Reconstitution Studies Show That Rifampicin Resistance Is Determined by the Largest Polypeptide of BaciZZus subtiZis RNA Polymerase*

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepha-rose column which bound the j3’ subunit. A phosphocellulose column was used to separate the (Y and j3 subunits. During purification, the enzyme eluted from the DNA-cel-lulose column in three separate forms in the order a&3’Fw1, @P’w’, and cu&3’w’a. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as p. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase. Escherichia is oligomer composed of two large nonidentical polypeptides

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be metalloenzymes containing 2 zinc atoms per core enzyme (9, 10). Wu et al. (11)  The gel was pre-run for 30 min at 25 mA. Samples were dialyzed against 0.0625 M Tris, 10% glycerol, 5% 2mercaptoethanol, 2% SDS, pH 6.8. They were then diluted 1:l with this same buffer including 0.002% bromphenol blue, heated for 5 min in boiling H,O and applied to the gel. It was then run between 15 and 25 mA for about 2l/2 h. It was stained and destained as discussed above.
The third system used was the anionic glycine system of Wu and Bruening (14). It was used as published, except that the acrylamide and N,N'-methylenebisacrylamide concentrations were altered as discussed in the above systems. Also, the buffer listed as upper reservoir buffer by Wu and Bruening was used as both upper and lower reservoir buffer. The system is as follows. The urea-SDS-polyacrylamide gel electrophoresis system of Wu and Bruening (14) was used. The core pattern (a) was observed in Fractions 31 to 42 and 35 to 39 of Fig. 1, a and b, respectively.
The holoenzyme pattern (b) was observed in Fractions 44 to 56 and 41 to 53 of Fig. 1, a and  good yields were required to optimally study reassociation of the subunits. The use of hemoglobin-Sepharose for the batch and column removal of proteases and peptidases which abound in extracts from B. subtilis has been described in detail under "Experimental Procedures" and by Nakayama et al. (18). The purification procedure was essentially that as described by Fukuda and Doi (22) except that the concentration of glycerol and KC1 were increased in the buffer used in chromatography on DNA-cellulose from 10% and 0.02 M to 20% and 0.1 M, respectively, and the column was washed with 0.3 M KC1 before gradient elution of the enzyme. While the yield of polymerase did not increase significantly, the modification of the buffer resulted in a much higher yield of holoenzyme relative to the core enzyme. This is shown in Fig. 1. When Fukuda and Doi (22) used 10% glycerol in their preparations about twice as much core (Fig. la, Fractions 32 to 42) as holoenzyme (Fig. la, Fractions 42 to 52)

In addition
to the subunits, (Y&P, the core fractions contained 0' and 6 (Fig. 3). These two subunits had been reported previously (29, 30). The holoenzyme fractions contained cu#pa and 0'; no 6 was present (Fig. 2c). It was also important during chromatography on DNA-cellulose to elute the column with a salt gradient to separate the core (Fig.  2~) and holoenzyme (Fig.  2b). The SDS-urea-gel electrophoresis analysis of the fractions containing the core and holoenzyme revealed additional subunit Another small polypeptide, probably o2 (51, was found loosely associated with both core and holoenzyme. While the polypeptide was found to be associated with the polymerase after sedimentation through a glycerol gradient (5), it was not associated in stoichiometric quantities with core after rechromatography on DNA-cellulose (Fig. 3). Core enzyme was rechromatographed on DNA-cellulose (Fig.  3~). The fractions containing the core enzyme were analyzed by urea-SDS-polyacrylamide gel electrophoresis (Fig. 3b). The stoichiometry of the ok' remained unchanged in the core fractions.
One o1  These conditions destabilized the holoenzyme and caused the release of (T and 6 factors.
Under these conditions, the v and 6 factors were found in the flow-through fractions from phosphocellulose while the core was eluted with Buffer E + 0.5 M KCl. The core obtained from the phosphocellulose column had a subunit composition of a&P'o'. The core was dissociated and fractionated into its subunits by two different methods.
The first method consisted of dissociating the core into subunits by treatment with urea followed by passage through a phosphocellulose column (Fig. 4). The order of elution of the subunits was (Y, /3, and /3'. Although the separation of the subunits was very effective, the recovery of the p and p' subunits was poor. The p subunit of polymerases from other bacterial species has been found to elute at a lower ionic strength than the /3' subunit (8). A more suitable method was developed which provided a rapid and effective purification of the subunits. Urea was added to core enzyme which was then dialyzed against Buffer G containing 6.5 M urea and applied to a blue dextran-Sepharose column (see "Experimental Procedures"). The (Y, wl, and j3 subunits did not bind to the column and were found in the flow-through fractions (Fig. 5) (Fig. 6b). The yield of the /3 subunit was 70 to 90% by this method.
A critical factor in obtaining good recovery of the p subunit was the immediate elution of the p subunit after the (Y subunit had eluted from the column. Recovery of the p subunit was very poor, if the a! and /3 subunits were applied slowly or allowed to remain on the column. While the fractions containing the /3 subunit were not contaminated with 01, a small amount of /? was sometimes found in the late cr fractions (Fig. 66, Fraction  19). The relative mobility of p and p' was compared by the use of three SDS-polyacrylamide gel electrophoresis systems (Fig.  7)  The circled subunits are from the rifampicin-resistant mutant. The a*/3 and p' subunits were obtained by blue dextran-Sepharose (24) column chromatography in the presence of urea. The reconstitution conditions and assay system are described under "Experimental Procedures." The subunit mixtures were kept at the temperatures for the time indicated under "Conditions" and then 5-~1 aliquots were added to 250 ~1 of the reaction mixture and incubated for 20 min at 37". When rifampicin (Rif) (1 pg) was included in the reaction mixture, it was introduced before the enzyme was added. All activities (cpm) are the averge of triplicate assays. Conditions ad3 + P' a*P + P' 2 +P' 2 -tP'  which simultaneously was asporogenous and had an altered mobility of the second largest RNA polymerase subunit in sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies. However, the nature of the mutation has not been established; some of the revertants to sporogeny remained rifampicin-resistant and had varied sporulation properties (32). Some of these revertants had core enzymes with subunits of wild type mobility. Thus from these studies alone it was not clear whether the second largest B. subtilis RNA polymerase polypeptide could unequivocally be labeled as p or /3'. Although our studies have shown that the electrophoretic properties were not a function of the specific method used, it is possible that bacterial strain differences could account for the faster mobility of the p polypeptide in the studies of Linn et al. (31).
A number of observations, summarized in Fig. 8, support labeling the largest polypeptide of B. subtilis RNA polymerase as p. The /3 subunit from each species is eluted at a lower ionic strength from phosphocellulose than the p' subunit ( Fig. 5) (12). Zinc has been reported to be associated with the p' subunit ofE. coli (11) and B. subtilis (10). The p' subunit from each species aggregates readily (5).4 The limited proteolysis of the /3' subunit of E. coli resulted in the appearance of a polypeptide with a molecular weight of 120,000, suggesting this subunit has an exposed loop (33). The proteolytic modification of the p' subunit ofB. subtilis results in the appearance of a polypeptide with the molecular weight of 110,000 (34). The p' subunit is the more basic subunit of the polymerase in all species of prokaryotes studied thus far; furthermore, the second largest polypeptide of B. cereus RNA polymerase is the most basic subunit and has been designated as /3' by Zillig (8).
The presence of 6 factor in fractions containing core but not holoenzyme is interesting since recent studies by Pero et al. (29) and Tjian et al. (35) have suggested that 6 plays an important role in promoter recognition during phage infection. The presence of 6 decreases the affinity of core in Buffer D for DNA-cellulose.
The 6 factor in vegetative cells appears to be different from the S1 and S* factors first reported in sporulating cells by Fukuda et al. (36) since the S1 and S* enzymes from sporulating cells had a much higher affinity for DNA-cellulose and had a high specific activity on several DNA templates in the absence of cr factor. The S-containing core from vegetative cells eluted at a lower ionic strength from DNA cellulose than holoenzyme (37) and had an extremely low specific activity on the poly[d(AT)] template. Although we and Plevani et al. (37) did not find 6 and (+ in the same core fractions eluted from the DNA-cellulose column, it is possible that D was dissociated from the S-containing core during DNA-cellu-

RNA Polymerase
Subunits 9031 lose chromatography. Whether o2 is an integral part of the polymerase or not is unclear as we found the association of this polypeptide to be variable. We are currently determining the relationship between the 6 factors which have been reported and the relationship between 6 and (T factor in vegetative holoenzyme.