Polyadenylic Acid Synthesis Activity of Purified DNA-dependent RNA Polymerase from CauZobacter*

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulohacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell DEAE-cellulose DNA-affinity chromatography, and low salt glycerol centrifugation. Holoenzyme can be resolved into core and o subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure o

. Previous studies have shown that transcription (Newton, 1972) and translation' are needed for normal development throughout most of the cell cycle. Furthermore, the pattern of protein synthesis changes sequentially during the development of these cells (Cheung and Newton, 1977). These results support the view that differential gene expression, presumably at the level of transcription, plays  The reaction was then chilled in ice and an aliquot of 0.2 ml was centrifuged for 9 h at 39,000 rpm on a 5 to 30% sucrose gradient. This is a sensitive assay for endonuclease activity because the gradient resolves the 16 S and 23 S species of RNA and any nicks or breaks alter the profile of the RNA. A second assay designed to measure solubilization of bulk RNA by nucleases was performed by incubation of [3HlRNA from C. crescentus with enzyme as above for 12 h at 30". Then the reaction mixture was chilled in ice and 500 pg of yeast tRNA in 0.1 ml of 0.3 M sodium acetate was added. RNA was precipitated by addition of 300 ~1 of absolute ethanol and centrifuged and the supernatant was counted in 3 ml of Bray's scintillation fluid (Bray, 1960).

RNA Synthesis Activity
For quantitation of RNase activity, 4 /*g of ["Hlpoly(U) in 50 /Al (1 mCi/mmol) was substituted for C. crescentus RNA and incubated with enzyme at 30" for 10 min, all in 4 IIIM phosphate buffer, pH 7.4. RNase activity was expressed as the radioactivity solubilized in 10 min under these conditions. The procedure for the assay was modified slightly from Burgess (1969 was started by the addition of labeled ATP after preincubation of the assay mixture plus enzyme for 10 min at 30". Assays were incubated at 30" for 10 min, chilled in ice, and precipitated with 3 ml of 5% trichloroacetic acid. After 30 min at 4", the precipitate was collected on a Whatman GF/A glass fiber filter paper disc, washed four times with 3 ml of 2% ice-cold trichloroacetic acid, and then three times with 2 ml of chilled absolute ethanol. All trichloroacetic acid solutions contained 0.01 M sodium pyrophosphate. The filters were dried in an oven at 70" for 30 min and then counted in 4 ml of toluene scintillation fluid (16 g of Z$diphenyloxazole and 0.4 g of 1,4-bis[2-(5.phenyloxazolyl)]benzene in 4 liters of toluene). One unit of RNA synthetic activity is defined as the amount of protein that catalyzes the incorporation of 1 nmol of AMP into acid-precipitable material in 10 min of incubation under the conditions described above. The specific activity is expressed as units of enzyme per mg of protein.
Protein was measured by the method of Lowry et al. (19511, using crystalline bovine serum albumin as standard. Deoxyribonuclease -Endonuclease activity was assayed according to Greene et al. (1974) with some modifications.
Reactions were carried out at 37" for 20 min in 30 ~1 of 0.1 M Tris (pH 7.51, 0.05 M NaCl, and 5 rnM MgCl,, containing 1 or 2 pg of enzyme and 0.5 /Lg of supercoiled PM2 DNA; the incubation was stopped by adding 5 ~1 of 5% SDS. Electrophoresis was then performed using 0.7% agarose in a Trisiborate buffer (Greene et al., 1974)  , 10 pg of enzyme and 1.5 pg of :'lPlabeled SV40 supercoiled DNA (about 10,000 cpm) were incubated at 30" for 1 h and the resultant DNA profile was analyzed on alkaline sucrose gradients as described by Meyer (1972).
The enzyme was preincubated in the assay mixture at 30" for 10 min without [llC]ATP, which was then added to start the reaction.
Assays were incubated for 2 h at 30", chilled in ice, and then precipitated with 3 ml of 5% trichloroacetic acid. The precipitate was processed for scintillation counting as described above. One unit of poly(A) synthetic activity is defined as the amount of protein that catalyzes the incorporation of 1 nmol of AMP into acid-precipitable material in 2 h of incubation under these conditions.
An assay for exonuclease and large amounts of endonuclease activities is based on solubilization of DNA. 32P-labeled SV40 DNA (3161 cpm in 0.5 pg) and 10 pg of enzyme were incubated at 30" for 12 h in 100 ~1 of nuclease buffer. The reaction was stopped by addition of 100 ~1 of 10% trichloroacetic acid and then centrifuged in a Beckman 152 microfuge for 5 min. The acid-soluble counts released were determined by counting the supernatant (100 ~1) in 3 ml of Bray's scintillation fluid.

Purification
The temperate Caulobacter bacteriophage, LC72, was purified by repeated centrifugation followed by equilibrium centrifugation through a CsCl density gradient. DNA from LC72 and C. crescentus, strain CB15, was purified by phenol extraction (Marmur, 1963;Melli and Bishop, 1970). C. crescentus was grown in Pye medium; a typical yield from 5 g of cells was 12 mg of DNA with an absorbance ratio (A,,,/A,,,) of 0.504. Purification procedure for the DNA-dependent RNA polymerase used in these studies is described in the miniprint supplement." during purification, the stability of enzyme activity in cell extracts was examined over the pH range 2 to 12 using citrate, phosphate, imidazole, and borate buffers. These results showed that the enzyme was most stable at pH 6.5 in imidazole buffer in the presence of 1 mM dithiothreitol and 20% (v/v) glycerol. All purification steps were carried out under these conditions.

Assays for Enzymatx
The purification procedure used in this study was developed independently of a method described recently by Amemiya et al. (1977). Both of the procedures use DEAE-cellulose and DNA-cellulose chromatography, but they differ in other respects. Among these are the avoidance of high salt concentrations at all stages and the use of glycerol density gradient centrifugation as a final purification step in the procedure described (see next section).

Resolution of CT Subunit
The core enzyme and (T subunit can be resolved in two different ways. As reported previously (Amemiya et al., 1977) holoenzyme and core polymerase can be recovered by DNAcellulose chromatography (Fig. 3). In addition, we have found that core enzyme and the putative v subunit can be isolated by glycerol gradient centrifugation.
When holoenzyme purified on a DNA-cellulose column was layered on a 15 to 30% (vl v) glycerol gradient in Buffer II containing 0.05 M KC1 and centrifuged, the peak fractions of activity contained core polymerase (see next section) and purified r~ was recovered from Fractions 15 to 20 (Fig. 4). SDS-gel electrophoresis of FRACTION NUMBER FIG. 4. Resolution ofC. crescentus core and a subunits by glycerol gradient centrifugation. Holoenzyme from DNA-cellulose column (7 mg in 3 ml) was applied to a 30-ml 15 to 30% (v/v) glycerol gradient in Buffer II + 0.05 M KCl, centrifuged at 63,000 x g for 24 h at 4" in a SW 25L. 1 rotor. The top of the gradient is at the rzght. Polymerase activity (0) was assayed and the peak was pooled (Fractions 5 to 15). Fractions were also analyzed by 12.5% SDS-polyacrylamide slab gels and the amount of proteins m each band was estimated by microdensitometer tracing (Joyce-Loebl). Quantitation was done by weighing the peaks; the relative abundance of each subunit in the indicated fractions was plotted, using arbitrary units. pp, A; (Y, A; and V, 0. these fractions (Fig. 5) and proteins of known molecular weight (not shown) shows characteristic core subunits of 165,000 (p'), 155,000 (p), 48,000 (a), and 96,000 (a). The 96,000dalton protein (a), as well as the other proteins, have been designated by analogy to the subunits of the E. coli RNA polymerase (Burgess, 1969).
The holoenzyme and core RNA polymerase purified as described above showed no difference in catalytic activity under the standard assay conditions when T4 or calf thymus DNA were used as templates (Table II). However, with Caulobacter phage LC72 DNA, the holoenzyme was 2-fold more active than the core polymerase (Table II). We have also been able to distinguish between holoenzyme and the core RNA polymerase of C. crescentus by differences in metal ion preferences (Table II). Although optimal RNA polymerase activity requires Mg *+, holoenzyme is 6-to 7-fold more active than core polymerase when Mn2+ was substituted for Mg*+ with bacteriophage T4 DNA as template. The holoenzyme was also more active with native CB15 DNA as template when Mn2+ was substituted for Mg2+ in the assay.
Attempts to reconstitute holoenzyme from purified core and cr subunit have not been successful. This may reflect the lability of isolated (T or suboptimal conditions for reconstitu-I234567 FIG. 5. SDS-polyacrylamide gel electrophoresis of C. crescentus RNA polymerase. Samples from the following stages of purification were analyzed on 10% polyacrylamide slab gels as described under "Materials and Methods." 1, peak from DEAE-cellulose column (10 pg); 2, holoenzyme after DNA-cellulose column (8.5 pg; Fig. 3, Fraction 40); 3, core polymerase after DNA-cellulose column (4 pg;  tion. The DNA from Caulobacter phage $CbK might be a preferred template in assaying for reconstitution since the holoenzyme is approximately g-fold more active than the core polymerase with this DNA (Amemiya et al., 1977).

RNA Polymerase
To assess the purity of the enzyme, samples from each step of the purification were analyzed by electrophoresis on 10% SDS-polyacrylamide slab gels (Fig. 5). The resolution of holoenzyme and core polymerase on a DNA-cellulose column is also shown.
The purity of the RNA polymerase preparation was determined from tracings of photographic negatives of stained SDSpolyacrylamide gels. The major contaminants in the holoenzyme after the DNA-cellulose column step are in two bands (cf. Slot 2, Fig. 5) that account for less than 5% of the total protein. Core polymerase at the same stage of purification has several minor contaminants (Slot 3, Fig. 5) that comprised about 15% of the total protein. However, after glycerol gradient centrifugation the core polymerase contained less than 2% contaminating proteins (Slot 4, Fig. 5) Enzymatic Purity Purified RNA polymerase was assayed for contamination by nucleases and polynucleotide phosphorylase as described below.

Ribonuclease
-The sedimentation profile of ribosomal RNA from C. crescentus was not altered after incubation for 1 h at 30" with 25 Kg of purified RNA polymerase holoenzyme; however, 0.01 yg of RNase A gave complete digestion of the RNA under similar conditions (Fig. 6). Thus, no endonuclease activity was detected in the purified enzyme under these conditions. The purified core polymerase (10 pg from glycerol gradient) also failed to solubilize radioactive CB15 in 10% trichloroacetic acid after incubation for 24 h at 30", whereas 0.01 pg of RNase A completely degraded the RNA after incubation for only 1 h (data not shown). Deoxyribonuclease -Since it was necessary to remove DNA by treatment with DNase during the purification (see miniprint supplement), the purified RNA polymerase was exam- ined for contaminating DNase activity. PM2 DNA, incubated for 20 min at 37" with 2 pg of purified enzyme (Fraction 4, Table I), exhibited the same agarose gel pattern as the control (no enzyme). DNase I (0.001 pg) under the same conditions nicked 90% of the DNA. Similarly, SV40 DNA retained the same sedimentation profile on alkaline sucrose gradient after incubation for 1 h at 30" with 10 pg of purified C. crescentus RNA polymerase (see "Materials and Methods"; data not shown). Thus, no contaminating endonuclease activity could be detected in the purified RNA polymerase preparation.
We also observed no solubilization of SV40 DNA even after incubation with 10 wg of polymerase for 12 h at 30", whereas 0.01 pg of DNase I rendered 1 pg of SV40 DNA completely soluble in trichloroacetic acid after 10 min of incubation at 30".
Polynucleotide Phosphorylase -Polynucleotide phosphorylase activity was assayed by the exchange of [32Plinorganic phosphate (New England Nuclear) into ADP (Reiner, 1969). E. coli polynucleotide phosphorylase (0.1 pg from P-L Biochemicals) exchanged about 10,000 cpm in 30 min at 30", while 10 pg of purified polymerase gave less than 1% of the above exchange.

Poly(A) Synthesis
Since a significant fraction of the unstable messenger RNA in C. crescentus is polyadenylated at the 3'-OH terminus6 (Ohta et al., 1975), we examined purified fractions of RNA polymerase for poly(A) synthetase. Traces of activity that catalyzed the synthesis of poly(A) from ATP in the absence of template were observed in RNA polymerase purified on DEAE-cellulose.
The total poly(A) synthetase activity in the RNA polymerase preparation increased significantly after DNA-cellulose chromatography, presumably because of re-  (Table  III). This metal ion preference contrasts with the polynucleotide phosphorylase from E. coli, which displays an almost absolute requirement for M$+ in the poly(A) synthesis reaction (Table  III).

Sensitivity of Poly(Ai Synthesis
The sensitivity of RNA polymerase to rifampicin was examined in the standard in vitro assay. This drug inhibits the E. coli RNA polymerase by binding to the p subunit and as expected from in uiuo transcription studies in C. crescentus (Newton, 19721, all fractions of the DNA-dependent RNA polymerase obtained during purification of the enzyme from these cells were inhibited by rifampicin.  polymerase was more sensitive than holoenzyme. The greater sensitivity of core polymerase to the drug has been observed previously in E. coli (Chamberlin, 1974) and C. crescentus (Amemiya et al., 19771. We determined the sensitivity of the same purified enzyme preparations to rifampicin in the synthesis of poly(A). The results (Table  V, Fig. 7) showed that this activity is also inhibited by rifampicin, although somewhat less so than the DNA-dependent RNA synthesis reaction.
Since rifampicin inhibits E. coli RNA polymerase by binding the /3 subunit of the enzyme (Chamberlin, 1974) this result suggests that at least the b subunit of C. crescentus RNA polymerase is involved in polyadenylic acid synthesis.    Table I) were obtained from rifampicin-resistant and wild type strains of C. crescentus (see text) and sedimented in low salt glycerol density gradients as described in Fig. 1. RNA polymerase from the peak fractions in each gradient was pooled and samples of 100 ~1 each were incubated with rifampicin as indicated for 5 min at 30 before standard polymerase assay mixture was added. Residual RNA polymerase activity at 30" was determined for CB15 (01, Cl-Rf3 (rifampicin-resistant mutant, A), and C12-Rf6 (the partially rifampicin-resistant mutant, 0) as described in text.

Poly(A) Synthesis in Rifampicin-resistant Mutants
To eliminate the possibility that poly(A) synthesis is catalyzed by a contaminating activity that is also sensitive to rifampicin, we have examined poly(A) and RNA synthesis in rifampicin-resistant mutants. The in vitro sensitivity of RNA polymerase in the mutants (see "Materials and Methods") was examined in cell extracts purified by high speed centrifugation (cf. Table I, Fraction 2) and sedimentation through low salt glycerol gradients; the profiles of RNA polymerase activity were similar to that of the wild type (cf. Fig. 1). Three rifampicin-resistant strains studied (Cl-Rfl, Cl-Rf2, and Cl-Rf3) exhibited complete resistance to 10 and 20 pglml of rifampicin in vitro, and the RNA polymerase activity from a fourth mutant, C12-Rf6, was only 50% inhibited by 10 pg/ml of rifampicin (Fig. 8). The wild type CB15 enzyme was completely inhibited at 0.2 yglml in the partially purified preparation.
Strains Cl-Rf3 and C12-Rf6 were examined in more detail. RNA polymerase from these strains was purified through the DNA-cellulose chromatography step (Table I, Fraction 4) to allow for detection of poly(A) synthetase activity, and the response of holoenzyme and core polymerase to rifampicin in RNA synthesis (Table IV) and poly(A) polymerization was determined (Table V). The purified enzyme from strain Cl-Rf3, which grew well in the presence of 50 pg/ml of rifampicin, was completely resistant to the drug, as assayed by both RNA synthesis and poly(A) polymerization activities. Purified RNA polymerase from the partially resistant mutant, C12-Rf6, was only partially inhibited by rifampicin in the two assays. These parallel sensitivities of the mutant enzymes to rifampicin in both assays strongly support the conclusion that core RNA polymerase of C. crescentus is responsible for both RNA synthesis and poly(A) synthesis.

DISCUSSION
Purified RNA polymerase from C. crescentus, strain CB15, catalyzes both polyadenylic acid synthesis in the absence of template and DNA-dependent RNA synthesis. This conclusion is supported by several lines of evidence: i) enzyme purified to 98% homogeneity on glycerol gradient catalyzes both of these activities; ii) poly(A) synthetic activity co-purifies with holoenzyme and core polymerase on DNA-cellulose columns; iii) both poly(A) synthesis and RNA polymerase activity exhibit in vitro sensitivity to rifampicin; and iv) purified RNA polymerases from partially rifampicin-sensitive strains show reduced sensitivity in both RNA synthesis and poly(A) synthesis. These results indicate that poly(A) synthesis is catalyzed by the core polymerase and that at least the p subunit is required for synthesis.
Although we do not know the physiological significance of the poly(A) synthesis activity, the result is interesting because a fraction of the polysomal RNA in C. crescentus is polyadenylated at the 3'-OH terminus" (Ohta et al., 1975). A fraction of the unstable RNA in E. coli also contains poly(A) tracts at the 3'-OH end (Srinivasan et ul., 1975) and Ramanarayanan and Srinivasan (1976) have characterized an enzyme which catalyzed the polymerization of ATP and ADP, as well as 32P, exchanges into ADP and ATP. This latter enzyme is distinct, however, from the DNA-dependent RNA polymerase of E. coli.
The poly(A) synthetase activity of C. crescentus described above also differs in its properties from other poly(A) polymerases present in E. coli. One of these is a ribosome-associated enzyme that catalyzes RNA-dependent poly(A) polymerization (August et al., 1962). This enzyme is more active with Mg'+ than with Mn"+, and it is apparently not associated with the DNA-dependent RNA polymerase in these cells. Chamberlin and Berg (1964) reported a template-dependent poly(A) synthesis activity in RNA polymerase; they attributed the activity to a slippage by the RNA polymerase on the DNA template when only adenosine triphosphate is present. The purification procedure for DNA-dependent RNA polymerase used in this study was developed for C. crescentus, strain CB15. The scheme described in the miniprint supplement results in a very high recovery of purified holoenzyme and core polymerase (Table I). Although DEAE-cellulose and DNA-affinity chromatography steps described in this procedure are also used in a procedure developed independently by Amemiya et al. (1977) for the purification of RNA polymerase