts suggest that early CMP incorporation is more important in determining T7 promoter efficiency than UMP incorporation.
The distributions of initiation products not only show that pyrimidine incorporation increases the relative frequency of abortive initiation, but they also show a tendency for abortive initiation to occur more frequently after the incorporation of AMP (41.1B and 43.8) than after incorporation of GMP. This would suggest that even transition mutations from +1 to +6, mutations changing the conserved G nucleotides to A, may increase abortive initiation from a T7 promoter.
Further examination of the distributions of initiation products reveals that the identical T7 class I11 promoters do not produce identical distributions of initiation products. The relative amounts of pentamer and hexamer produced by the class I11 promoters 46.5,410, and 413 differ considerably even though all of the pentamers are GGGAG and all of the hexamers are GGGAGA. This suggests that sequences outside the conserved T7 promoter can influence processes that occur within the conserved sequences of a T7 promoter.
Finally, for all of the promoters assayed, the abundance of short initiation products decreases dramatically after the incorporation of the 8th nucleotide, but in all cases initiation products longer than 8 nucleotides are present (partially visible in Fig. 2). The abundance of these longer initiation products appears to decrease regularly as the length of products increase (data not shown), but at a product length of approximately 13-16 nucleotides, the abundance of the longer initiation products again decreases dramatically, and the even longer initiation products fade into the background. The dramatic decrease in the production of abortive products after the incorporation of the 8th nucleotide has been observed previously, and it has been suggested that a highly processive ternary complex is formed by the T7 RNA polymerase, the DNA template, and the nascent RNA after incorporation of the 8th nucleotide (19). The distributions of initiation products seen here seems to indicate that a processive ternary complex starts to form after the incorporation of the 8th base in a transcript, but it appears that the complex is not fully formed until a transcript 13-16 long has been synthesized.
Components of Promoter Strength-Since promoter strength is the net combination of the rate of promoter bindinglopening, the rate of initiation, and the efficiency of the conversion of the initiation complex to a stable transcription complex (25) then relative promoter strength divided by the percentage of initiations that produce full length tran-scripts should equal the relative maximum velocity for the production of small initiation products. Division of the relative promoter strengths measured here by their respective efficiencies of run-off transcription yields a relative ranking of maximum velocities of initiation that are consistent with the maximum velocities of initiation ( VmaX) that were measured previously (24). This confirms that all three measurements (relative promoter strength, the percentage of initiations that produce full length transcripts, and Vmax) are selfconsistent and suggests that the three measurements are a reliable reflection of the factors that influence the strengths of T7 promoters.
In conclusion, measurements of the relative strengths of three T7 class I1 promoters and three T7 class I11 promoters confirm that the class I1 promoters are generally weaker than the conserved class I11 promoters; however, the data also show that the relative strengths of class I1 promoters vary widely and can range from activities approaching a class I11 promoter to activities near zero. Interestingly, this distinctive class difference in the strengths of the T7 promoters 41.1B, 41.3, 43.8, 46.5, $10, and 413 is not fully reflected in their the maximum rates of initiation. This suggested that promoter clearance might be a contributing factor to the class differences in the strengths of T7 promoters. It is apparent from the measurements of the efficiencies of run-off transcription that promoter clearance is a major factor in determining the class differences in the strengths of T7 promoters, and that T7 promoter strength is determined by both the maximum rate of initiation of a promoter and its efficiency of promoter clearance.