INTRODUCTION

The two largest subunits of Escherichia coli RNA polymerase, (M_r 155,063) and  (M_r 150,538) are homologous to the largest and second largest subunits, respectively, of all multisubunit RNA polymerases (Allison et al., 1985; Briggs et al., 1985; Falkenberg et al., 1987; Hudson et al., 1988; Leffers et al., 1989; Patel and Pickup, 1989). Most catalytic and regulatory functions of RNA polymerase appear to involve these subunits. Thus  and are likely to form an enzymatic platform for RNA synthesis common to all forms of life, making elucidation of structure/function relationships in these subunits in a well studied prokaryote an ideal approach to understanding the mechanism of transcription. Sequence comparisons and recent crystallographic studies of single subunit DNA and RNA polymerases reveal broadly similar catalytic regions for both classes of enzyme (Steitz et al., 1994; Pelletier et al., 1994; Beese et al., 1993; Sousa et al., 1993; Kohlstaedt et al., 1992). Although E. coli RNA polymerase has proven refractory to study by x-ray crystallography, owing principally to its large size and multisubunit complexity, low-resolution electron crystallography of E. coli RNA polymerase and yeast RNA polymerase II suggests that the multisubunit RNA polymerases also share this basic architecture, most notably a central nucleic acid binding channel large enough to encompass the DNA double helix (Darst et al., 1989, 1991; Polyakov et al., 1995). Most progress in identifying functionally important regions of the enzyme, however, has come through study of altered function mutants and sites of cross-linking between the subunits and nucleotide analogs.

Of the three core subunits, most is known about , largely because it is the target of a widely studied antibiotic that inhibits transcription, rifampicin (Rif).¹ Rif^r mutants in particular have been a focus of study for decades since they exhibit a number of pleiotropic phenotypes, most notably effects on pausing and termination (Fisher and Yanofsky, 1983; Jin et al., 1988). Several more recent genetic studies have identified sites throughout rpoB, generally in evolutionarily conserved regions, that affect pausing and termination (Landick et al., 1990; Tavormina et al., 1996). In addition, substitutions in  have been isolated that suppress substitutions in Rho and NusA, suggesting that  is a site of functional, and possibly physical interaction with these transcriptional regulators (Guarente and Beckwith, 1978; Guarente, 1979; Jin and Gross, 1989; Ito et al., 1991; Ito and Nakamura, 1993). Others have used reverse genetics and high resolution cross-linking to identify domains in  that affect a number of aspects of polymerase function (Grachev et al., 1987, 1989; Mustaev et al., 1991, 1995; Kashlev et al., 1990; Lee et al., 1991; Martin et al., 1992) and thus suggest the functional importance of highly conserved regions.

Several lines of evidence suggest that the subunit also is intimately involved in RNA chain synthesis. Early physical studies showed that purified subunit is capable of binding DNA and heparin (Zillig et al., 1970), and several cross-linking studies have demonstrated that is in the immediate vicinity of short and intermediate length transcripts (Hanna and Meares, 1983; Dissinger and Hanna, 1991; Borukhov et al., 1991a), as well the DNA template (Okada et al., 1978; Chenchik et al., 1982). Substitutions in affecting termination, suppression of nusA and rho mutants, DNA replication, and phage growth have been described, although, with a few exceptions, the particular amino acids affected have not been identified (Nomura et al., 1984; Rasmussen et al., 1983; Tanaka et al., 1983; Petersen and Hansen, 1991; Robledo et al., 1991). More recently we reported several clusters of substitutions in conserved regions of that alter pausing and termination (Weilbaecher et al., 1994), streptolydigin-resistance was found to result from amino acid substitutions in one of these clusters that corresponds to the amanitin-resistance region of the largest subunit of RNA polymerase II (Severinov et al., 1995), and conserved aspartic acid residues that appear to chelate active-site Mg²⁺ ions were identified using the homolog in yeast RNA polymerase III (Dieci et al., 1995).

To understand the roles of  and in transcription better, we have studied mutations isolated as allele-specific suppressors of rho201 by Guarente (1979) and mapped to the rpoBC locus. These mutations were localized to either rpoB or rpoC in a previous study and renamed rpoB211, rpoB212, and rpoC214 (Jin and Gross, 1989). Unlike the rpoB8(Rif^r) mutant that was also isolated as a suppressor of rho201 (Guarente and Beckwith, 1978) as well as independently by several other criteria (reviewed in Jin and Gross (1988)), these mutations do not cause general defects in termination, but rather alter termination only subtly at a few -dependent and -independent terminators (Jin and Gross, 1989).

To continue this investigation, we identified the precise amino acid substitutions in rpoB211, rpoB212, and rpoC214, finding that both rpoB211 and rpoB212 specify N1072H and that rpoC214 specifies R352C, and purified both mutant RNA polymerases. A current model of Rho-dependent termination (Jin et al., 1992; Platt, 1994) postulates that the extent of termination is determined by ``kinetic'' coupling between extrusion of the nascent RNA by the elongating RNA polymerase molecule and movement of Rho along the RNA toward the complex. That this relationship is the basis of at least some modes of rho-suppression was demonstrated for rpoB8(Q513P): Q513P RNA polymerase elongates at a significantly slower rate than wild type, and its hypertermination defect is correlated to its elongation rate (Jin and Gross, 1991). We wanted to determine whether a reduction in elongation rate was a plausible basis of isolation of these RNA polymerase mutants, or if they suppress rho201 by a different mechanism (such as disruption of interaction with Rho). If these mutants are defective in transcription itself, we wanted to ask whether they and Q513P exhibit similar or distinct transcriptional defects.

Here we show that N1072H and R352C elongate more slowly in vitro and argue, based on their effects on abortive initiation and open complex stability, that they probably do so by principally affecting distinct interactions of RNA polymerase with different components of the transcription complex.

MATERIALS AND METHODS

Chemicals and Enzymes

Adenosyl 5-uridine (ApU) and isopropyl--D-thiogalactopyranoside were from Sigma. -ANS-UTP was a kind gift of M. Thomas Record. NTPs were purchased from Boehringer Mannheim or Pharmacia (high performance liquid chromatography-purified). Radioactive [-³²P]UTP (800 Ci/mmol) and [-³²P]ATP (3000 Ci/mmol) were obtained from DuPont NEN, [-³²P]CTP (410 Ci/mmol) was from Amersham. Polynucleotide kinase and restriction enzymes were from New England BioLabs. Sequenase Version 2.0 was purchased from U. S. Biochemical Corp. AmpliTaq was obtained from Perkin-Elmer Instruments and Tfl polymerase from Epicentre.

Single-stranded DNA-agarose and Q-Sepharose FF were purchased from Pharmacia and S-300HR from Sigma. Magic Miniprep columns were from Promega. Nitrocellulose filters (BA85, 24 mm, 0.45-m pore size were purchased from Schleicher and Schuell.

Bacterial Strains, Plasmids, and Bacteriophage

The bacterial strains used in this study are E. coli K12 derivatives and are listed in Table I. Plasmids and bacteriophage used in this study are listed in Table II. T7D111 phage stock was from Studier and phage DNA was prepared as described (Studier, 1975).

Table I. Strains used in this study Strain Relevant genotype Source/reference MG1655 Wild-type K12 CGSC MG1655rpoB211 btuB::Tn10 rpoB211 Jin and Gross (1989) MG1655rpoB212 btuB::Tn10 rpoB212 Jin and Gross (1989) MG1655rpoC214 btuB::Tn10 rpoC214 Jin and Gross (1989) CAG14179 MG1655 +pPLT13 +pJH76 This work CAG14180 CAG 14179rpoB211 This work CAG14181 CAG 14179rpoC214 This work RL676 W3110 rpoC::S. aureus protein A, trpR tnaA2 zja::kan (recA-srl)306 srl-301::Tn10 Weilbaecher et al. (1994) RL3215 RL676 pRW308(S350F) This work RL3216 RL676 pRW308(G351D) This work

Table II. Plasmids used in this study Plasmid Relevant markers Source/reference pCL185 Contains T7A1 promoter with modified downstream sequence at +17 from start site of transcription Feng et al. (1994) pJH76 Contains rpoD under control of Ptac on pBR322 Hu (1987) pPLT13 Contains lacIq on mini F Tavormina (1994) pRW308 Contains rpoC expressed from Ptrc and controlled by lac repressor encoded on the same pBR322-based plasmid Weilbaecher et al. (1994) pRL611 Derivative of pBR322 carrying only rpoC codons 1-292 This work; see also Clerget et al. (1995) pRL612 As pRL611 with codons 293-544 This work; see also Clerget et al. (1995) pRL613 As pRL611 with codons 546-876 This work; see also Clerget et al. (1995) pRL614 As pRL611 with codons 878-1214 This work; see also Clerget et al. (1995) pRL615 As pRL611 with codons 1214-1407 This work; see also Clerget et al. (1995) pRL616 As pRL611 with codons 293-544 (opposite orientation of pRL612) This work; see also Clerget et al. (1995) pRL617 As pRL611 with codons 546-877 (opposite orientation of pRL613) This work; see also Clerget et al. (1995)

Bacterial Techniques

Cells were grown in L broth (Difco Laboratories). LB plates were prepared as described (Miller, 1992). Antibiotics were added at the following concentrations, unless otherwise indicated: ampicillin (Amp) 50 µg/ml; kanamycin (Kan) 30 µg/ml; tetracyline (Tet) 20 µg/ml. Competent cells were prepared by CaCl₂ shock (Mandel and Higa, 1970), or by electroporation (Dower et al., 1988) and were stored at 80 °C. Electroporation was performed with a Bio-Rad GenePulser using 0.2-cm Bio-Rad GenePulser cuvettes.

Marker rescue of the cold-sensitive growth phenotype of rpoC214 was as described (Jin and Gross, 1989) using plasmids pRL611-pRL617 (Table II).

DNA Techniques

Restriction enzyme digests and agarose gel electrophoresis were as described by Sambrook et al. (1989). Plasmid DNA purification was as described by Sambrook et al. (1989) or by purification on Qiagen or Magic Miniprep columns.

End-labeled DNA was prepared by PCR essentially as described by Dombroski et al. (1992), using 30 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C and purifying the DNA by elution from nondenaturing 10% polyacrylamide gels as described by Sambrook et al. (1989). Quantitation of labeled DNA was as described by Dombroski et al. (1992).

DNA binding was determined by measuring retention of polymerase-DNA complexes on nitrocellulose filters. All data represent the average of at least 3 duplicate experiments, unless stated otherwise. Binding buffer (BB: 50 mM KCl, 40 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 1 mM -mercaptoethanol) was made according to the specifications of Pfeffer et al. (1977) for the heparin competition experiments; for the excess DNA competition experiments, the KCl concentration was increased to 100 mM (see ``Results''). The composition of the wash buffer (WB) was identical to that of BB, except that KCl was omitted. The experimental procedure was essentially as described (Pfeffer et al., 1977; Hinkle and Chamberlin, 1972). Briefly, 0.25-1 nmol of end-labeled DNA (2 × 10⁵ cpm/pmol) was preincubated with a 4-fold molar excess of active RNA polymerase in 1 × BB at 37 °C for 10 min. Duplicate 25-µl aliquots were filtered and washed with 250 µl of 1 × WB prior to the addition of competitor (t = 0). The reaction mixture was split into two tubes: competitor was added to one, the other was the no competitor control. Duplicate aliquots were removed at the indicated times after competitor (or at regular intervals in the no competitor controls) and filtered at 10-15 mm Hg. Filters were washed once with 250 µl of 1 × WB, dried under an infrared heat source, and subjected to liquid scintillation counting. Background retention was typically <3% of the total counts and was subtracted from the experimental values. Experimentally determined dissociation rates were corrected for baseline dissociation when necessary (calculated from the control reactions described above) by subtracting the dissociation rate of the no-competitor control.

Marker-rescue of the cold-sensitive growth phenotype of rpoC214 was done as described for rpoB211 and rpoB212 (Jin and Gross, 1989). Mutations rpoB211, -212, and rpoC214 were sequenced by PCR amplification of the identified regions. Primers were made flanking the regions, and PCR reactions were essentially as described above. Double-stranded PCR product was gel purified from 2% agarose gels in Tris acetate-EDTA buffer and eluted according to Sambrook et al. (1989). Purified fragment was sequenced according to the Sequenase procedure for sequencing double-stranded plasmid DNA.

Biochemical Techniques

Strains CAG 14179, 14180, and 14181 (Table I) were grown in LB + antibiotics in a 6-liter fermenter (University of Wisconsin Pilot Plant) and harvested in mid-log phase (OD₆₀₀  1). Protein purification of 25 g of cells (wet weight) was essentially as described by Lowe et al. (1979) and Burgess and Jendrisak (1975), with several modifications: cell suspensions were sonicated in several 30-s intervals instead of being subjected to high speed sheer (described in Thompson et al. (1992)); the TGED + 1 M NaCl polymin P eluate was precipitated with ammonium sulfate as described (Burgess and Jendrisak, 1975) and resuspended in TGED to a conductivity equivalent to 0.25 M NaCl (in TGED), then loaded on a 10-ml single-stranded DNA-agarose column. Holoenzyme, eluted in TGED + 1 M NaCl, was precipitated, resuspended, and loaded on a 120-ml S-300 HR sizing column. The holoenzyme fractions were pooled and eluted from an 8-ml Q-Sepharose FF column in TGED over a gradient from 0.25 to 0.75 M NaCl; the polymerase fraction typically eluted at 0.5 M NaCl.² Highly pure fractions of holoenzyme (>95%) were pooled and dialyzed against polymerase storage buffer (TGED, containing 50% glycerol + 0.1 M NaCl) and stored in aliquots at 80 °C.

Crude preparations of S350F and G351D RNA polymerases from strains RL676 were from strain RL676 transformed with appropriate plasmids (Weilbaecher et al., 1994) using a variation of the RNA polymerase microprep protocol of Gross et al. (1976) described by Tavormina et al. (1996). Similar preparations of G1074D and H526Y (rpoB2) RNA polymerases were generously provided by P. Tavormina.

Activity of polymerase preparations was determined both by the Chamberlin T7D111 assay and by the fluorescence abortive initiation assay of Bertrand-Bergraff et al. (1994), using -ANS-UTP (data not shown). The results of both assays indicated that the wild-type preparation was 45-50% active and the N1072H and R352C preparations were between 20 and 25% active. Q513P RNA polymerase was purified from strain CY15023 (Horn and Yanofsky, 1981) by the method of Hager et al. (1990) and determined as described above to be ~25% active.

T7D111 assays (Chamberlin et al., 1979) were carried out at 30 °C and at 0.4 mM NTPs. The P_R-t_R1 assays, originally described by Chen and Richardson (1987), were done according to the modifications described by Heisler et al. (1993). Reactions were carried out at 37 °C; 1 × NTPs equals 0.2 mM ATP, CTP, GTP, and 0.02 mM UTP.

We used an abortive initiation assay similar to that originally developed by McClure et al. (1978), a more detailed description of which will be published separately.³ 0.25 pmol of the 142-bp fragment of pCL185 (T7A1 promoter; Feng et al. (1994); see Table II) was incubated with 1 pmol of active RNA polymerase in 1 × T7A1 buffer (20 mM Tris acetate, pH 8.0, 20 mM NaCl, 14 mM MgCl₂, 14 mM -mercaptoethanol; Levin et al. (1987)) and 25 µg of acetylated bovine serum albumin/ml at 37 °C for 10 min. Reactions were initiated by the addition of ApU and [-³²P]CTP (final specific activity 6 Ci/mmol) at the concentrations indicated. Aliquots were withdrawn at 2-min intervals (from 2 to 10 min) into a 4-fold excess of transcription stop buffer (final concentration, 1 × TBE, 3.5 M urea, 0.025% xylene cyanol, 0.025% bromphenol blue). 4.5-µl samples were electrophoresed on 15% denaturing PAGE, and wet gels were exposed to a Molecular Dynamics PhosphorImager screen at 80 °C to prevent diffusion of the bands. The results were quantitated on a Molecular Dynamics PhosphorImager and normalized to ¹⁴C standards. Apparent substrate constants for nucleotides were determined assuming a random-order, rapid-equilibrium mechanism (Siegel, 1975). Since only the pathway in which NTP-binding preceded ApU-binding was significant, only the apparent equilibrium constants for binding NTP first (K_NTP) and for binding ApU second (K_ApU) are reported. The apparent equilibrium constant for binding GTP (K_GTP) was determined by varying [GTP] at high [ApU].

Transcription assays and PAGE were essentially as described for abortive initiation assays, except that ApU was added at 0.5 mM in all reactions, and GTP, ATP, [-³²P]CTP, and 3-deoxy-UTP were added at the concentrations indicated. Aliquots were withdrawn into stop solution at the times indicated and electrophoresed on 15% urea-polyacrylamide gel, as described above. Control experiments done in the absence of 3-dUTP (data not shown) demonstrated that the results were unchanged in the presence of the chain terminating NTP, except that there was no readthrough of the 17 nucleotide product (see also Sagitov et al. (1993)). The identities of the products as indicated in Fig. 4 were confirmed by Feng et al. (1994) using 5-AUC, 5-AUCG, and 5-AUCGA generated in transcription reactions with ApU and [-³²P]CTP only, ApU, CTP, and [-³²P]GTP only, or ApU, CTP, [-³²P]GTP, and 3-deoxy-ATP only, and showing (i) that they co-migrated with the abortive products as assigned in Fig. 6 and (ii) that addition of a 5 PO₄ increased the mobility of these short transcripts to that of 5-pAUC, 5-pAUCG, and 5-pAUCGA, confirming that they were initiated with ApU. Note that the mobilities of short RNAs (less than 5 nucleotides in length) lacking a 5 PO₄ are reversed on a 15% urea-polyacrylamide gel (Feng et al., 1994; Krummel, 1990; Levin et al., 1987; Cai and Luse, 1987).

Escape from the T7 A1 promoter by WT, N1072H, and R352C RNA polymerases.

NTP concentration dependence of productive and abortive transcripts as a function of [NTP] for WT, N1072H, and R352C RNA polymerases.

RESULTS

Jin and Gross (1989) describe the localization of rpoB211 and rpoB212 to a 650-bp region in the C-terminal portion of rpoB. We amplified this region by PCR and determined the sequence changes of these chromosomal mutations (see ``Materials and Methods''). These two mutations contain identical single base pair substitutions and may have been isolated as siblings in the original selection, or as two independent isolates (Guarente, 1979). The amino acid change, N1072H, occurs in conserved region H, which has been subjected to extensive mutational analysis (Sagitov et al., 1993; Fig. 1). Mutations in this region affect many aspects of transcription.

Conserved regions of  and subunits and locations of significant features and substitutions examined.

Prior to this study, rpoC214 had not been mapped within rpoC. Marker rescue of the cold-sensitive phenotype of this mutant by plasmids containing internal fragments of rpoC (see ``Materials and Methods'') allowed us to localize this mutation to an interval of rpoC between amino acids 305 and 695 (region 2; Weilbaecher et al. (1994)). PCR amplification and sequencing identified an amino acid change from Arg to Cys at amino acid 352. This amino acid lies in a region of weak sequence similarity to a putative DNA-binding region in the Klenow fragment of DNA polymerase I (Ollis et al., 1985) and is adjacent to several rpoC mutations isolated for their effects on termination (Weilbaecher et al. (1994); Fig. 1).

N1072H and R352C Decreased Elongation Rate in Vitro

We purified the mutant enzymes according to variations in the procedures of Burgess and Jendrisak (1975) and Lowe et al. (1979) (see ``Materials and Methods'') and examined their rates of elongation on T7D111, a deletion of T7 phage DNA that contains only one E. coli E⁷⁰ promoter, T7 A1 (Studier et al., 1975). In this assay, wild type RNA polymerase reached the T7 Te terminator 6.5 min after addition of nucleotides and heparin or at 15 nucleotides/s, whereas N1072H and R352C elongated RNA at 6 and 8 nucleotides/s, respectively (Fig. 2). We also isolated RNA polymerase from two rpoC mutants containing amino acid changes adjacent to R352C: S350F and G351S (Weilbaecher et al., 1994). These mutant enzymes elongated at rates comparable to that of R352C (S350F, 10.5 nucleotides/s and G351S, 8 nucleotides/s; data not shown).

Transcript elongation of WT, N1072H, and R352C RNA polymerases on a T7D111 template.

To confirm the elongation defect of mutants N1072H and R352C, we tested transcription on a P_r-t_R1 template. This template contains a number of well characterized pause sites in addition to the t_R1 terminator (Chen and Richardson, 1987). At 1× NTPs (0.2 mM), wild type reached the terminator by 2 min, while N1072H required 4× NTPs and R352C, 2× NTPs, to match the transcription rate of wild type at 1× NTPs (Fig. 3). Neither mutant exhibited fundamental differences in the pattern of pausing at any concentration of nucleotides (the patterns are far more similar that different, especially since apparently new minor pauses in the mutant lanes may be difficult to detect in the 0.5-min wild-type sample; Fig. 3). Thus, the reduced elongation rates of these mutants result from slower elongation at pause sites that also are recognized by the wild-type enzyme, rather than recognition of new pause sites. Furthermore, these defects in elongation rate are magnified at lower NTP concentrations and do not appear to be template-specific since the magnitudes of their elongation defects were similar on the two templates studied. In these regards, both mutants resemble Q513P(rpoB8) (Q513P elongates at ~0.25 the rate of wild-type; Jin and Gross (1991)).

Transcript elongation from P

of WT, N1072H, and R352C RNA polymerases.

N1072H and R352C Increased the Apparent K_m for both Priming and Substrate Nucleotides during Initiation

In principle, the defects in elongation we found could either reflect a fundamental catalytic defect, which should be evident at all nucleotide addition events, or might be specific to the elongation phase of transcription, such as would be the case if they affected movement of the DNA through the enzyme. In an attempt to distinguish these possibilities, we examined the mutant RNA polymerases using a steady-state abortive initiation assay first described by McClure et al. (1978). This assay takes advantage of the cycling of RNA polymerase in an open complex when the presence of a limited set of nucleotides forces the enzyme to synthesize and release a single short transcript (usually a dimer or trimer) repetitively. By restricting nucleotide addition to a single new phosphodiester bond in the open complex, we hoped to learn both whether the defects affected the catalytic center and if they also were manifest during initiation. We measured the synthesis of the trimers ApUpC or ApUpG on the T7A1 promoter (or a single bp variant, C + 3  G) by systematically varying the concentrations of the priming dinucleotide ApU and the substrate NTP (either CTP or GTP; see ``Materials and Methods''). Double reciprocal plots of reaction velocity versus substrate concentration were linear over the entire range tested, indicating that there was no substrate activation or inhibition (data not shown). However, the kinetic parameters determined must be regarded as apparent substrate constants, rather than true dissociation constants for binding of ApU and NTPs since we cannot be certain the reactants bound in rapid equilibrium (see McClure et al., 1978; Smagowicz and Scheit, 1978).³ On both templates, the mutant RNA polymerases exhibited substantially high apparent substrate constants for both ApU primer and NTP substrates (Table III). K_ApU for wild-type was ~0.325 mM, in agreement with values determined for K_ATP on this promoter (Smagowicz and Scheit, 1978). Both mutants yielded slightly increased K_ApU values (~1 mM; Table III), suggesting that these amino acid changes may somewhat perturb the primer sub-site of the catalytic center (site i, see Fig. 8; Erie et al. (1992)), either directly or indirectly.

Table III. Apparent binding constants of wild-type and mutant enzymes for substrates during abortive formation of trinucleotides Enzyme KApUa KCTP KGTP mM µM Wild type 0.34 47 80  N1072H 1.1 257 500  R352C 1.4 132 140 a See ``Materials and Methods'' for description of the analysis used to derive apparent equilibrium binding constants.

In agreement with determinations on this and other promoters, the wild-type K_NTP values were approximately an order of magnitude smaller than K_ApU (K_CTP = 47 µM, K_GTP = 80 µM, Table III). Although both the  and substitutions increased these values, interestingly, they were approximately 6-fold greater than wild-type for N1072H and about 2-3-fold greater for R352C, depending on the nucleotide in question (Table III).

Thus, the abortive initiation assay uncovered at least one component of the transcriptional defect of these mutant RNA polymerases, namely a decrease in apparent affinities for both priming nucleotide and substrate NTP that is consistent with their reduced elongation rate. We suspected, however, that the mutants might affect other aspects of RNA polymerase function because, although N1072H has a somewhat greater defect in elongation rate and K_NTP during abortive initiation, the growth rate of R352C is slower than that of N1072H. If elongation were the only aspect of transcription affected, N1072H would be the more deleterious allele. Furthermore, R352C demonstrated a more severe hypertermination phenotype in vivo (Jin and Gross, 1989), suggesting that its reduced elongation rate is not the sole determinant in its suppression of the rho-201 phenotype.

Promoter escape, or the transition from abortive to productive synthesis, is another component of the transcription cycle that is sometimes altered by amino acid changes in RNA polymerase. Reductions in promoter escape appear to be correlated with increased pausing and a reduced elongation rate (Kashlev et al., 1990; Sagitov et al., 1993). Interestingly, during initial assays of the N1072H and R352C RNA polymerases, we found that both were defective in forming halted elongation complexes at position 16 of a T7 A1 promoter template at low nucleotide concentrations (pCL185 G16 complexes; data not shown; see Feng et al. (1994)). To characterize the NTP concentration dependence of promoter escape, we monitored the appearance of abortive and productive transcripts as a function of time in the presence of ApU, 3-dUTP, and several different concentrations of ATP, CTP, and GTP (see ``Materials and Methods''). These conditions limited productive transcription to the formation of a dU17 complex. Representative time courses for the wild-type and two mutant polymerases at low (5 µM) and high (50 µM) NTP concentrations are shown in Fig. 4 and complete quantified data from these experiments in Fig. 5. Productive initiation is reflected by appearance of the dU17 transcript, whereas abortive initiation yielded three short RNAs, ApUpC (the major product), ApUpCpG, and ApUpCpGpA (Fig. 4; see ``Materials and Methods'' and Feng et al. (1994)). Other bands between the abortive and full-length products are most likely paused intermediates (Krummel and Chamberlin, 1989), since they chased through the time course of the experiment; these have not been included in the quantification. We first discuss the transition to productive initiation exhibited by wild-type polymerase and then consider how the mutants alter this pattern.

Amounts of productive and abortive transcripts produced by WT, N1072H, and R352C RNA polymerases.

Quantification of the accumulation of productive and abortive products by wild type reveals that the transition from abortive to productive initiation is a time-dependent process (Fig. 5A). The lag observed in attaining the plateau level reflects the time required for escape from abortive cycling; with each round of initiation, a certain fraction of complexes clears the promoter. This switch between abortive and productive mode is demonstrated graphically by the biphasic accumulation of abortive transcripts. At early times (often <10 s), accumulation of abortive transcripts was rapid due to the relatively large number of complexes engaged in synthesizing abortive products. The inflection points in the accumulation of abortive products and full-length transcripts correspond to the point at which the limit of productive initiation was reached (less than 1 mol of transcript/mol of template, indicating that transcription has been limited to a single round). The small amount of abortive transcript that continued to be synthesized at a constant rate beyond the inflection point probably arose from a subpopulation of templates that somehow form another open complex at high ratio of active RNA polymerase to DNA (4:1 in our experiments) or from a subpopulation of polymerase molecules that are slow to clear the promoter. Kubori and Shimamoto (1996) recently reported detection of the latter class of complexes using heparin to block initiation by multiple polymerases. However, rigorously establishing that initiating complexes can become trapped in abortive mode will require showing unambiguously that the template DNA does not harbor a second polymerase molecule that inhibits promoter escape by the first.

Nucleotide concentration influences the transition to productive synthesis in two ways, which are probably not independent. First, it takes longer to make the transition to productive transcription at lower [NTP] (Fig. 5, compare 5 and 50 µM). Second, many more abortive transcripts are made per productive transcript at lower [NTP]. These two points are demonstrated graphically in Fig. 6. The transition to productive mode is efficient only at higher nucleotide concentrations, requiring the production of >3 abortive transcripts/productive transcript at the lowest nucleotide concentration (5 µM; Fig. 6A). However, essentially every polymerase-promoter complex eventually made a productive transcript, even at the lowest nucleotide concentration (5 µM, Fig. 6B).

N1072H is altered in its nucleotide dependence for the abortive to productive transition. This mutant dramatically over-synthesized abortive products (Fig. 5B) relative to the wild type (Fig. 5A). At the lowest nucleotide concentrations, N1072H synthesized approximately 60 abortive products for every productive product, which is more than an order of magnitude greater than the abortive/productive ratio of wild-type polymerase (Fig. 6A). As a consequence, N1072H was significantly slower than wild type in making the transition to productive initiation (compare Fig. 5, panels A and B). Indeed, at 5 µM NTP only one-fourth of the mutant complexes (as compared to wild type) succeeded in making the productive transcript (Fig. 6B). We note that the fraction of polymerases repetitively engaged in producing abortive transcripts at low NTP concentrations is not a dead-end population; addition of high nucleotide concentrations as much as 10 min after the reaction is started allows their transition to productive initiation (data not shown). These aberrant responses of N1072H were ameliorated by higher nucleotide concentrations; higher NTP concentrations increased the fraction of polymerases that make productive transcripts (Fig. 6A) and decreased the ratio of abortive to productive transcripts (Fig. 6B) such that at the highest NTP concentration, the fraction of N1072H complexes that made productive transcripts approached that made by the wild-type complexes.

R352C is also defective in the abortive to productive transition (Fig. 5C); however, in this case the effect of increasing nucleotides is less straightforward. At low NTPs, a smaller fraction of mutant than wild-type polymerases made the transition to productive initiation (5 µM, Fig. 6A), and the abortive/productive ratio for the mutant was slightly greater than for wild type (Fig. 6B). Although these deficiencies were slightly ameliorated by increased nucleotide concentrations, much of the defect of R352C for promoter escape did not appear to be responsive to increased nucleotides (Fig. 6A). We return to this point under ``Discussion.''

The rpoC mutations we investigated lie in a region of weak sequence similarity to a region in E. coli DNA polymerase I that may be in the vicinity of the DNA-binding channel. Because of the possible impact of DNA-binding defects on termination as well as on the promoter escape defect noted above, we examined the DNA-binding properties of these mutants. We used nitrocellulose filter binding to measure stability of RNA polymerase binding to an end-labeled DNA fragment containing the T7 A1 promoter. Chamberlin and co-workers (Pfeffer et al., 1977) have demonstrated that polymerase-promoter complexes at this promoter exhibit very different kinetics of dissociation depending on the choice of competitor; T7 A1 complexes are extremely sensitive to heparin, most likely owing to invasion of the open complex by the polyanion, while these same complexes are stable in the presence of passive competitors such as excess DNA (Hinkle and Chamberlin, 1972). In light of these observations, we examined the properties of wild-type and mutant complexes in the presence of each type of competitor. To test for possible correlations with an altered termination phenotype, we also tested the stability of complexes formed by Rif^r mutants with known termination defects: Q513P(rpoB8), which hyperterminates on all terminators examined, and H526Y(rpoB2), which hypoterminates.

We present the results of the DNA-binding properties of wild type and mutants using heparin as a competitor in Table IV, second column. Results of a representative set of experiments are shown in Fig. 7. As demonstrated by Pfeffer et al. (1977), the wild type complexes were disrupted with a t1/2 of approximately 30 min in the presence of 10 µg/ml heparin (Table IV). The mutants examined fell into 3 categories with respect to their stability in this assay. N1072H and G1074D, another elongation defective mutant in region H (Tavormina et al., 1996), exhibited t1/2 values that are only slightly decreased relative to wild type (less than 2-fold faster), consistent with their primary defect being on the process of nucleotide addition. Two of the mutations in rpoC, R352C, S350F, as well as H526Y, caused pronounced changes in t1/2 of disruption, reducing the stability of the binary complex by approximately a factor of 5. We will discuss the implications of the magnitude of the defect for H526Y below. Q513P and G351S exhibited intermediate defects (t1/2 approximately twice as fast as wild type). G351S, located between the two rpoC mutants that had a large effect in this assay, exhibited a t1/2 that is similar to that seen for Q513P. G351S may be an amino acid substitution that does not significantly affect RNAP:DNA binding. That both Rif^r mutants affect DNA binding, and that it is the hypoterminating allele H526Y that is less stable, suggests that there is no straightforward correlation between promoter binding and termination. The mutants that have a large effect in this assay, however, may profoundly affect the architecture of the open complex such that it is more susceptible to invasion by heparin.

Table IV. Effects of mutants on dissociation of polymerase-T7A1 binary complexes Enzyme Heparin t1/2 (min)a Poly(dA-dT)t1/2 (min)b WT 29  (±4.5) 37  (±8)  R352C 8  (±1) 9  (±1.5)  G351S 17  (±2)c 14  (±2.5)  S350F 6  (±0.5)c 8  (±2)  N1072H 20  (±2) 40  (±4.5)  G1074D 30d NDe  Q513P(B8) 14  (±1) 14  (±1)  H526Y(B2) 4  (±0.5) 8  (±0.1) a 50 mM KCl. b 100 mM KCl. c Done twice; duplicates typically varied <10%. d Done once. e Not done.

Dissociation of open complexes formed by WT, N1072H, and R352C RNA polymerases at the T7 A1 promoter.

Qualitatively similar results were seen when approximately 10 µg poly(dA-dT)/ml was used as a cold competitor (Table IV, third column; also Fig. 7). When these experiments were done under the same solution conditions as the heparin challenge experiments, we observed much slower dissociation rates for wild type than those measured when heparin was used as competitor, consistent with the hypothesis that heparin actively invades the open complex, while the DNA competitor acts as a trap for enzyme molecules that dissociate (data not shown). Because of the high degree of inaccuracy in these measurements, we increased the salt concentration (from 50 to 100 mm), thus increasing K_d (Hinkle et al., 1972). Again, the mutants fell into essentially the same classes, with the same two rpoC mutants exhibiting the most profound effects on K_d, the region H mutant showing little deviation from the wild-type value, and Q513P and G351S falling between these two extremes (Table IV, third column).

DISCUSSION

We identified the amino acid substitutions of three mutant alleles of rpoB and rpoC that were originally isolated by Guarente (1979) as suppressors of rho201. rpoB211 and -212 encode a change from Asn to His at amino acid 1072 in conserved region H of rpoB, and rpoC214 encodes a change from Arg to Cys at amino acid 352 in conserved region C of rpoC. Unlike another suppressor of rho201, rpoB8(Q513P), neither N1072H nor R352C greatly increase termination at most -dependent and -independent terminators, but both appear to be somewhat specific for the -dependent terminator, trpt, used for their isolation. Furthermore, all three of the rho201-suppressing mutants, Q513P, N1072H, and R352C fail to suppress another conditional lethal rho mutation, rho-15 (Das et al., 1978), whereas another class of polymerase mutants, is specific for rho-15 (Jin and Gross, 1989). We have purified the N1072H and R352C mutant enzymes and investigated their properties in vitro. Our most important findings are that (i) although their phenotypes might have been consistent with specific interactions at trpt, N1072H and R352C exhibit general defects in elongation and transcriptional activity that are consistent with their compensating for a reduced activity of Rho by slower elongation, (ii) the precise nature of the defects caused by these substitutions (and thus the likely role in transcription of the corresponding conserved regions of the  and subunits) differ; (iii) the specificity of N1072H and R352C for trpt and rho201 most probably reflect differences in the rate-limiting steps for termination in these cases that are affected differently by the particular structural perturbations caused by these two substitutions. To discuss these points, we refer to a simple diagram of the structure of a transcription elongation complex (Fig. 8).

N1072H and Conserved Region H in  May Be Located Near the Catalytic Center

Our studies of N1072H are consistent with the idea that conserved region H may be in the proximity of the catalytic center (C, Fig. 8). Our failure to detect significant effects of this mutation on the stability of the open complex (Table IV; Fig. 7) and of the ternary complex (data not shown) suggests that DNA binding is not perturbed in this mutant. Although this region could interact with the nascent transcript, at least some of the mutant defect is manifest even at the initial nucleotide binding step. The initiating N1072H mutant encounters the same decision points as the wild-type enzyme but releases abortive transcripts much more often, resulting in a higher ratio of abortive to productive transcripts and inefficient conversion to an elongation complex. The decreased affinity of the mutant for NTPs clearly could contribute to favoring release over elongation since the defect is greatly reduced at higher concentrations of NTPs. Finally, N1072H increases apparent K_ApU ~3-fold and K_NTP ~6-fold during abortive initiation. Perturbation of the catalytic center appears able to explain all of these effects of this mutant. However, apparent, but only moderate, perturbation of binding sites both for the priming nucleotide and the substrate NTP argues against direct contacts between N1072 and either reactant. Instead, a general deformation of the active site caused by N1072H could be an indirect effect of the mutation. For example, N1072H could alter an amino acid contact that positions one of the secondary structure elements that actually form the active site.

This interpretation is consistent with other studies of conserved region H, one of nine colinear regions of sequence conservation found in  homologs from bacteria to humans (Sagitov et al. (1993) and references therein). Cross-linking studies indicate that conserved region H is likely to be positioned close to the 5 face of the priming nucleotide; an evolutionarily invariant lysine residue within this segment (Lys-1065 in  and probably Lys-979 in Saccharomyces cerevisae polII) is within 2 Å of the -phosphate of the initiating NTP (Grachev et al., 1987, 1989; Mustaev et al., 1991; Riva et al., 1990). The observation that the nascent chain can be elongated by only two residues after cross-linking to this site is further evidence for its proximity to the nucleotide binding site (Mustaev et al., 1993).

Studies of site-directed mutants in this region by the Goldfarb group also support the location of this region of  near the catalytic center. The K1065R mutation exhibits a severe lethal phenotype. Although it forms open complexes, it catalyzes the formation of only one or two phosphodiester bonds, an effect similar to inhibition by Rif. However, replacement of a number of conserved amino acids between 1063 and 1073, including Lys-1065, with alanine caused a less severe phenotype (Sagitov et al., 1993). Single alanine substitutions all retained significant transcriptional activity on natural templates (15% of the wild-type polymerase in multiround assays), although they deviated from wild type in a number of steps in transcription, including promoter escape and the extent and pattern of pausing. The mild phenotypes of most Ala substitutions in region H are consistent with the view that it does not form the nucleotide addition site per se, but is probably in its immediate vicinity and is likely to affect RNA polymerase by perturbing the catalytic center indirectly. For instance, changes in charged residues (e.g. Lys-1065 to Arg and Asn-1072 to His) could disrupt catalytic function by forming new H-bonds or charge-charge interactions that reposition side chains near the active center.

Satigov et al. (1993) have suggested that mutations in this region distort the active center, possibly by affecting the coupling of nucleotide addition and translocation. Although our experiments do not define the function of the region, they are most consistent with this idea. Jin and Turnbough have recently described a Rif^r polymerase (R529C in ) that exhibits an increased apparent K_NTP for pyrimidines in promoter escape (Jin and Turnbough, 1994). However, the fact that the N1072H mutant is more defective than R352C in the process of promoter escape, but exhibits a comparable increase in K_NTP (cf. 6-fold for N1072H and 2-3-fold for R352C), suggests that additional factors, such as context-dependent conformational changes, may contribute to the phenotype of N1072H at such decision points. A more thorough investigation of the block to elongation at these points is necessary to better articulate the nature of the N1072H defect.

R352C and Conserved Region C in May Contact DNA Like a Weakly Similar Sequence in a DNA-binding Region of DNA Polymerase I

Our selection of experiments on R352C were motivated in part by finding it located adjacent to two termination-altering mutations at amino acids 350 and 351 (Weilbaecher et al., 1994) in a region of weak sequence similarity to a putative DNA-binding region in DNA polymerase I. Our results support the view that this weak sequence similarity reflects functional similarity of these two sequences: substitutions in this region profoundly destabilize polymerase-promoter complexes in the presence of competitor molecules.

In DNA polymerase I, this region comprises -sheets 7 and 8 of the thumb domain of the DNA-binding cleft and is immediately upstream of helices J and K. The region homologous to amino acids 350-352 in forms part of a loop between these two  sheets. Polesky et al. (1990) have generated an alanine substitution of the arginine (R668A) corresponding to Arg-352 in rpoC. This mutation has profound effects on k_cat (400-fold decrease), K_d for DNA (20-fold decrease), and slight effects on K_m for dNTPs, suggesting that this arginine side chain forms part of the catalytic site of polymerase I and may also be in close proximity to the phosphate backbone of the DNA (Polesky et al., 1990).

Our results suggest that this region of may, like the R688A mutation in DNA polymerase I, be involved in more than one important function of polymerase. The convergence of effects on K_NTP with those on DNA binding might be predicted in a region involved in nucleotide addition; at some point in the process, addition of NTPs must be tightly coupled to DNA contacts. Similarly, the failure of R352C to convert efficiently from the abortive to productive mode (Figs. 4, 5, 6), while not due to an obvious increase in the release of abortive products, could reflect aberrant contacts between the polymerase and either the transcript or the template. These defects could, however, also be the indirect result of alterations in the conformational changes that accompany the switch to productive initiation.

It is premature to conclude that this region of is involved in nucleotide addition or stabilization of contacts to the template during this process: the mutants that are described in this study do not have strong enough phenotypes to unequivocally determine what aspects of transcription are primarily altered. However, further study of this region of is warranted, particularly with a view to uncovering the relationship between defects in DNA binding and nucleotide addition. The relatively weak phenotype of R352C could stem from its having been isolated as a haploid, altered-function mutation, necessitating that it retain significant function. We are currently generating the R352A mutation in rpoC as well as several other site-directed changes to evaluate more stringently the function of this region. Interestingly, no significant differences were detected between R352C and wild type in an elongation assay designed to measure dissociation of the ternary complex during elongation (Arndt and Chamberlin, 1988; data not shown). It is conceivable that in the ternary complex additional contacts between the polymerase and the RNA or DNA mask the putative destabilizing effect of the mutation at R352C but that the structural pertubation still affects transcript elongation.

DNA-binding Defects of Substitutions in and in the Rif^r Region of  Suggest the Existence of DNA-binding Domains in Each Subunit

We have presented results implying that regions in both rpoB and rpoC are important in DNA binding, although we do not yet have sufficient evidence to suggest that these regions contact DNA directly. Prior to this study, only a few workers had addressed the DNA binding properties of the core subunits of RNA polymerase. Martin et al. (1992) describe the DNA binding defect of a deletion mutant of rpoB (in conserved region C, Fig. 1) that fails to form stable complexes at the promoter. Several groups have described the isolation and partial in vitro characterization of cold-sensitive mutations in  and that appear to be defective in open complex formation (Larionov et al., 1979; Gragerov et al., 1980; Panny et al., 1974). Interestingly, Nomura et al. (1984) describe mutations in rpoC, as well as a double mutation in  which includes the H526Y change, discussed below, that appear to be important in promoter selectivity. Unfortunately, none of the rpoC mutations examined in these earlier studies have been mapped or sequenced.

The three mutations in rpoC and two mutations in the Rif^r region of rpoB that we analyzed significantly affected the stability of the open complex to challenge by both heparin and poly(dA-dT) (Table IV). In both cases, R352C and S350F, as well as H526Y, formed open complexes that were significantly less stable than wild type in the presence of competitors, consistent with, but not proving that these residues stabilize contacts to the DNA.

The result obtained with H526Y is particularly interesting. The T7 A1 open complex containing H526Y is as susceptible to both types of competition as the rpoC mutants. Recent experiments by Goldfarb and co-workers (Mustaev et al., 1995) in which Rif, substituted with a cross-linkable side chain, is bound to RNA polymerase at the T7 A1 promoter indicate that the bound Rif molecule cross-links to the template strand of the DNA between positions 2 and 3 relative to the start site of transcription. These observations, taken with our findings for H526Y and Q513P, suggest that the Rif^r region of rpoB is in the immediate vicinity of the DNA when polymerase is bound at the promoter and may be involved in stabilizing contacts to the DNA in the open complex. In another study,³ we found evidence suggesting that the Rif^r region, or at least the residue affected by the Q513P mutation, is important in correctly positioning the 3 end of the nascent transcript in the ternary complex (i.e. makes contacts in the promoter distal segment of region B, Fig. 8). This same region could be involved in contacting the DNA in the absence of transcription (i.e. in the binary promoter complex). An interaction between the Rif^r region and DNA is also consistent with observations by Chamberlin and co-workers (Hinkle et al., 1972) that the affinity of RNA polymerase for Rif is reduced by 2 orders of magnitude when polymerase is bound to DNA. Whether or not these regions contact DNA directly, their effects on the stability of binary complexes suggest that they must at least communicate with regions that stabilize polymerase-DNA interactions.

Jin et al. (1992) previously described a model to explain the kinetic interplay between RNA polymerase and Rho. In this view, the movements of polymerase and Rho on DNA and RNA, respectively, are kinetically coupled such that alterations in the translocation rate of either polymerase or Rho affect termination efficiency. This model has been invoked to explain the hypertermination phenotype of rpoB8 (Q513P), which elongates more slowly in vivo and in vitro, and exhibits increased termination on all Rho-dependent (and intrinsic) terminators tested. Indeed, rpoB8 was also isolated (as rpoB203) as a suppressor of rho201 (Guarente and Beckwith, 1978).

Both Guarente and Beckwith (1978) and our previous study (Jin and Gross, 1989) concluded that the rpoB211 and rpoC214 mutations do not exhibit a general termination defect such as that observed for rpoB8. Although reconstruction experiments verified that both mutants increased termination at the trptt terminators used in their selection, only rpoC214 (R352C) increased termination on other terminators, and this defect was not particularly pronounced. Furthermore, in contrast to Q513P, these mutants show only a marginal increase in termination at intrinsic terminators in vitro (data not shown). These differences in phenotype suggested that a study of the defects of these two mutants might reveal additional characteristics of the termination decision.

We were therefore not surprised to discover that while both N1072H and R352C elongate more slowly in vitro, neither demonstrates an elongation defect as severe as that measured for Q513P. Furthermore, although N1072H demonstrates the greater elongation defect of the two, R352C exhibits the more significant termination-altering properties. We suggest that the specific effects of the mutants on the trptt terminator can best be understood in the context of the mutant defects elaborated above for the transition between abortive synthesis and productive elongation. Termination versus elongation is a decision similar to abortive versus productive initiation. In both, release competes with RNA chain elongation. The same properties of the mutants invoked to explain their defects in making the transition to productive elongation may account for their altered termination properties. Thus, the defect of N1072H in nucleotide addition, and the altered template binding properties of R352C may affect their ability to terminate. Moreover, such defects are likely to be manifest only at those terminators whose particular rate-limiting steps are affected by this defect, giving rise to the terminator specific properties of the mutants. For similar reasons, region H mutants exhibit severe defects in forming productive elongation complexes only at certain promoters (Sagitov et al., 1993). Our findings complement other recent studies examining the elongation properties of termination-altering mutants that suggests that elongation rate is not the sole determinant of the probability of termination (Landick et al., 1990; Weilbaecher et al., 1994). Furthermore, it is becoming increasingly clear that contacts to additional protein factors, particularly NusG, play an important role in determining the efficiency of Rho-dependent termination, at least at some terminators. Further studies are underway, both in our groups as well as several others, to unravel the intricacies of the elongation to termination decision at both Rho-dependent and intrinsic termination sites.