Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes*

(Received for publication, October 11, 1996, and in revised form, January 16, 1997)

Srinivas S. Sastry Dagger and Barbara M. Ross

From the Laboratory of Molecular Genetics, Box 174, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.


INTRODUCTION

Transcription by procaryotic DNA-dependent RNA polymerases can be represented as follows (Scheme I).
<B><UP>R</UP></B><UP>NAP</UP>+P <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> RP<SUB><UP>c</UP></SUB><LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>−2</SUB></LL><UL>k<SUB>2</SUB></UL></LIM> RP<SUB><UP>o</UP></SUB><UP> </UP><LIM><OP><ARROW>→</ARROW></OP><UL><UP>NTPs</UP></UL></LIM> R<UP>-DNA-RNA</UP><AR><R><C>↗<UP>RNA</UP></C></R><R><C></C></R><R><C><UP>&drarr;DNA</UP></C></R></AR>
↑                                                         ‖
<UP>--------------------RNAP--------------------</UP>
<OVL>     </OVL><UP>Initiation</UP><OVL>   </OVL><UP>Elongation</UP><OVL>   </OVL><UP>Termination</UP>
<UP><SC>Scheme I. </SC></UP><UP><B>Representation of a transcription cycle.</B></UP>
RNAP1 is RNA polymerase, P is promoter, RPc is a closed complex, RPo is an open complex, k is a rate constant for the various steps, and NTPs are ribonucleoside triphosphates.

Initiation involves binding of RNAP holoenzyme to promoter DNA (RPc) and the isomerization to open complexes (RPo) (for a review, see Ref. 1). Open complexes synthesize short RNAs (up to 10 nts) during abortive initiation. After clearing the promoter, RNAP enters the elongation phase. Termination occurs at certain DNA sequences in either a factor-dependent or a factor-independent manner (2). The current view of transcription elongation has been possible because of our ability to arrest elongation at specific sites on DNA templates, partial purification of arrested complexes, and the enzymatic and chemical probing of their structures (3-17).

The unexpected discovery of an RNA cleavage reaction in arrested Escherichia coli complexes (18) was followed by documentation of analogous cleavage reactions in other RNAPs (19-23). E. coli RNAP and eucaryotic RNAP II are capable of RNA cleavage in binary and ternary complexes (24-26). GreA and GreB of E. coli enhance the intrinsic cleavage by E. coli RNAP (25, 27, 28). GreA and GreB (and a eucaryotic counterpart, SII) prevent elongation arrest (29). GreA may participate in the fidelity of RNA synthesis (30).

T7 RNAP (98.8 kDa) belongs to a class of single-subunit RNAPs that includes T3 and SP6 phage RNAPs (31-33). The three-dimensional structure of T7 RNAP shows high alpha -helicity with a deep cleft. Using a novel photochemical cross-linking technique, we have identified the T7 RNAP cleft as the site of promoter binding (34). The polymerase shows a striking structural similarity to E. coli DNA polymerase Klenow fragment and HIV reverse transcriptase (35). In vitro, T7 RNAP transcribes DNA without additional protein factors. Promoter complexes have been characterized by footprinting and low resolution NMR (36, 37). T7 RNAP has a higher rate of elongation (~250 nts/s) than E. coli RNAP (40-50 nts/s). Elongation complexes are remarkably resilient in their elongating phase. T7 RNAP can transcribe past noncovalently bound proteins (38), DNA triple helices (39), and some base-specific adducts (40), whereas other roadblocks such as psoralens (3, 4), acetoaminofluorine (40), and Z-DNA antibodies (41) can temporarily block elongation. RNA cleavage by bacteriophage T7-like RNAPs has not been demonstrated so far. Part of the problem has been the difficulty of isolating partially purified T7 RNAP elongation complexes in a stable register.

Here, we show that T7 RNAP also exhibits a novel DNA and RNA cleaving property. RNase activity occurs in ternary elongation complexes and in binary complexes. In combination with an unusual 3'-end-initiated polymerase activity, the RNase activity produces heterogeneity in stalled/arrested T7 RNAP elongation complexes.


EXPERIMENTAL PROCEDURES

Nucleic Acids and Proteins

DNA and RNA oligonucleotides were commercially synthesized by automated solid phase procedures. DNAs were purchased from Midland Certified Reagent Co. (Midland, TX), and RNAs were purchased from the Rockefeller University biotechnology facility. The plasmids pBluescript and pBR322 were prepared using standard protocols (42). The concentrations of nucleic acids were calculated from their respective molar extinction coefficients at 260 nm (~104 M-1·cm-1/nt). rNTPs and dNTPs were purchased from Pharmacia Biotech Inc. at a concentration of 100 mM. T4 polynucleotide kinase and calf terminal transferase were from New England Biolabs and Boerhinger Mannheim, respectively. Heparin sulfate was from Sigma. T7 lysozyme and anti-T7 RNAP antiserum were a kind gift from Dr. F. W. Studier (Brookhaven National Laboratory, Upton, NY).

Nuclease Assays

T7 RNAP was prepared locally according to the procedure of Grodberg and Dunn (43) and also by a procedure that was modified from Ref. 44. We also used preparations of T7 RNAP that were prepared by a procedure from the Uhlenbeck laboratory (45). His-tagged T7 RNAP was overexpressed from a plasmid (pBH161) in E. coli cells (BL21). The enzyme was purified using a Ni2+-agarose column. The bacterial strains overexpressing the His-tagged T7 RNAP, the strains containing the insertion mutations in the T7 RNAP structural gene, and the procedure for purification of His-tagged T7 RNAP were a generous gift from Dr. Bill McAllister (State University of New York at Brooklyn). Other sources of T7 RNAP are mentioned under "Results." The concentration of the purified enzyme was determined using epsilon 280 = 1.4 ± 0.1 × 105 (43). For nuclease assays, 2 pmol of 5'- or 3'-end-labeled DNAs or RNAs were mixed with 5-10 pmol of T7 RNAP in a 25-µl reaction containing transcription buffer (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM spermidine, 5% (v/v) glycerol). The reaction mixture was incubated at 37 °C for various lengths of time during which 2- or 4-µl aliquots of the reaction were taken out at different times and mixed with 4 or 10 µl of 8 M urea-Tris borate, 20 mM EDTA plus tracking dyes and heated in a boiling water bath for 5 min and run on a 24% acrylamide, 8 M urea gel (19:1 acrylamide:bisacrylamide). The cleavage reaction was visualized by autoradiography with an x-ray film or phosphor screen. Where necessary, the kinetics of the cleavage reaction was measured by quantitation of the residual full-length 23-mer DNA band or 20-mer RNA band at specific intervals relative to the band representing the starting material, i.e. the 0-min time point. Semiquantitative data were obtained using the ImageQuant program of a PhosphorImager.

Preparation of 66-mer DNA Templates

Two templates were prepared, one unmodified and the other with a psoralen cross-link (between T at the +36 position on the top strand and T at the +37 position of the bottom strand (66XL); see Fig. 7). The 66XL template was prepared as described previously (Ref. 3; see Ref. 46 for methods of adduct preparation). Briefly, a 14-mer furanside monoadduct (CGAAGCTACGAGCA) was ligated to a synthetic, kinased 52-mer (5'-GAGGCCATCGATAAAGGTCTAGATCTCCCTATAGTGAGTCGTATTAATTAGC-3') and a 13-mer (5'-GGCCTCTGCTCGT-3'). The 13-mer DNA served as a ligation "bridge" between the 14-mer and the 52-mer. An equimolar amount of a synthetic 66-mer nontemplate strand was then added and the 66-mer furanside monoadduct bottom strand (the ligation product) was cross-linked to the nontemplate strand with 320-380 nm light. The 66XL was then purified from preparative denaturing gels.


Fig. 7. Sequence of the 66-mer template used for the analysis of elongation complexes. The box represents the polymerase molecule. The arrows show the DNase I footprints (3).
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Analysis of Elongation Complexes

Unmodified 66-mer ds template was constructed from two complementary strands that were synthesized individually and purified by high performance liquid chromatography. The sequence of the template is the same as the one previously used (see Fig. 7 for sequence) (3, 4). To construct an unmodified 66-mer template equimolar amounts of a mixture of the two complementary strands (1 µM each) were heated at 70 °C for 10 min and slowly cooled to room temperature over a period of 2 h. When needed, the template strand was 32P-labeled with the aid of [gamma -32P]ATP and T4 polynucleotide kinase (42). Psoralen cross-linked templates were constructed as described above. The psoralen site specifically cross-linked the T at the +36 position on the top strand with the T at the +37 position on the bottom strand (see Fig. 7). Typical transcription reactions were carried out by first mixing ~80 pmol of template DNA (either 32P-labeled at the 5'-end of the template strand or unlabeled) in transcription buffer containing 1 mM each of cold ATP, GTP, and UTP, cold CTP at 50 µM, [alpha -32P]CTP (specific activity, 3000 Ci/mmol) at 0.3 µM, and 60 units of human placental RNase inhibitor. Transcription was initiated by the addition of 400 pmol of T7 RNAP and incubation at 37 °C. After 15 min of transcription, 300 mM NaCl or 90 µg/ml heparin was added. When necessary, sodium pyrophosphate (Sigma) was added at a final concentration of 2 mM after the addition of NaCl. After 3 min at room temperature, the entire reaction was passed through a Sephadex G-25 spin column. The flow-through was concentrated by SpeedVac centrifugation at room temperature (~10 min) to about 50-100 µl, and glycerol was added to 5% final concentration. The reaction was loaded on an 8% acrylamide nondenaturing gel (14 cm × 16 cm) and run at 10 V/cm. For some reactions, the spin column step was eliminated to achieve a better yield of the complexes. The gel was run until the blank xylene cyanol dye was at the bottom of the gel. C1 and C2 bands were identified by autoradiography of the wet gel, and the respective complexes were eluted from gel pieces by electroelution. The nucleic acids were recovered by precipitation with EtOH and run on a 24% acrylamide, 8 M urea-TBE gel. The sizes of the RNAs were measured using markers generated by diethyl pyrocarbonate plus piperidine (purines) cleavage of DNA oligonucleotides whose sequences are known. Additional markers were a synthetic DNA ladder ranging from 8 to 75 nts. RNA migrated about 2-2.5 nts slower than DNA of the same length on 24% denaturing acrylamide gels.


RESULTS

Single-stranded DNA Cleavage by T7 RNAP

Fig. 1 shows that purified T7 RNAP from overexpressing E. coli cells cleaved a 5'-32P end-labeled 23-mer ss DNA (5'-TAATACGACTCACTATAGGGAAG-3', promoter top strand) and a 20-mer RNA (5'-UUUUUUUUUUCUGACUUAGC-3'). The cleavage occurred from the 3'-end (see below) in steps of mono- and dinucleotides (Fig. 1, lanes 2-8). After 60 min of DNA digestion, major products of 2 or 3 nts were seen. The pattern of DNA and RNA fragments was the same, except that the DNA oligonucleotide was cleaved to smaller products faster than the RNA oligonucleotide. The 20-mer RNA migrates about 2-2.5 nts slower compared with the same-sized DNA. Hence, in Fig. 1, the RNA and DNA bands appear to migrate approximately at the same positions on the high resolution gel (24% acrylamide). To rule out the possibility that the nuclease activity was due to an adventitious contaminant in the enzyme preparations, we purified mutant T7 RNAPs containing insertion and point mutations in the structural gene of T7 RNAP. These mutants were cloned in Dr. McAllister's laboratory (see Refs. 47 and 48 for details). We prepared T7 RNAPs from four mutant strains containing plasmids pLG12, pLLG22, pWJC22, and pCAR27. All four mutant T7 RNAPs were defective in DNA and RNA cleavage to different extents. In Fig. 1 we show an example of a cleavage pattern that was observed with the pLG12 T7 RNAP insertion mutant (MUT). This mutant had a 6-base pair linker insertion within or immediately after codon 566 in bacteriophage T7 gene 1 (the structural gene for T7 RNAP) (47). This mutant was defective in promoter binding and catalysis (47). In our assay, the mutant enzyme was defective in both DNA and RNA cleavage (Fig. 1). Surprisingly, the mutant T7 RNAP behaved somewhat differently with DNA and RNA oligonucleotides. Whereas DNA cleavage occurred to some extent, albeit at a slower rate than with wild type T7 RNAP, RNA cleavage was almost completely absent (Fig. 1). For example, with the mutant T7 RNAP, after 60 min of incubation with ss DNA, bands extending up to 17-mer were visible, the most intense bands being the 11-mer and 12-mer (Fig. 1, MUT, lane 8). Whereas with wild type T7 RNAP, at 60 min a much smaller distribution was observed (Fig. 1, WT, lane 8). This experiment showed that 1) the nuclease activity is indeed due to the polymerase itself; 2) because the overall pattern of cleavage of both ss RNA and ss DNA appears to be the same, a similar mechanism of cleavage probably occurred; 3) the cleavage rates are different for ss DNA versus ss RNA for both wild type and mutant, perhaps due to differential affinities of the polymerase for ss DNA versus ss RNA (indeed, using gel-shift assays, we found that T7 RNAP binds ss DNA better than ss RNA; not shown); and 4) because the mutant T7 RNAP is defective in promoter binding and catalysis (47) and is also defective in our cleavage assay here, cleavage is probably catalyzed by the active center of the enzyme.


Fig. 1. Nuclease activity of T7 RNAP on a 5'-end-labeled ss DNA and ss RNA. The autoradiogram of a 24% acrylamide gel shows the cleavage patterns of DNA and RNA at different intervals of time. Aliquots were removed at indicated time intervals from reaction mixtures containing purified T7 RNAP plus a 23-mer DNA or a 20-mer RNA. Lane 1 shows the oligonucleotides before polymerase addition, whereas lane 2 (0.5 min), lane 3 (1 min), lane 4 (5 min), lane 5 (10 min), lane 6 (20 min), lane 7 (30 min), and lane 8 (60 min) show the oligonucleotides after polymerase addition. WT is wild type polymerase and MUT is mutant polymerase containing an insertion (pLG12; see Ref. 47). The two less intense shorter bands aside from the full-length DNA oligonucleotide band in lane 1 are failure sequences during solid phase synthesis. The presence of these minor bands does not compromise the interpretation of our data.
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Four other T7 RNAP preparations were also tested for nuclease (data not shown): T7 RNAP that was prepared using the procedure described in Ref. 45; His-tagged T7 RNAP that was prepared using a Ni2+-agarose column (bacterial strains and procedure from Dr. W. T. McAllister); T7 RNAP that was purchased from Epicenter Technologies (Madison, WI) (43) and modified by Dr. J. J. Jendrisak (Epicenter Technologies); and T7 RNAP from Dr. J. J. Dunn (Brookhaven National Laboratory, Upton, NY) (43). All these purified preparations displayed the same patterns of cleavage as in Fig. 1. Cleavage activity also occurred on a 66-mer ss DNA or a 66-mer ds DNA with a 3' nucleotide overhang (not shown). All these oligonucleotides contained a T7 RNAP promoter sequence. Nuclease assays using a 5' 32P-end-labeled 34-mer ss DNA without promoter sequence (5'-CGAAGCTACGAGCGGTAGCCATCGATAAATAGCT-3') gave the same result (not shown). Therefore, promoter sequence is not required for T7 RNAP nuclease activity.

To further rule out the possibility of contaminating nuclease in our T7 RNAP preparations we carried out the following experiments. SDS-polyacrylamide gel electrophoresis showed the presence of a single band migrating at ~99 kDa after staining with Coomassie Brilliant Blue (Fig. 2A) or with silver. We estimated that these preparations were >90-95% pure T7 RNAP. When T7 RNAP was run on a native (i.e. without SDS) polyacrylamide gel only two distinct bands were seen after Coomassie Brilliant Blue staining (Fig. 2B). A multimeric complex (MC) was observed. Since T7 RNAP is known to form aggregates in low salt,2 it is possible that a large fraction of T7 RNAP migrates close to a 669-kDa marker apparently as a multimer in native gels containing low concentrations of salt. However, it is difficult to assign a mass to this multimer based solely on native gels. Hence, we shall simply refer to this as a MC. A second, fainter band, apparently corresponding to a monomer (Fig. 2B, M), based on its electrophoretic mobility, was also observed. Because this was a native gel (i.e. without SDS), we are not certain whether band M represented the uncleaved ~99-kDa T7 RNAP molecule or the "nicked" 80-kDa fragment that is often seen in polymerase preparations (43). There was no visible amount of the 80-kDa fragment in our T7 RNAP in SDS gels (Fig. 2A). This is because we prepared T7 RNAPs from E. coli strain BL21 (without Omp T protease; see Ref. 43). Since the same T7 RNAP preparation was run on SDS and native gels, and because the ~20 kDa (~99-80 kDa) was not seen on native gels, we believe that the lower band probably represents a monomer of T7 RNAP. However, for the interpretation of the data below, whether band M represents the full-length monomer or the nicked polymerase is not relevant because the material in band M did not bind DNA.


Fig. 2. In situ assay for nuclease activity. A, 10% acrylamide-SDS gel showing an example of T7 RNAP preparation used in this work. The protein was stained with Coomassie Brilliant Blue. B, native (without SDS) 10% acrylamide gel stained with Coomassie Brilliant Blue. MC is a multimeric complex of T7 RNAP and M is the monomer. S contains native gel standards from Pharmacia. The standards are: 669 kDa, thyroglobulin; 440 kDa, ferritin; 232 kDa, catalase; 140 kDa, lactate dehydrogenase; 67 kDa, albumin. C, binding of 32P-labeled 23-mer to multimeric complex of T7 RNAP. After electrophoresis at 4 °C, the native gel was rinsed at 4 °C by agitating in three changes of transcription buffer for 3 h. The final rinse was discarded, and the gel was shaken overnight at room temperature in 30 ml of transcription buffer containing 32P-labeled 23-mer (1 pmol/ml). The gel was then rinsed three times (15 min each) in transcription buffer and autoradiographed. The MC band was identified by aligning the autoradiogram with the gel after staining with Coomassie Brilliant Blue (as in B). D, denaturing gel showing the isolated DNA from C. Lane 1 contains an aliquot of the 32P-labeled 23-mer DNA from the binding solution used in C. Lane 2 contains DNA eluted from band MC from the gel in C. Lane S contains synthetic DNA oligonucleotide standards.
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To assay for DNA binding we soaked a gel identical to that in Fig. 2B in nuclease assay buffer containing 5' 32P-end-labeled 23-mer DNA. Only the MC band showed a prominent 32P signal (Fig. 2C). Overexposed autoradiograms (not shown) of the gel did not reveal DNA binding to band M. The band representing MC was excised from the gel, and the DNA was eluted and subsequently run on a denaturing 24% acrylamide, 8 M urea gel (Fig. 2D, lane 2). It is clear that cleavage of the 23-mer DNA occurred in the T7 RNAP complex. The in situ cleavage pattern is qualitatively similar to the cleavage pattern in solution (compare Fig. 1, WT, lanes 2 and 3, with Fig. 2D, lane 2). These experiments show that T7 RNAP may exist as a multimer that cleaves 32P ss DNA. Next, we did a corollary experiment to demonstrate that T7 RNAP indeed binds to the ss DNA oligonucleotide in stoichiometric amounts. Fig. 3 is a gel-shift assay demonstrating that increasing amounts of T7 RNAP bind to the 23-nt ss DNA. A single major upper band (U) and two faster migrating minor bands (L) are seen (Fig. 3). The faster migrating shifted bands may represent the 5' cleavage products that are still bound to the enzyme, consistent with the results from the in situ cleavage experiment. Alternatively, they may be faster migrating conformational isomers of the enzyme-ss DNA complexes. These experiments demonstrate that T7 RNAP binds and cleaves ss DNA in a stepwise manner reminiscent of a 3' to 5' exonuclease. Gel-shift assays with 5' 32P-end-labeled 20-mer RNA also showed that T7 RNAP was bound to RNA in binary complexes (not shown).


Fig. 3. Gel-shift assay for DNA binding to T7 RNAP. 32P-Labeled 23-mer DNA was mixed with T7 RNAP at different molar ratios of T7 RNAP:DNA in transcription buffer. The reaction mixture was incubated at 37 °C for 10 min and made 5% glycerol. The reaction was loaded on an 8% acrylamide, TBE gel (20 cm × 20 cm) and run at room temperature (7 V/cm). The gel was run until the bromphenol blue dye was 9 cm from the bottom of the gel and then fixed in 5% methanol, 5% acetic acid, 3% glycerol for 20 min, dried, and autoradiographed. 0 indicates no polymerase. 1, 3, 5, and 15 indicate -fold excess of T7 RNAP over DNA.
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Additional criteria were used to show that the cleavage activity is a property of T7 RNAP. 1) The kinetics of heat inactivation of T7 RNAP at 70 °C indicated that the destruction of nuclease activity paralleled the destruction of transcription activity (not shown). 2) Amino acid composition analyses of two of our preparations matched the known amino acid composition of T7 RNAP. 3) Western blotting of gel shifts of binary complexes of T7 RNAP and 23-nt DNA (such as those in Fig. 3) and transcription complexes (see Fig. 8) with anti-T7 RNAP antibody indicated that these complexes contained T7 RNAP. 4) DNA cleavage activity during T7 RNAP purification coincided with T7 RNAP peak fractions from the cation-exchange column.


Fig. 8. Gel-shift assay for the isolation of T7 RNAP elongation complexes paused at the end of a template (A) or those arrested by a psoralen cross-link (B). RNA was labeled with [alpha -32P]CTP. See "Experimental Procedures" for details. For a better band resolution, we ran the free RNA off the gels (down-arrow ). A, unmodified 66-mer was the template. B, the template was a psoralen cross-linked 66-mer DNA. C, high resolution gel analysis of RNAs in end-paused elongation complexes. Lane C1 contains 32P-labeled RNA isolated from NaCl-treated complexes. Lane C2 contains 32P-labeled RNA isolated from heparin-treated complexes. RF is an aliquot of labeled RNA from transcription reactions before spin column gel separation. D, high resolution gel analysis of RNAs in elongation complexes arrested at a site-specific psoralen cross-link. Lanes C1 and C2 contain 32P-labeled RNA isolated from untreated or NaCl-treated complexes.
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ss DNA Cleavage Activity Starts at the 3'-End and Is Mg2+-dependent

When the 23-mer ss DNA was 32P-labeled at the 3'-end, the nuclease immediately released a mononucleotide (Fig. 4). This was deduced using 32P-labeled dinucleotide ([alpha -32P]GpG) and 32P-labeled ATP as markers on a 24% acrylamide denaturing gel. The product size remained the same even after prolonged incubation. This indicated that the initial product of the cleavage is probably a mononucleotide. (Within the scope of the present paper we do not think it is necessary to unambiguously identify this product.) The same result was seen with 3' 32P-end-labeled 34-mer nonpromoter ss oligonucleotide DNA (not shown). Putting together the 5'- and 3'-end labeling experiments, T7 RNAP nuclease acts by a stepwise removal of nucleotides starting from the 3'-end and going toward the 5'-end with the limit product at the 5'-end being 3-5 nucleotides. That cleavage left a 3'-OH end was verified by 32P labeling the cleaved unlabeled 3-mer and 20-mer with 3' terminal transferase and [alpha -32P]ddATP after the nuclease assay. Omission of MgCl2 or inclusion of molar equivalents of EDTA with Mg2+ blocked cleavage, indicating complete Mg2+ dependence of the nuclease (not shown).


Fig. 4. Cleavage of 3'-end-labeled 23-mer DNA by T7 RNAP. The 23-mer DNA was labeled at its 3'-end with [alpha -32P]ddATP (+1) and terminal deoxynucleotidyl transferase. Cleavage reactions were carried out as described under "Experimental Procedures." 0 indicates no polymerase. 0.5, 2, 5, and 10 indicate min after addition of polymerase.
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Nuclease Activity Is Inhibited by T7 Lysozyme or Heparin

A 20× excess (w/w) of T7 lysozyme over T7 RNAP or heparin (250 µg/ml) inhibited the rate of nuclease activity (Fig. 5). After 5-30 min of incubation with T7 lysozyme, 21-mer and 20-mer or 10-12-mers were still seen, whereas without lysozyme the DNA was cleaved faster to much shorter products. Prolonged incubation with these reagents showed smaller products, indicating that these reagents did not completely block nuclease activity. The inhibition by T7 lysozyme or heparin was concentration-dependent (not shown). Half-maximal inhibition by heparin was at 50 µg/ml. T7 lysozyme is a specific inhibitor of T7 RNAP (49, 50), again indicating that T7 RNAP is the source of nuclease activity. Because the polyanion heparin is a competitive inhibitor of DNA binding, DNA binding is a prerequisite for T7 RNAP nuclease activity.


Fig. 5. Inhibition of cleavage activity by T7 lysozyme and heparin. T7 lysozyme or heparin were mixed with labeled 23-mer DNA in transcription buffer. To this mixture, kept at 37 °C, T7 RNAP was added, and aliquots were removed at different intervals of time and run on 24% acrylamide-urea denaturing gel. 0 indicates no polymerase. 5, 15, and 30 indicate min after addition of polymerase. None indicates reactions that contained neither lysozyme nor heparin.
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Polymerase Activity Suppresses Nuclease Activity

Addition of rNTPs suppresses the nuclease activity while unmasking an unusual polymerase activity (compare lanes without NTPs and with rNTPs, Fig. 6). The 3'-end extension of DNA (or RNA, not shown) by T7 RNAP with rNTPs has been observed previously (51-53). Prolonged incubation in the presence of rNTPs shows evidence of nuclease activity on the 40-mer DNA-RNA chimeras (e.g. see 30-min lane of Fig. 6). Consistent with T7 RNAP being the nuclease, dNTPs do not elicit the same effect as rNTPs (Fig. 6). This showed that the active site of T7 RNAP is involved in nuclease activity, consistent with our earlier results with mutant T7 RNAPs (Fig. 1). Fig. 6 shows that nuclease activity and polymerase activity can manifest independently. In the presence of NTPs, polymerase activity predominates over nuclease activity (see below).


Fig. 6. Polymerase activity suppresses nuclease activity in the presence of rNTPs. Reaction mixtures contained either rNTPs or dNTPs before the addition of T7 RNAP. 0 indicates no polymerase. 1, 5, and 30 indicate min after addition of polymerase.
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Nuclease Activity Is Absent during Normal Transcription

The plasmid pBluescript (Stratagene Corp., San Diego, CA) contains a class III promoter but not a T7 terminator. In the presence of T7 RNAP and rNTPs, a large amount of RNA was produced by multiple rounds of transcription, indicating that our T7 RNAP was transcriptionally active. No small cleavage products originating from the DNA were seen. In the absence of NTPs, no cleavage of the plasmid was seen either (data not shown). To confirm that no cleavage of the plasmid occurred, we isolated the DNA from gel bands from agarose gel and restricted the DNA with HindIII and PstI separately. Re-examining the restricted DNA separately on agarose and denaturing polyacrylamide gels revealed no indication that T7 RNAP cleaved the plasmid with or without NTPs. We observed similar results with the plasmid pBR322 that has no consensus T7 promoter, although in this case very little RNA was made. We examined transcription initiation on a short oligonucleotide template containing a 23-base pair core promoter sequence (5'-TAATACGACTCACTATAGGGAAG-3'). In the presence of GTP as the sole NTP, T7 RNAP produces a G-ladder that extends to ~14 nts and then tapers off (71). The G-ladder is thought to be produced by multiple rounds of slippage synthesis at the three cytidines in the promoter (54). The G-ladder accumulated in a time-dependent manner, indicating that the ds template was not destroyed and the RNA was not degraded (71). We also examined transcription on an unmodified 66-mer DNA template (71). During abortive transcription, there was a time-dependent accumulation of transcripts but no evidence of template cleavage (71). These observations indicate that in the presence of NTPs during normal transcription no T7 RNAP DNase activity is seen.

Isolation of Elongation Complexes

Next, we examined RNA cleavage activity in isolated ternary elongation complexes. Because T7 RNAP elongation complexes are relatively unstable (dwell times of ~5-15 min) we were not able to "walk" discrete and isolated T7 RNAP elongation complexes in a manner that was done with E. coli and eucaryotic RNAP elongation complexes. To circumvent these problems, an alternative protocol (albeit a less "clean" strategy compared with traditional walking methods) to examine the RNA component in elongation complexes was developed. Transcription was initiated by the addition of T7 RNAP to a synthetic oligonucleotide template in the presence of NTPs. Following 15 min of transcription, NaCl (to 300 mM) or heparin was added. Addition of high salt or heparin disrupted all existing promoter complexes and inhibited new rounds of initiation. Footprinting and other experiments have shown that 300 mM NaCl disrupted promoter-initiation complexes but not elongation complexes (3, 4, 55-57). To isolate stable complexes, we first passed the transcription reactions through a Sephadex G-25 spin column to remove NTPs and other low molecular weight components. The radiolabeled complexes were resolved on a low percentage nondenaturing acrylamide gel. The 32P label was present either in the nascent RNA or on the 5'-end of the template strand. To ensure that the resolved complexes seen on nondenaturing gels contained T7 RNAP, we probed the complexes with anti-T7 RNAP antiserum using Western blotting techniques (not shown). The same bands that were visible in autoradiograms when the RNA or the DNA templates were labeled with 32P were also recognized by anti-T7 RNAP antibody, attesting that the complexes indeed contained T7 RNAP. Omission of the spin column step increased the yield of the complexes without changing the pattern of bands on the nondenaturing gel. Following autoradiography of the native gels, the radioactive RNAs were recovered and subsequently resolved on a 24% denaturing acrylamide gel (see "Experimental Procedures"). This protocol selected high salt or heparin-resistant stable elongation complexes.

We examined the RNA composition in two different T7 RNAP ternary complexes: 1) elongation complexes apparently paused at the end of an unmodified 66-mer ds DNA template; and 2) elongation complexes arrested by a psoralen cross-link site specifically placed toward the 3'-end of the same 66-mer (66XL) template (3).

Cleavage Reactions in End-paused Elongation Complexes

Fig. 7 shows the sequence of the 66-mer DNA for examining end-paused and psoralen-arrested complexes. Footprinting demonstrated that elongating T7 RNAP, as indicated by the box in Fig. 7, was blocked by the psoralen cross-link (3). Here, we made the unmodified as well as the psoralen cross-linked template to study cleavage and end-polymerization reactions. On the unmodified 66-mer template, the elongation complexes paused at the end of the template (end-paused complexes). The position of the polymerase molecule in end-paused complexes is suggested by the box in Fig. 7. Although we have not footprinted the end-paused elongation complexes, we assume that these complexes occupied a similar overall position at the end of the template as the psoralen-arrested complex (Fig. 7). This assumption is justified because the majority of the RNA in the isolated complexes is a runoff transcript (43 nts; Fig. 8C).

Using the protocol described in the previous section, we examined the 32P-labeled RNA in end-paused and psoralen-blocked complexes. Fig. 8A (with unmodified template) and 8B (with cross-linked template) show complex 1 and complex 2 with different mobilities. Identical bands were seen when only the DNA was labeled (not shown). From the specific radioactivity of [32P]RNA or [32P]DNA in gel bands seen in the absence of high salt, we estimated that complexes contained approximately an equimolar ratio of DNA and RNA. Two bands were seen after addition of 300 mM NaCl, which was added after 15 min of transcription. Only elongation complexes were stable during gel electrophoresis, and they have different mobilities on nondenaturing gels. When heparin was added, only complex 2 was visible. This is in agreement with previous observations that heparin caused disruption of some elongation complexes (4, 58). The ratio of [32P]CMP-labeled C1 to C2 with 66XL was almost equal (Fig. 8B), whereas with unmodified 66-mer ds DNA template, C1 accounted for >90% of the total radioactivity (Fig. 8A). Clearly, the presence of the psoralen cross-link influenced the relative populations of C1 and C2. The basis for this difference is unknown.

Examination of 32P-labeled RNA in NaCl- (C1) or heparin-resistant (C2) complexes revealed a heterogeneous pattern of transcripts (Fig. 8C). In C1, the major cleavage products were of 12, 17, 20, 26, and 31 nts, etc., and were interspersed with minor products with 1- or 2-nt differences. Longer than +1-initiated transcripts (up to ~60-mers) were also seen. These transcripts were not due to end initiation (or internal initiations at sites other than +1) because they were observed even when [alpha -32P]GpG was used, which forces T7 RNAP to correctly initiate at +1 (not shown). The 12-nt transcripts are not abortive transcripts because the latter are usually only up to 10 nts and are generally more abundant relative to the longer RNAs. Moreover, just after addition of NaCl or heparin and prior to passage through the spin column, most of the complexes were end-paused, as indicated by the abundance of the 43-nt runoff species (Fig. 8C, lane RF). We believe that the heterogeneity is due to RNA cleavage and end-polymerization reactions analogous to those seen earlier (see Figs. 1 and 6). Heterogeneity cannot be due simply to the trapping of polymerase complexes in various registers of elongation by the spin column/gel. This would have produced a more or less stochastic distribution of RNAs. There are other compelling reasons to eliminate the latter explanation. 1) Addition of 300 mM NaCl or heparin precluded any further polymerase binding after 15 min of transcription. 2) Even in heparin-resistant complexes (C2 in Fig. 8C) the cleavage and end-extended RNAs are observed. In fact, more or less the same pattern (down to 22 nts) is seen with both NaCl and heparin (because of the lower yield of heparin-resistant complexes, the band intensities were lower). 3) Furthermore, examination of the transcription products before spin column/gel purification (Fig. 8C, lane RF) showed RNAs that were overwhelmingly in the larger sizes (35-43 nts), indicating that just before spin column passage most of the complexes in solution were close to the end of the template. Because the NTPs were removed after the passage through the spin column gel, RNA cleavage and end polymerization must have occurred prior to the spin column step and/or before native gel electrophoresis. The high salt- or heparin-resistant heterogeneous complexes were trapped by the gel. We believe the heterogeneity of transcripts is due to a fraction of the end-paused complexes undergoing end-polymerization and cleavage reactions.

Cleavage Reactions in Psoralen Cross-link-arrested Elongation Complexes

Next, we examined the RNA composition of T7 RNAP complexes arrested by an authentic roadblock (psoralen cross-link) (3, 56). Fig. 8D shows that the patterns of cleavage of nascent RNA transcripts from C1 and C2 complexes is quite different from the end-paused ones on noncross-linked template (Fig. 8C). Surprisingly, RNA transcripts were extremely heterogeneous in both C1 and C2, occurring in a wide range in 1- or 2-nt steps (11-28 nts; Fig. 8D). The pattern of cleavage is reminiscent of that in Fig. 1. Transcripts extending up to ~75 nts were seen in abundance in C2. The 36-mers expected from transcription up to the psoralen cross-link were only a minor species. Longer RNAs than those specified by +1 initiation were due to RNA 3'-end polymerization, whereas the shorter RNAs were due to cleavage. Pyrophosphorolysis is the chemical reversal of nucleotide incorporation by nucleic acid polymerases. Pyrophosphorolysis is usually accomplished in vitro by millimolar concentrations of PPi (13, 23, 59-61). We did not observe any significant differences in the pattern of transcripts from psoralen-arrested elongation C1 and C2 complexes following addition of PPi, suggesting that PPi has no effect on RNA cleavage. In general the yield of complexes was lower in the presence of NaPPi (data not shown).


DISCUSSION

Nuclease Activity of T7 RNAP

We have discovered that T7 RNAP prepared from overexpressing E. coli cells exhibits DNase and RNase activity. This is a new enzymatic activity of T7 RNAP. Many experiments have ruled out the probability of other contaminating nucleases in our T7 RNAP preparations (see "Results"). Moreover, T7 RNAP prepared using different methods and from different sources exhibited nuclease activity. We cannot completely rule out the possibility that a low molecular weight protein factor (analogous to GreA) is tightly bound to T7 RNAP. It is, however, highly unlikely that there was an adventitious contaminant nuclease unrelated to T7 RNAP. Hydrolytic cleavage of RNAs has been documented with all tested RNAPs. These include E. coli RNAP, higher eucaryotic pol II and pol III, vaccinia RNAP, and yeast RNAP (see Introduction for Refs.). In at least two cases (E. coli and yeast pol II) the cleavage of RNA has been demonstrated in binary complexes as well as in ternary complexes. Here, both binary and ternary complexes of T7 RNAP exhibit nuclease activity. The DNase activity of T7 RNAP leaves a 3'-OH end and a 5'-PO4 and is absolutely dependent on Mg2+. The nuclease appears to be a 3' to 5' exonuclease on single-stranded oligonucleotide. Although we have not isolated and analyzed the first cleavage product to the 3'-end, based on the pattern of cleavage of 5'-end- or 3'-end-labeled DNA and RNA we can only surmise that the cleavage occurs in mono- or dinucleotide steps (Figs. 1 and 4). The 5' cleavage products appear to be bound to the enzyme as indicated by the results from the in situ cleavage assay (Fig. 2). Multiple T7 RNAP molecules may be involved in the cleavage of each DNA (distributive mechanism) as indicated by the binding stoichiometry (Fig. 3) and by the fact that only the multimeric form of the T7 RNAP showed binding and cleavage of oligonucleotide DNA (Fig. 2). Since T7 RNAP nuclease cannot act on covalently closed circular plasmid DNA or linear oligonucleotide ds DNA, it is not an endonuclease. We could not detect T7 RNAP nuclease activity on linear blunt ended ds DNA oligonucleotides in the absence of transcription. The time course of T7 RNAP RNase activity is slower than that of the DNase. We believe that DNase and RNase activities are carried out by the same catalytic pocket because the cleavage ladders from a 5'-end-labeled DNA or RNA are of the same pattern, in both cases polymerase activity competes with and suppresses nuclease activity, and T7 RNAP mutants are defective in cleavage. It is possible that the same binding site can accommodate both ss RNA and ss DNA. This proposal is consistent with the ability of T7 RNAP to strongly bind ss DNA and ss RNA oligonucleotides (5, 62) (Fig. 3). Recently, one of us (S. S.) has shown by photochemical cross-linking that the ss DNA (and also perhaps ss RNA) binding activity lies in the fingers (or fingers-palm junction) domain (34).

Previous reports suggest that 3'-end NMP addition to RNA was due to the presence of a self-complementary secondary structure or partial intermolecular complementarity in the nascent RNAs (51, 53). These authors suggested that T7 RNAP binds to free RNA in transcription reactions to form binary complexes in which end polymerization occurred. In our experiments, we have eliminated this possibility by a multistep procedure. 1) We passed the transcription reaction through a Sephadex spin column to remove NTPs. 2) We ran the complexes on native polyacrylamide gels. In this step, we have carefully identified ternary complexes by 32P labeling either the DNA or RNA and ensuring that the complexes contained T7 RNAP using Western blotting with anti-T7 RNAP antibody. We also made sure that when the template DNA or RNA were independently 32P-labeled, the reactions were run on the same gel and bands with identical mobilities were recovered from the gels. 3) We recovered the complexes from gel pieces by electroelution and then ran the radiolabeled RNAs from disrupted complexes on high resolution denaturing acrylamide gels. These procedures assured us that the complexes isolated from native gels were indeed ternary complexes. Some elongation complexes are resistant to heparin, whereas others are not (4, 55, 58, 63). Therefore, we believe that the observed cleavage and end-polymerization reactions occurred in ternary transcription complexes and not simply in binary complexes of free RNA and DNA in transcription reactions. During elongation, ternary complexes of T7 RNAP are highly heterogeneous. This heterogeneity is clearly seen in isolated elongation complexes paused/arrested by a psoralen roadblock or at the end of a template (Fig. 8). End pausing has been observed with eucaryotic pol II during transcription on blunt-ended templates (64).

Models for Cleavage and Unusual Polymerization during Elongation and the Heterogeneity of Complexes

Fig. 9A is a diagrammatic representation of an elongation complex paused at the end of the template or arrested by a psoralen roadblock. The RNAP encloses a "bubble" with a 3-base pair (or up to 7-base pair) RNA-DNA hybrid (3, 37, 55, 65, 71). The size of the bubble and hybrid in elongation complexes of T7 RNAP have not been directly measured. However, they can be inferred from published reports (55, 71). A fraction of the paused/arrested polymerases may "back up" (Fig. 8B). Backup of the polymerase was probably facilitated by the inhibition of enzyme turnover by high salt/heparin. Backing up may be associated with conformational rearrangements in the catalytic core of RNAP. The 3' nascent RNA product binding site in the arrested/paused RNAP may be reorganized such that 3'-end RNA-DNA contacts are lost but the 3' distal contacts are maintained (Fig. 8B). A part of the 3'-end is now partially single-stranded and is held in a new RNAP site. The binding of the RNA in the new RNAP site may facilitate or effect cleavage (Fig. 8B). Recent work with E. coli RNAP indicated that RNA product binding site reorientation and conformational changes occur in paused and arrested complexes (67). We visualize a stalled/paused T7 RNAP as having two activities, viz. 3'-end-initiated polymerization (Refs. 51, 53, and 66, and this work) and 3' to 5' RNase activity (this work). These are opposing activities that generate heterogeneity in paused/arrested complexes. Although the catalytic pocket is probably the site of both nuclease and polymerase activities, in the presence of NTPs the polymerase activity is kinetically dominant. This explains why addition of NTP to the ss DNA oligonucleotide suppresses the nuclease (Fig. 6) and why during normal processive transcription the nuclease activity is not observed. The nuclease activity is only kinetically unmasked when the polymerase is paused or arrested by a roadblock.


Fig. 9. Model for events leading to transcription arrest/pause and subsequent cleavage/polymerization reactions. A-C represent stages in the transcription cycle. See text for a description.
[View Larger Version of this Image (20K GIF file)]


Why does cleavage of ss RNA oligonucleotide occur with higher than a 1:1 molar ratio of enzyme to RNA, whereas cleavage in ternary elongation complex occurs at an equimolar ratio? The two events, binding and catalytic cleavage, occur in that sequence. In paused/arrested ternary elongation complexes, the RNA is held very tightly by the enzyme. The RNA and RNAP in these complexes are not in equilibrium with free RNA and free polymerase, unlike in binary complexes, where they are in equilibrium with free RNA and free RNAP. Hence, higher concentrations of polymerase are required to drive by mass action the equilibrium toward binding and cleavage in binary complexes.

Role of Nuclease Activity

Comparison of the T7 RNAP nuclease activity with the known nuclease activity of other RNAPs or HIV reverse transcriptase reveals many differences and some similarities. At first glance, the ss DNA-ss RNA nuclease activity of T7 RNAP appears to be somewhat different from the known GreB-enhanced intrinsic cleavage activity of E. coli RNAP. The GreB-enhanced E. coli RNAP cleaves the nascent RNA in arrested/paused ternary complexes into fragments larger than T7 RNAP (24, 25). The T7 RNAP nuclease pattern resembles that of GreA-enhanced E. coli RNAP, eucaryotic pol II, or vaccinia RNAP in that these enzymes seem to cut in mono- or dinucleotide steps (23, 61). T7 RNAP cleavage is reminiscent of SII-mediated "minor" mode cleavage in yeast pol II binary complexes (26) because cleavage starts at the 3'-end releasing 1-3 nts at a time. It is not known if E. coli RNAP or the eucaryotic RNAPs have a single-stranded DNase activity. T7 RNAP nuclease activity resembles the HIV reverse transcriptase nuclease in that arrest of template-specified polymerization is needed (72). The dual activities, viz. the 3'-end-initiated nontemplated polymerization and the nuclease activity, are common to T7, E. coli, and pol II RNAPs and to HIV reverse transcriptase (73). Other common features are the complete requirement for a divalent cation and the apparent independence from pyrophosphorolysis. Although we have not demonstrated that cleavage is accompanied by a backward movement of the T7 RNAP from the end of the template or from a stalled site, the patterns of the cleavage sites (Fig. 8) are consistent with this possibility. Because of the relative instability (dwell times of <5-10 min) and heterogeneity of T7 RNAP elongation complexes, it is technically difficult or perhaps impossible to walk T7 RNAP from one register to another in isolated homogeneous complexes as was done with E. coli RNAP. In the case of E. coli RNAP and eucaryotic pol II, it has been speculated that hydrolytic nuclease activity may be an error-correcting mechanism or a way of rescuing paused/arrested complexes. Similar mechanisms may operate during T7 RNAP transcription. What role might the ss DNase activity play in vivo? During T7 phage DNA replication in vivo, linear T7 DNA concatemers are formed through base pairing of terminal repeats. This is accompanied by digestion of surplus single-stranded DNA (68). The mechanisms of these processes are not clearly understood. We speculate that digestion of surplus single-stranded DNA serves two functions: 1) production of dNTPs to augment cellular pools that are channeled for rapid T7 DNA replication; and 2) reduction of the competition from ss DNA for T7 DNA polymerase activity (69). There are three known nucleases produced by the T7 phage. 1) Gene 6 exonuclease (5' to 3'), which is normally involved in removal of primers after lagging strand synthesis, in recombination, and in the destruction of bacterial DNA. This is a ds DNA-specific exonuclease (70). 2) Gene 3 endonuclease, which is specific to single-stranded DNA and may be a resolvase involved in homologous recombination. 3) 3' to 5' exonuclease of T7 DNA polymerase, which is an error-correcting activity during T7 DNA synthesis. T7 RNAP nuclease, the fourth nuclease activity, may serve as an additional nuclease in the digestion of the surplus single-stranded DNA during T7 DNA replication and maturation. Since T7 RNAP nuclease is a single-stranded 3' to 5' exonuclease and is suppressed during normal transcription elongation, the double-stranded form of T7 DNA would not be its target.


FOOTNOTES

*   This work was supported in part by a Louis B. Mayer Foundation grant and a Hewlett-Packard Company Foundation grant for a high pressure liquid chromatography work station and related service contracts. This work was presented at a FASEB Summer Research Conference, July, 1995.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.
Dagger    A Louis B. Mayer Foundation Fellow. To whom correspondence should be addressed: Laboratory of Molecular Genetics, Box 174, The Rockefeller University, New York, NY 10021. Tel.: 212-327-8987; Fax: 212-327-8651; E-mail: sastrys{at}rockvax.rockefeller.edu.
1   The abbreviations used are: RNAP, RNA polymerase; T7 RNAP, bacteriophage T7 RNA polymerase; nt(s), nucleotide(s); XL, interstrand psoralen-DNA cross-link; TBE, 180 mM Tris borate, pH 7.5, 2 mM EDTA buffer; HIV, human immunodeficiency virus; ss, single-stranded; ds, double-stranded; MC, multimeric complex; pol II, polymerase II; GpG, guanylyl-3'-5'-guanosine phosphate; NMP, nucleoside monophosphate.
2   J. J. Dunn, personal communication.

ACKNOWLEDGEMENTS

We thank Prof. F. W. Studier for the kind gifts of anti-T7 RNAP antisera and T7 lysozyme and Dr. Bill McAllister for bacterial strains and suggestions. We appreciate the very helpful suggestions from Profs. Mike Chamberlin and Charles Richardson. We also thank Dr. Stewart Shuman for critical comments on the manuscript and Prof. Joshua Lederberg for interest in the project.


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