THE JOURNAL or BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vd. 258, No. 5, Issue of March 10, pp, 32363241, 1983

Purification of Yeast RNA Polymerases Using Heparin Agarose Affinity Chromatography TRANSCRIPTIONAL PROPERTIES OF THE PURIFIED ENZYMES ON DEFINED TEMPLATES*

(Received for publication, August 9, 1982)

Charlotte I. Hammond+ and Michael J. Holland8 From the Department of Biochemistry, University of Connecticut Health Center, Farnzington, Connecticut 06032

A rapid procedure for the simultaneous purification of yeast RNA polymerases I, 11, and Ill is described. The procedure involves direct fractionation of a yeast cell extract by heparin agarose affinity chromatogra- phy, followed by glycerol gradient centrifugation and DEAE-Sephadex chromatography. The purification can be completed in 3-4 days using 20-200 g of yeast cells. Two forms each of RNA polymerases I, 11, and I11 are resolved after DEAE-Sephadex chromatography. In the cases of RNA polymerases I and 11, these forms differ in subunit structure. The transcriptional properties of the isolated enzymes were determined using hybrid plasmid DNA templates containing yeast ribosomal and glycolytic structural genes. Both forms of RNA polym- erases I and I1 transcribe plasmid DNA templates with low efficiency and no evidence for selective initiation of transcription was found for these enzymes using a wide variety of templates. Both forms of RNA polym- erase 111 transcribe plasmid DNA templates with high efficiency and direct the synthesis of discrete tran- scripts. Sites for initiation and termination of transcrip- tion by RNA polymerase 111 within defined plasmid DNA templates were determined. The data show that RNA polymerase 111-dependent synthesis of discrete transcripts from restriction endonuclease-digested plasmid DNA templates is initiated from selected ends of the templates and terminates at discrete sites down- stream from the site of initiation. RNA polymerase 211 initiates synthesis at many sites within supercoiled plasmid DNA templates.

Eucaryotic cells contain three forms of RNA polymerase which are involved in transcribing defined classes of structural genes in the cellular genome (1, 2) . The purified enzymes fail to selectively transcribe isolated genes in vitro; however, in some cases they are capable of selective transcription when reconstituted with crude cellular extracts (3-7). It is likely that the enzymes play a role in determining transcriptional specificity, although this role has not been defined.

We have been studying the transcription of isolated yeast ribosomal and glycolytic genes in vitro. A central component

*This research was supported by United States Public Health Services Grants GM 23109 and GM 30307 and a grant from the March of Dimes Birth Defects Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. + Present address, Department of Microbiology, University of Cal- ifornia, San Francisco, CA 94143.

5 American Heart Association Established Investigator. Present address, Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616.

-

for these studies are the yeast RNA polymerases. Several fractionation schemes for isolating these enzymes from yeast have been described (8-12). These methods yield pure polym- erases which are similar with respect to apparent subunit structure as well as transcriptional properties. In most cases, however, these methods are lengthy and are not well suited to isolations from small quantities of cells. Davison et al. (13) report the isolation of RNA polymerase from Bacillus subtilis using heparin agarose affinity chromatography. Their method is rapid and avoids cumbersome steps for removing nucleic acid early in the fractionation. The specific activity of the isolated B. subtiLis enzyme is significantly higher than ob- served with other preparation procedures (13) suggesting that a higher proportion of the active enzyme molecules in the extract is recovered after heparin agarose chromatography. We have used a similar approach for the isolation of the three yeast RNA polymerases from small amounts of cells.

We report here a rapid procedure for the simultaneous isolation of yeast RNA polymerases I, 11, and 111. The tran- scriptional properties of the isolated enzymes using isolated yeast ribosomal and glycolytic genes as templates are de- scribed.

EXPERIMENTAL PROCEDURES‘

RESULTS

Purification of Yeast RNA Polymerases Using Heparin Agarose Chromatography-Yeast RNA polymerases can be resolved after heparin agarose chromatography without prior removal of nucleic acid from the cell extract. A crude S35 fraction from disrupted cells is applied directly to a heparin agarose column. The purification scheme follows that, de- scribed €or the isolation of RNA polymerase from Bacillus subtilis except that DEAE-Sephadex chromatography was routinely used as a final step rather than DNA-agarose chro- matography (13). The three polymerases have been purified from as little as 20 g of cells using this procedure and the entire purification can be completed in 3-4 days. Purification of the yeast polymerases from 200 g of cells is summarized in Table I. The enzymes are recovered in approximately 20% yield and are 95% pure as judged by SDSS-polyacrylamide gel electrophoresis. The specific activities of RNA polymerases I

I The “Experimental Procedures” and additional references are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814 U. S. A. Request Document No. 82“ 2167, cite the authors, and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

‘The abbreviations used are: SDS, sodium dodecyl sulfate; kb, kilobase pair.

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TABLE I Purification of yeast RNA polymerases I, II, and III

Enzyme activity units were determined as described under “Ex- perimental Procedures.” The values reported are an average of two - preparations from 200 g of yeast cells.

Fractionation step RNtrE:2m- y2i Units Protein Specific Yi activity ml mg ‘I,

s35 315 1189 5726 0.21 Heparin agarose I1 216 607 101 6 100

I and 111 211 511 60 8.5 100 Glycerol gradient I1 74 372 14 26 61

1 and 111 58 195 6.3 31 38 DEAE-Sephadex Ila and IIb 34 141 0.82 172 23

Ia and Ib 52 80 0.77 104 16 IIIb 18 13 <0.01 ND“ 2

‘‘ ND, not determined.

and I1 isolated by this procedure are comparable to values reported for other procedures (8-12). Insufficient quantities of protein were obtained in fractions containing RNA polymer- ase I11 for accurate determination of specific activity. It is clear, however, that the specific activity of the isolated RNA polymerase I11 is at least two orders of magnitude higher than those determined for RNA polymerases I and IT (discussed below). The general properties of the isolated enzymes are similar to those reported previously (8-12). Only RNA polym- erase I1 is inhibited when 50 pg/ml of a-amanitin is included in the assay reaction. RNA polymerases I and I1 are inactive when assayed in the presence of 0.2 M (NH&S04, while RNA polymerase 111 retains 90% of its activity under these condi- tions. These latter properties have been used to distinguish the three enzymes at early stages in the purification.

Preparation of the Cellular Extract-Midlog phase yeast cells (200 g) are disrupted with an Eaton press in buffer A which does not contain either (NHJ2SO4 or KCI. Greater than 85% of the RNA polymerase activity present in this crude cell lysate is recovered in the supernatant fraction after centrifu- gation at 35,000 X g for 30 min. An additional 10% of the RNA polymerase activity can be recovered from the pellet after extraction with an equal volume of buffer A. Small amounts of RNA polymerase activity can be obtained on further ex- traction of the pellet with buffer A containing 0.35 M (NH4)2S04. Aliquots of supernatants obtained after two ex- tractions with buffer A and after an extraction with buffer A containing 0.35 M (NH4)2S0, were assayed in the presence of 0.2 M (NH&S04 and in the presence of 50 pg/ml of a-amanitin. The proportions of polymerase activity which are inhibited by a-amanitin or 0.2 M (NH&S04 were the same in all three aliquots indicating that all three forms of yeast RNA polym- erase are extracted by this procedure with similar efficiency. The observed proportions of activity for RNA polymerases I, 11, and I11 in the S35 fraction are 45:45:10, respectively.

The RNA polymerases present in the S35 fraction are associated with nucleic acid since they can be quantitatively precipitated by adjusting the extract to 0.25% protamine sul- fate. The three forms of RNA polymerase can be recovered from the protamine sulfate precipitate by extraction with buffer containing 0.25 M (NH4)2S04. It is important to note that removal of nucleic acid with protamine sulfate or by ion fitration chromatography on DEAE-Sephadex prior to hep- arin agarose chromatography leads to extremely poor resolu- tion of the polymerases and alterations in the order of elution of the different forms of polymerase on heparin agarose. Based on these observations, it is likely that competition for binding RNA polymerases between nucleic acid in the extract and heparin residues on the column plays a significant role in the

observed resolution of the enzymes after heparin agarose chromatography.

Heparin Agarose Chromatography-The supernatant frac- tion (S35) is applied directly to a heparin agarose column and the RNA polymerases are eluted with a linear KC1 gradient. Routinely, 90-95% of the RNA polymerase activity in the supernatant fraction binds to the column. The activity which does not bind the column is resistant to 50 pg/ml of the toxin a-amanitin and is 90% active when assayed in the presence of 0.2 M (NH&S04. The amount of activity present in the flow through fraction is also inversely proportional to the amount of RNA polymerase I11 recovered after DEAE-Sephadex chro- matography as described below. Based on these data, it is likely that this unbound fraction contains RNA polymerase 111.

Two fractions of RNA polymerase are reproducibly resolved after heparin agarose chromatography (Fig. 1). The first frac- tion, which elutes at 0.3 M KCl, is completely inhibited in assays containing 50 pg/ml of a-amanitin and in assays con- taining 0.2 M (NH4)2S04. Based on these data and those described below, this fraction contains RNA polymerase 11. The second fraction, eluting a t 0.45 M KCl, contains RNA polymerase activity which is resistant to inhibition by 50 pg/ ml of a-amanitin. Approximately 10-158 of the activity deter- mined in assays containing 50 mM (NH4)zS04 is retained in assays containing 0.2 M (NH4)2S04. These data as well as those presented below indicate that this fraction contains a mixture of RNA polymerases I and 111. Heparin agarose chromatography results in a 50-100-fold purification of the RNA polymerase which binds the column. The two fractions of RNA polymerase are pooled separately and concentrated by ultrafitration. The concentrated fractions are then further purified by glycerol gradient centrifugation.

Glycerol Gradient Centrifugation-A 10-fold purification of each of the fractions from heparin agarose is obtained after glycerol gradient centrifugation. Representative gradients of the fractions containing RNA polymerase I1 and RNA polym- erases I and I11 are shown in Fig. 2, A and B, respectively. A 40-60% recovery of polymerase activity is obtained after this purification step. Fractions containing RNA polymerase are pooled and diluted with buffer B in order to bring the KC1 concentration to 50 mM. The diluted fractions are applied directly to DEAE-Sephadex or DNA-cellulose columns.

DEAE-Sephadex Chromatography-A chromatogram of

- 210

1 I50 - M I O

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-I 5 60 20 - Y)

0

5 30 I O 3

0 5 0 5

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0 20 40 60 80 I d 0 I20 I40 1 6 0 i o

FRACTION NUMBER

FIG. 1. Heparin agarose chromatography. The supernatant of a crude extract (S35) from 200 g of yeast cells was chromatographed on a heparin agarose column (32 X 3.2 cm) as described under “Experimental Procedures.” RNA polymerases were eluted with a linear (0-1.0 M) KC1 gradient. RNA polymerase activity was measured using a mixture of denatured and native salmon sperm DNA (M) and in the presence of 50 p g / d of a-amanitin (W) or 0.2 M (NH4)2S04 (M).

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3232 Yeast RNA Polymerases

A.

450

3.0

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2 250-

-I

-

z 0

I s! N 2.0

t 3 200-

I50

50

4

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i I I I I , I I , I I I

2 4 6 8 IO I2 14 16 18 20 22 24

FRACTION NUMBER

enzyme is identified as RNA polymerase I1 and is a contami- nant derived from the pooled fractions after heparin agarose chromatography. The fourth fraction of RNA polymerase is not inhibited by a-amanitin and is active when 0.2 M (NH4)&304 is included in the assay. Based on these properties as well as subunit structure analysis, this fraction contains RNA polymerase 111. Rechromatography of fractions contain- ing RNA polymerase Ia or Ib on DEAE-Sephadex does not result in changes in the relative proportions or chromato- graphic properties of the two forms of RNA polymerase I in the respective fractions. These data argue against the possi- bility that multiple forms of the enzyme are generated as a consequence of chromatography on DEAE-Sephadex.

RNA polymerase activity which is not inhibited when 0.2 M (NH4)d304 is included in the assay is routinely observed eluting from the DEAE-Sephadex column at a slightly higher (NH4),S04 concentration than RNA polymerase Ib. Co-chro- matography of yeast RNA polymerases I and I11 on DEAE-

A.

140

120- - 1 0.6-

100- 2 - a a

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80- 5 0.4- w 0

60- 8 - 0

40- $02-

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700 1 1 P

FRACTION NUMBER

FIG. 2. Glycerol gradient centrifugation. Glycerol gradients were centrifuged for 36 h at 24,000 rpm in a Beckman SW27 rotor at 4 “C. A , sedimentation profile of fractions containing RNA polymer- ase I1 activity after heparin agarose chromatography. B , sedlmenta- tion profie of fractions containing a mixture of RNA polymerases I and 111 after heparin agarose chromatography.

the fraction containing RNA polymerases I and 111 after DEAE-Sephadex chromatography is shown in Fig. 3A. Four forms of RNA polymerase are reproducibly obtained at this stage of the purification. The major forms of polymerase elute at 0.1 M and 0.14 M (NH4)2S04. The RNA polymerase activity contained in these fractions is resistant to inhibition by 50 pg/ ml of a-amanitin. Based on their apparent subunit structures described below, these forms have been designated RNA polymerases Ia and Ib, respectively. A third form of RNA polymerase eluting at 0.18 M (NH&SO4 is completely inacti- vated when 50 pg/ml of a-amanitin is included in the assay. Based on these observations and the chromatographic prop- erties of RNA polymerase I1 shown below, this form of the

x 300

-1 200

u) c z 100

0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

FRACTION NUMBER

FIG. 3. DEAE-Sephadex chromatography. Fractions contain- ing RNA polymerase activity after glycerol gradient centrifugation were chromatographed on DEAE-Sephadex columns (11 X 2.5 cm) as described under “Experimental Procedures.” RNA polymerases were eluted with linear (0.05 “0.72 M ) (NH4)2S04 gradients. RNA polym- erase activity was measured using a mixture of denatured and native salmon sperm DNA (W) and in the presence of 50 pg/ml of a- amanitin (.”--.) or 0.2 M (NH,),SO4 (-). A, chromatogram of fractions containing RNA polymerases I and 111. B, chromatogram of fractions containing RNA polymerase 11.

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Sephadex has been observed previously (12). Since activity is retained in the presence of 0.2 M (NH&S04, this activity was tentatively identified as RNA polymerase 111. This identifi- cation was confi ied by further chromatography of this frac- tion on DNA-cellulose. The activity which is not inhibited by 0.2 M (NH4)2S04 in this fraction is completely resolved from RNA polymerase I after DNA-cellulose chromatography and elutes at a position in the gradient expected for RNA polym- erase I11 (data not shown). It is concluded, therefore, that two forms of RNA polymerase 111 are resolved after DEAE-Seph- adex chromatography. They are designated RNA polymerases IIIa and IIIb based on their order of elution from the column.

DEAE-Sephadex chromatography of the fraction contain- ing RNA polymerase I1 after glycerol gradient centrifugation results in the resolution of two forms of RNA polymerase (Fig. 323). Both are inactivated by 50 p g / d of a-amanitin and 0.2 M (NH4)&04. Based on these data and the subunit struc- tures described below, both forms are RNA polymerase IT. These forms of RNA polymerase I1 are designated IIa and IIb based on their order of elution from the column.

DNA-cellulose Chromatography-RNA polymerases I and 111 are resolved after DNA-cellulose chromatography of pooled fractions from glycerol gradients (Fig. 4A). RNA po- l-merase I elutes at 0.13 M KC1 and RNA polymerase 111 elutes at 0.32 M KCI. In contrast to the results after DEAE- Sephadex chromatography, multiple forms of RNA polymer- ases I and I11 are not resolved on DNA-cellulose. DNA- cellulose chromatography of fractions after glycerol gradient centrifugation containing RNA polymerase I1 (Fig. 4B) also results in resolution of a single form of RNA polymerase I1 which binds the column weakly eluting at 0.075 M KC1.

RNA polymerases I and I1 isolated after DEAE-Sephadex or DNA-cellulose chromatography are stable for 1-2 months when stored at concentrations 1 mg/ml or greater in buffer A containing 50% glycerol at -20 “C. RNA polymerase IIIb loses activity under these buffer conditions with a half-life of several days. It is likely that this instability is related to the low concentrations of protein in the preparation since RNA po- lymerase IIIa activity contained in fractions with RNA polym- erase Ib is stable for several weeks under these conditions.

Subunit Structures of Purified Yeast RNA PoZymerases- Fractions containing RNA polymerase activity after DEAE- Sephadex chromatography were analyzed on polyacrylamide gels in the presence of SDS. The elution profiles from the DEAE-Sephadex columns used for this analysis are illustrated in Fig. 5, A and C. SDS-polyacrylamide gels of fractions containing RNA polymerases la, Ib, IIIa, and IIIb are shown in Fig, 5B. The apparent subunit structures of RNA polym- erases Ia and Ib are extremely similar to those reported using several different purification procedures (9, 11, 12, 14). Both forms of the enzyme contain the 44,000- and 36,000-dalton polypeptides which are present in variable amounts in differ- ent preparations of the enzyme from yeast. The only obvious difference in subunit structure between Ia and Ib appears to be the presence of a 48,000-dalton polypeptide associated with Ib but not Ia. This subunit of the enzyme is absent from some preparations of the enzyme (12) and is thought to be required for efficient transcription of native DNA templates (11, 15).

No evidence of subunits characteristic of RNA polymerase 111 are observed in fractions containing Ib and IIIa. It is likely that these subunits are not present at sufficiently high con- centrations to be detected. It was necessary to analyze ap- proximately 40% of the RNA polymerase IIIb isolated from 200 g of cells on a single lane of the slab gel (Fig. 5B)in order to visualize the subunits of RNA polymerase IIIb. Three subunits characteristic of RNA polymerase 111 (1fjO,ooo, 130,- 000, and 85,000 daltons) are detected confirming that this

A.

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0 10 20 30 40 50 60 70 80 90 1 0 0 FRACTION NUMBER

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0 IO 20 30 40 50 60 TO 80 90 100

FRACTION NUMBER

FIG. 4. DNA-cellulose chromatography. Fractions containing RNA polymerase activity after glycerol gradient centrifugation were chromatographed on DNA-cellulose columns as described under “Experimental Procedures.” RNA polymerases were eluted with lin- ear (0.02-2.0 M) KC1 gradients. RNA polymerase activity was meas- ured using a mixture of denatured and native salmon sperm DNA (o”--o) and in the presence of 50 pg/ml of u-amanitin (w) or 0.2 M (NH4)&30, (M). A, chromatogram of fractions containing RNA polymerases I and III. B, chromatogram of fractions containing RNA polymerase 11.

form of the enzyme is RNA polymerase 111 (10, 12). There was insufficient protein in the preparation to make a complete analysis of the subunit structure of the enzyme. As noted below, we are unable to accurately measure the specific activ- ity of RNA polymerase IIIb because of the low levels of protein present in the Preparation. Based on the amounts of stainable protein observed on SDS gels, it is likely that the specific activity of RNA polymerase I11 isolated by this method is at least two orders of magnitude greater than determined for RNA polymerases I or 11.

SDS-polyacrylamide gels of fractions containing RNA po- lymerases IIa and IIb are shown in Fig. 50. The apparent subunit structures of these forms of the enzyme are extremely similar to those reported for other preparations of yeast RNA polymerase I1 (8, 12). Two polypeptides appear to be associ- ated with both forms of the enzyme which have not been described previously. The location of these polypeptides, 45,000 and 11,000 daltons, respectively, are indicated in Fig. 5 0 . The single striking difference between the subunit struc- tures of the two forms of the enzyme is the presence of the 24,000-dalton polypeptide in fractions containing RNA polym-

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A

FRACTION NUMBER

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FRACTION NUMBER

MW

185- 137-

-85

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12-

44 45 46 47 48 49 50 51 52 I IIb Fraction No.

k

14.5- 17-

12.5- (11)-

62 63 64 65 66 67 68 69 Fraction No.

FIG. 5. SDS-polyacrylamide gel electrophoresis of RNA polymerases after DEAE-Sephadex chroma- tography. Fractions containing RNA polymerase activity after DEAE-Sephadex chromatography were analyzed on SDS-polyacrylamide gels as described under "Experimental Procedures." The arrows indicate the interface between the 8.75% and 12.5% polyacrylamide portions of the gels. A, chromatogram of RNA polymerases I and 111 after DEAE-Sephadex chromatography. B, SDS-polyacrylamide gel of fractions containing RNA polymerase I activity after DEAE-Sephadex chromatography and a pooled fraction containing RNA polymerase 111 activity. This later fraction contains approximately 40% of the RNA polymerase IIIb activity recovered from 200 g of yeast cells. C, chromatogram of RNA polymerase I1 after DEAE-Sephadex chromatography. D, SDS-polyacrylamide gel of fractions containing RNA polymerase I1 activity after DEAE-Sephadex chromatography. The molecular weights of the polypeptides were determined using RNA polymerase purified by the method of Hager et al. (12) as markers. The molecular weights of polypeptides associated with RNA polymerase I1 which have not been described previously are indicated in parentheses. All molecular weights are times

erase IIb but not IIa. Although this latter polypeptide has DEAE-Sephadex chromatography. These latter polypeptides always been identified in RNA polymerase I1 preparations are not present in amounts proportional to the known subunits isolated by different methods, we observed no significant of the enzyme and are presumed to be contaminants. The difference in specific activity for IIa and IIb when assayed largest subunit of RNA polymerases IIa and IIb (170,000 with salmon sperm DNA. Three high molecular weight poly- daltons) is likely to be a proteolyzed form of a larger subunit peptides in addition to the 170,000- and 145,000-dalton sub- described by Dezelee et al. (16). Although there are small units of RNA polymerase I1 are present in the fractions after amounts of a polypeptide which is larger than the 170,000-

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Yeast RNA Polymerases 3235

dalton polypeptide in the preparation, it is clear that the majority of the enzyme isolated here contains a proteolyzed subunit despite the fact that log-phase cells were used and phenylmethylsulfonyl fluoride was included in the prepara- tion.

The subunit structures of RNA polymerase I isolated after DNA-cellulose chromatography are similar to those described above. RNA polymerase I1 isolated after DNA-cellulose con- tains the same apparent subunit structure as the enzyme isolated after DEAE-Sephadex chromatography except that the major high molecular weight contaminant present in the latter preparation (95,000 daltons) was absent from the prep- aration after DNA-cellulose chromatography. RNA polymer- ase I isolated after DNA-cellulose chromatography contains all of the polypeptides described here except that the 48,000- dalton polypeptide is present in substoichiometric amounts.

Isolation of Defined Templates for Yeast RNA Polymer- ases I, 11, and 111-Yeast 5 S, 18 s, 5.8 S, and 25 S rRNAs are encoded by a 9.7-kb cistron which is repeated tandemly in the yeast genome (17-20). The physical structure and location of sequences encoding each rRNA within the cistron has been determined (19, 20). Sites for initiation of transcription by RNA polymerases I and I11 are contained within the cistron. For this reason, we isolated hybrid plasmids containing yeast rDNA to serve as templates for purified RNA polymerases I and 111. Plasmids containing large segments of yeast rDNA were isolated from a collection of transformants in which randomly sheared yeast DNA was joined into the Eco RI site of the vector pSF2124 using the A:T joining method (21). Two of the isolated plasmids, designated pribl05 and prib20, were analyzed by restriction endonuclease mapping (Fig. 6). In the case of pribl05, a segment of rDNA extending from a site within a 2.4-kb Eco RI fragment to a second 2.4-kb Eco RI fragment was isolated. The plasmid prib20 contains yeast rDNA between sites within two 2.1-kb Eco RI fragments in adjacent cistrons. These two plasmids were analyzed in par- allel since they contain all of the sequences present in a complete cistron.

Three plasmids containing small portions of yeast rDNA were generated from these larger plasmids. The plasmid pribBl was constructed by ligating the 2.4-kb Eco RI fragment from prib20 into the Eco RI site of pSF2124 (Fig. 6). The 2.4- kb Eco RI fragment contains the 5 S rRNA structural gene (19, 20) as well as the putative initiation site for RNA polym- erase I. The plasmid pribl05R was derived from pribl05 by limit digestion with Eco RI followed by ligation to join the two distal Eco RI sites in prib105. The plasmid prib105H was derived from pribl05 by limit digestion with Hind111 followed by ligation to join the two distal Hind111 sites in prib 105. The plasmid pgap49 1 contains a yeast glyceraldehyde-3-phosphate dehydrogenase structural gene (22) and was used as a template for purified RNA polymerase 11.

Transcription of Defined Templates by Purified Yeast RNA Polymerases I, 11, and 111-RNA polymerases IIa and IIb isolated after DEAE-Sephadex chromatography as well as RNA polymerase 11 isolated after DNA-cellulose chromatog- raphy transcribe supercoiled as well as linear plasmid DNA templates with low efficiency. The template efficiency with defined templates was less than 5% that observed with salmon sperm DNA in assays containing M e or Mn’+. Transcripts synthesized from supercoiled pgap491, Sma I-digested pgap491, Sma I-digested pSF2124, and Bum HI-digested SV40 DNA were analyzed on polyacrylamide gels in the presence of 7 M urea. No discrete transcripts were observed with the linear DNA templates. Minor discrete transcripts were observed with supercoiled templates; however, the ma- jority of the transcripts synthesized from the supercoiled

pSF2124 k : : : I 4 8 8 3 2 8 s 4 8 8 1

k t 4

2 Eco RI I. earn H I

3 Srna I

5 Sac I 4 891 I

3 9 711 69 311674

priJ 20 %2 2 2 2 2

J’D’ 1 A ‘;.E“ 18

priJ 8,

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FIG. 6. Restriction endonuclease cleavage maps of plasmids containing yeast ribosomal DNA and a yeast glyceraldehyde- 3-phosphate dehydrogenase structural gene. Yeast DNA se- quences were cloned into the Eco RI cleavage site in the vector pSF2124 as described under “Experimental Procedures.” The plasmid pgap491 contains a yeast glyceraldehyde-3-phosphate dehydrogenase structural gene. The location of this gene and the direction of tran- scription are indicated by the shaded arrow. The plasmids pribl05 and pri620 contain segments of yeast rDNA. The restriction endo- nuclease cleavage maps of the rDNA portions of these two plasmids have been aligned to indicate overlapping rDNA sequences. The plasmid prib B1 contains the 2.4-kb Eco RI fragment of yeast rDNA isolated from prib20 ligated into the Eco RI site of pSF2124. The location of the gene coding for 5 S rRNA in prib B1 and its direction of transcription are indicated by a shaded arrow.

templates were not of discrete size. No discrete truncated transcripts were synthesized from pgap491 after digestion with restriction endonucleases which cleave downstream from the transcriptional initiation site for the glyceraldehyde-3- phosphate dehydrogenase gene contained within the plasmid. We conclude from these data that RNA polymerase TI pre- pared by the method described above does not differ signifi- cantly in its transcriptional properties from previous prepa- rations of this enzyme from yeast.

The transcriptional properties of RNA polymerase Ia after DEAE-Sephadex chromatography and RNA polymerase I isolated after DNA-cellulose chromatography are similar to those described above for RNA polymerase 11. The purified enzyme transcribes supercoiled and linear plasmid DNA tem- plates containing yeast rDNA with 5-10% the efficiency ob- served with salmon sperm DNA in the presence of either Mg“+ or MnZ+. No discrete transcripts were observed on polyacrylamide gels in the presence of 7 M urea when super- coiled pribl05, przb20, przbBl, or pSF2124 was used as tem- plate. Cleavage of these templates at a number of sites within the ribosomal cistron did not result in synthesis of discrete truncated transcripts.

In contrast to the results obtained with RNA polymerases I and 11, RNA polymerase IIIa (a mixture of RNA polymerases Ib and IIIa) and IIIb direct the efficient synthesis of discrete transcripts from plasmid templates which are linearized by digestion with restriction endonucleases. Minor discrete tran- scripts were also synthesized from supercoiled templates. The size and number of discrete transcripts was dependent on the template used in the assay and the restriction endonuclease used to cleave the template. The transcripts synthesized by RNA polymerases IIIa and IIIb when Eco RI-digested przbBl is used as template are illustrated in Fig. 7A. Several discrete transcripts are synthesized. The predominant transcript is approximately 110 nucleotides in length. The transcripts syn- thesized by the two forms of RNA polymerase I11 are quan-

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3236 Yeast RNA Polymerases

titatively indistinguishable suggesting that although they dif- fer in chromatographic properties, their transcriptional prop- erties are similar. These data also demonstrate that RNA polymerase Ib which is present in the RNA polymerase IIIa preparation contributes little if anything to the pattern of transcripts synthesized.

Since pribBl and prib20 contain a 5 S rRNA structural gene, the possibility that transcripts synthesized by RNA polymerase I11 are initiated in this gene was tested. The plasmid prib20 and the vector pSF2124 were digested with the restriction endonuclease Pst I. Both plasmids contain six Pst I cleavage sites which are all located within the vector sequences. As illustrated in Fig. 7B, both templates direct the synthesis of the same array of discrete transcripts when tran- scribed by RNA polymerase IIIa. These data demonstrate that none of the discrete transcripts observed is dependent on yeast rDNA sequences.

The transcriptional properties of the RNA polymerases were analyzed after each step in the fractionation described above in order to determine if these properties changed as a function of purification. Only those fractions containing RNA polymerase I11 directed the synthesis of discrete transcripts from plasmid DNA templates. The transcripts synthesized by fractions containing RNA polymerase I11 after each step in the purification procedure from a number of supercoiled and restriction endonuclease digested plasmids containing yeast are shown in Fig. 8. The same array of discrete transcripts was synthesized from each template after each step in the fractionation. These data suggest strongly that the transcrip- tional properties of RNA polymerase I11 observed here are not altered significantly during the fractionation procedure. RNA polymerase IIIa isolated after DEAE Sephadex chro-

A.

* I i’

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- 4s-

A B p2124J LQrd20

FIG. 7. RNA polymerase 111-dependent synthesis of discrete transcripts from restriction endonuclease-cleaved plasmid DNA templates. Transcripts were synthesized in the presence of [a-’”PJUTI’ and analyzed on polyacrylamide gels in the presence of 7 M urea as described under “Experimental Procedures.” A, an auto- radiogram of transcripts synthesized from Eco HI-digested prib B1 by RNA polymerase IIIa ( A ) and IIIb ( B ) isolated after DEAE- Sephadex chromatography. The location of unlabeled yeast 5 S rRNA and tRNA (4 S ) markers is indicated. B, an autoradiogram of tran- scripts synthesized from Pst I-digested pSF2124 and Pst I-digested prib20 by RNA polymerase IIIa isolated after DEAE-Sephadex chro- matography. The arrow indicates the interface between the 2.5% and 4% polyacrylamide portions of the gel. The locations of unlabeled yeast 25 S, 18 S , and 5 S rRNA and tHNA (4 S ) markers are indicated.

matography does synthesize a number of minor discrete tran- scripts when supercoiled pribl05R and supercoiled pribl05H were used as template (Fig. 8). These latter transcripts were not reproducibly observed when enzyme fractions after hep- arin agarose chromatography or glycerol gradient centrifuga- tion were assayed. It is likely that this difference in apparent transcriptional properties is due to the fact that the fractions after DEAE-Sephadex chromatography do not contain nu- cleases which convert the supercoiled template to nicked circular DNA during the reaction. Nucleases capable of this conversion of the template are present in fractions after hep- arin agarose chromatography and glycerol gradient centrifu- gation.

RNA polymerase 111-dependent synthesis of discrete tran- scripts from supercoiled and restriction endonuclease-digested plasmid DNA templates is linear for a t least 2 h at 30 “C. These data suggest that the enzyme is capable of reinitiating synthesis on template DNA. Synthesis increased linearly over a 20-fold range of RNA polymerase I11 concentration and activity was not saturated a t 100 pg/ml of template in the reaction mixture. The template efficiency with plasmid DNA (supercoiled or linear) was in all cases comparable to or greater than that observed with denatured plus native salmon sperm DNA when assayed under the same conditions.

RNA Polymerase ZZZ Initiates Synthesis from the Ends of Restriction Endonuclease-digested Plasmid DNA Tem- plates-RNA polymerase 111-dependent synthesis of discrete transcripts from restriction endonuclease-digested templates requires that initiation and termination of RNA synthesis occur a t discrete sites within the template. Synthesis could be initiated from sites within the template followed by termina- tion at restriction endonuclease cleavage sites (truncation). Alternatively, transcription may be initiated from the ends of the template DNA followed by termination at a discrete site within the template. The data described above do not distin- guish these possibilities. In order to define sites of initiation and termination of transcription by RNA polymerase 111, transcripts were synthesized and analyzed from the plasmid prib105R after cleavage with Eco RI, HindIII, and Hpa I. Reactions contained RNA polymerase IIIa isolated after DEAE-Sephadex chromatography. The plasmid pribl05R is cleaved once by each enzyme. The three cleavage sites are present within a 300-base-pair portion of the yeast rDNA sequences within the plasmid (a restriction endonuclease cleavage map of this region is shown in Fig. 11).

Each template directs the synthesis of major discrete tran- scripts (Fig. 9). Cleavage with Eco RI gives rise to a 110- nucleotide transcript. HindIII-digested plasmid directs the synthesis of 160-, 250-, 315-, and 375-nucleotide transcripts. Hpa I-digested plasmid directs the synthesis of 115-, 150-, 290-, 385-, and 440-nucleotide transcripts. To determine the approximate location of sequences in the template which encode these transcripts. pribl05R was transcribed after cleavage with two restriction endonucleases (Fig. 9). Template digested with Eco RI and HindIII directs the synthesis of the 110-, 160-, and 250-nucleotide transcripts but not the 315- and 375-nucleotide transcripts synthesized from HindIII-digested template. These data suggest that Eco RI cleaves within sequences which encode these latter transcripts. Eco RI- and Hpa I-digested pribl05R directs the synthesis of the 110-, 115-, 150-, and 290-nucleotide transcripts, but not the 385 and 440 nucleotide transcripts synthesized from Hind I11 digested pribl05R. These data are,consistent with Eco RI cleavage within sequences encoding these latter transcripts. A new transcript (300 nucleotides) is synthesized after cleavage of the template with Eco RI and Hpa I. Finally, cleavage of the template with HindIII Hpa I results in the synthesis of the

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FIG. 8. Synthesis of discrete transcripts from plasmid DNA templates by RNA polymerase 111 after heparin agarose chromatography, glycerol gradient centrifugation, and DEAE-Sephadex chromatog- raphy. Transcripts were synthesized in the presence of [a-:"P]UTP, electrophoresed on polyacrylamide gels in the presence of 7 M urea, and analyzed by autoradiography as described under "Experimental Procedures." Fractions containing RNA polymerase 111 after heparin agarose chromatography and glycerol gradient centrifugation were compared to RNA polymerase IIIa isolated after DEAE-Sephadex chromatography. The templates used were: A, pribl05K digested with Eco HI; a, pribl05R digested with Ero HI and Sma I; B, pribl05H digested with HindIII; 6, pribl05H digested with HindIII and Sma I; C, supercoiled pribl05H; D, supercoiled pribl05H; E , pSF2124 digested with Bam HI; F, pribl05H digested with Barn HI; and C, the purified 2.4-kb h'ro HI fragment of yeast rDNA. The arrows indicate the interfaces between the 2.5% and 4% polyacrylamide portions of gels. The locations of unlabeled yeast 25 S, 18 S, and 5 S rRNA and tRNA (4 S) markers are indicated.

115-, 160-, 315-, and 375-nucleotide transcripts but not the 150-, 290-, 385-, or 440-nucleotide transcripts synthesized from Hpa I-digested template or the 250-nucleotide transcript syn- thesized from HindIII-digested prib105R. A new 130-nucleo- tide transcript is synthesized from the template. In the case of template digested with Eco RI and HindIII, the new transcript synthesized (300 nucleotides) corresponds in size to the dis- tance between the Eco RI and HindIII cleavage sites in the template. The new 130-nucleotide transcript synthesized from pribl05R after digestion with HindIII and Hpa I also corre- sponds to the distance between the HindIII and Hpu I cleav- age sites in the template.

The data described above are consistent with the hypothesis that the 385- and 440-nucleotide transcripts synthesized from Hpa I-digested pribl05R are initiated from one end of the Hpa I-cleaved template and that Eco RI digestion truncates these transcripts to form the new 300-nucleotide transcript. Similarly, the 150-, 290-, 385-, and 440-nucleotide transcripts synthesized from Hpa I-digested pribl05R appear to be trun- cated to form the 130-nucleotide transcript when the template is further digested with HindIII. The 250-nucleotide transcript synthesized from HindIII-digested pribl05K is lost after cleav- age with Hpa I. Since this transcript was not abolished by digestion of HindIII-digested template with Eco RI, it is likely that synthesis of this transcript is initiated from one end of the HindIII-cleaved template and is truncated by cleavage with Hpa I to form a 130-nucleotide transcript. A similar argument can be made for the loss of the 315- and 375- nucleotide transcripts synthesized from HindIII-digested prihl05R after further digestion with Eco RI. In this latter case, no new truncated transcripts were detected, however. Since the distance between the Eco RI and HindIII cleavage sites in the templates is approximately 160 base pairs, it is possible that the putative truncated transcript co-electropho-

reses with the 160-nucleotide transcript synthesized from HindIII-digested pribl05R.

It is clear that digestion of pribl05R with specific restriction endonucleases gives rise to the synthesis of a unique array of discrete transcripts by RNA polymerase 111. The fact that new transcripts synthesized from templates digested with two of the restriction endonucleases correspond in size to the distance between the two cleavage sites in the templates strongly suggests that transcription is initiated from the ends of the template. To confirm this interpretation of the data, "'P-labeled discrete transcripts synthesized from each tem- plate were isolated and hybridized against DNA filter blots containing separated strands of the 2.4-kb Eco RI fragment of yeast rDNA in order to determine the direction of synthesis of each transcript. The transcripts were also hybridized against DNA filters containing Hue I11 fragments derived from the 2.4-kb Eco RI fragment of yeast rDNA or fragments generated after cleavage of pribl05R with HindIII and Smu I to determine the location of sequences in the template which encode each transcript.

Examples of the blotting data are shown in Fig. 10. The strand of the 2.4-kb Eco RI fragment which encodes yeast 5 S rRNA was identified by hybridization against '"P-labeled yeast 5 S rKNA (Fig. 10) and is arbitrarily designated the minus strand. As predicted from the analysis described above, the 250-nucleotide transcript synthesized from HindIII-di- gested prihl05R hybridizes to the plus strand of the fragment. The 290-nucleotide transcript synthesized from Hap I-di- gested pribl05R hybridizes to the minus strand of the frag- ment. Cleavage of the 2.4-kb Eco RI fragment of yeast rDNA with Hae I11 generates three fragments, 1.1,0.9, and 0.4 kb in size. Only the 1.1- and 0.9-kb fragments were analyzed since the 0.4-kb fragment transferred poorly to nitrocellulose. The 110-nucleotide transcript synthesized from the purified 2.4-kb

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FIG. 9. RNA polymerase 111-dependent synthesis of discrete transcripts from restriction endonuclease-cleaved pribl05R. Transcripts were synthesized in the presence of [a-’”P]UTI’ using RNA polymerase IIIa isolated after DEAE-Sephadex chromatogra- phy, electrophoresed on a polyacrylamide gel in the presence of 7 M urea, and analyzed by autoradiography as described under “Experimental Procedures.” The templates analyzed were: A, pribl05H digested with Eco RI; B, prihl05R digested with Eco RI and HindIII; C, pribl05R digested with HindIII; D, pribl05H digested with Hpa I; E , pribl05H digested with Eco RI and Hpa I; and F, pribl05H digested with HindIII and Hpa I. The molecular weights of the transcripts were determined using a series of restriction endonu- clease cleavage fragments as markers. The location of unlabeled yeast 25 S, 18 S, and 5 S rRNA markers is indicated. The arrow indicates the location of the interface between the 2.5b and 4 8 polyacrylamide portions of the gel. The molecular weights of new truncated tran- scripts are shown in parentheses.

Eco RI fragment hybridized only to the 1.1-kb fragment demonstration, together with the data shown in Fig. 9, that sequences encoding this transcript are located between the Eco RI and HindIII cleavage sites in the template. Analysis of total ’”P-labeled RNA synthesized from the purified 2.4-kb Eco RI fragment showed that the majority of the RNA synthesized hybridizes to the 1.1-kb Hue I11 fragment. These latter data confirm that the majority of the RNA synthesized from this template is the 110-nucleotide transcript. The 110- nucleotide transcript hybridizes to the plus strand of the 2.4- kb Eco RI fragment.

The transcripts synthesized from pribl05R after digestion with either HindIII or Hpa I were isolated and hybridized against the separated strands of the 2.4-kb Eco RI fragment and the three fragments generated after cleavage of pribl05R with HindIII and Srna I. The data are summarized in Table 11. All of the transcripts tested hybridized with the minus strand of the 2.4-kb Eco RI fragment except the 250-nucleo- tide transcript synthesized from HindIII-digested pribl05R which hybridizes with the plus strand. These data establish the direction of transcription of these transcripts. The 160-, and 315, 375-nucleotide transcripts synthesized from the HindIII-digested template hybridized only with the 0.4-kb fragment which extends from the HindIII site to a Srna I site within the yeast rDNA sequences in pribI05R. The 250-nu- cleotide transcript hybridizes to the 7.2-kb fragment which extends from the HindIII site to an Srna I cleavage site within

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by RNA polymerase 111. Transcripts were synthesized in the pres- ence of [u-,’”’P1UTP using HNA polymerase IIIa and isolated as described under “Experimental Procedures.” The isolated transcripts were hybridized against DNA filter blots containing the terminal 1.1- kb and 0.9-kb fragments which extend from Kc0 HI sites to Hae I11 sites in the 2.4-kb Isco RI fragment of yeast rDNA and the separated strands of the 2.4-kb Eco RI fragment of yeast rDNA. a, the separated strands of the 2.4-kb Eco HI fragment visualized by ethidium bromide staining before transfer to nitrocellulose. 6, ‘“1’-labeled yeast 5 S rRNA hybridized against a nitrocellulose filter containing the strands of the 2.4-kb Eco HI fragment. c, “’P-labeled 250-nucleotide transcript synthesized from HindIII-digested pribl05H hybridized against the separated strands of the 2.4-kb Eco RI fragment. d. ‘”P-labeled 290- nucleotide transcript synthesized from Hpa I digested pribl05R hy- bridized against the separated strands of the 2.4-kb Eco RI fragment. A, the 1.1-kb and 0.9-kb Eco KI/Hae 111 fragments from the 2.4-kb Eco RI fragment visualized by ethidium bromide staining before transfer to nitrocellulose. B, hybridization of the ‘”P-labeled 110- nucleotide transcript synthesized from the 2.4-kb Eco RI fragment against a DNA filter blot containing the Eco KI/Hae 111 fragments. C, hybridization of total ‘’YP-labeled transcripts synthesized from the 2.4-kb Eco HI fragment against a DNA filter blot containing the Kc0 KI/Hae 111 fragments.

TABLE I1 Mapping of discrete transcripts synthesized from HindIII and Hpa

I-digested pribl05R by RNA polymerase I I I ‘”P-labeled transcripts were isolated as described under “EX-

perimental Procedures.” The transcripts were hybridized against DNA filter blots containing restriction endonuclease fragments gen- erated after cleavage of pribl05K with HindIII and Sma I or the seDarated strands of the 2.4-kb Eco RI fragment of yeast rDNA.

Enzyme used to molecular $ ~ ~ ~ ~ ~ L ~ l ~ ~ ~ d strand of the 2.4- Transcript Complementary Complementary

cleave prihl05R weight kh Eco 131 (nucleotides) with Srna I and fragment

HindIII 160 250 315 375

Hpa I 115 150 290 385 440

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N I) Minus

“ ND, not determined.

the vector sequences. All of the transcripts synthesized from pribl05R digested with Hpa I hybridize to the 7.2-kb frag- ment. The 290-, 385-, and 40-nucleotide transcripts also hy- bridize with the 0.4-kb fragment. Hybridization of the 160- nucleotide transcript to the 0.4-kb fragment was not detected. If synthesis of this latter transcript is initiated from the Hpa

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Yeast RNA Polymerases 3239

I cleavage site, it would contain only 30 nucleotides which are complementary to the 0.4-kb fragment.

The most obvious interpretation of the data described above is that transcription by RNA polymerase 111 is initiated from certain ends of the cleaved template and that the discrete transcripts observed arise as a consequence of a series of termination events at discrete points within the template. The map shown in Fig. 11 summarizes all of the data on the direction of synthesis, location of complementary sequences in the template, and truncation with a second restriction endonuclease cleavage. The possibility that some of the smaller transcripts which were not truncated by digestion with a second restriction endonuclease cleavage of the tem- plate do initiate from sites other than the ends of the template cannot be excluded. This possibility seems remote, however, in light of the results with the larger transcripts.

RNA Polymerase III Initiates Synthesis from Many Sites on Supercoiled Plasmid DNA Templates-Purified RNA polymerase 111 does direct the synthesis of discrete transcripts from supercoiled plasmid DNA templates. To test the possi- bility that these transcripts are initiated from a few discrete sites within the template, 32P-labeled transcripts were synthe- sized from supercoiled pSF2124 and prib20 in the presence of cordycepin triphosphate as described under “Experimental Procedures.” The transcripts were analyzed by sucrose density gradient centrifugation in parallel with transcripts synthesized in the absence of cordycepin triphosphate. Transcripts syn- thesized in the presence of the ATP analog were reduced in size and had an average size corresponding to approximately 100 nucleotides. These latter transcripts were hybridized against immobilized fragments of the templates generated after digestion with Pst I which cleaves the vector sequences six times or Eco RI which cleaves yeast rDNA sequences in prib20 seven times. Transcripts synthesized in the presence of cordycepin triphosphate from both templates hybridized ran- domly with all of the restriction fragments generated from the templates. These data demonstrate that RNA polymerase 111 initiates synthesis from many sites within the template rather than from a few discrete sites.

Srna I Eco R, HlndlU Hpo I Pvu II

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synthesized by RNA polymerase I11 from pribl05R cleaved FIG. 11. Transcription map of the major discrete transcripts

with Eco RI, HindIII, or Hpa I. A restriction endonuclease cleavage map of the yeast rDNA portion of prib105R. The arrows indicate the direction of transcription and sites of initiation and termination for each transcript synthesized. Transcrzpt a, synthesized from pribl05R digested with Eco RI, is 110 bases. The sizes of transcripts a, b, c, and d synthesized from HindIII-digested pribl05R are 375, 315, 250, and 160 bases, respectively. The sizes of transcripts a, b, c, d, and e synthesized from Hpa I-digested przbl05R are 440, 385,290, 150, and 115 bases, respectively.

DISCUSSION

Purification of yeast RNA polymerase by the procedure described in this report is rapid and should facilitate analyses which require these enzymes. Yeast RNA polymerase II i s resolved from a mixture of RNA polymerases I and I11 after heparin agarose chromatography of an S35 fraction prepared from disrupted cells. Approximately 95% of the enzyme activ- ity present in the S35 fraction is retained on the column. Davison et al. (13) suggest that B. subtilis RNA polymerase present in crude cell lysates is displaced from endogenous nucleic acid by heparin residues on the column. It appears that the same mechanism occurs with the yeast RNA polym- erases since they are bound to nucleic acid in the 535 fraction as shown by their precipitability with protamine sulfate and since the resolution reported here is lost when nucleic acid is removed from the S35 fraction prior to heparin agarose chro- matography. Because it is not necessary to remove nucleic acid before heparin agarose chromatography and the fact that this chromatographic step results in a 50-100-fold purification of the yeast polymerases, this purification procedure is rela- tively simple and rapid compared to other methods (8-12). RNA polymerase 111 activity in the S35 fraction is not quan- titatively bound to the heparin agarose column. This is prob- ably related to the fact that yeast RNA polymerase I11 binds nucleic acid more tightly than do RNA polymerases I and I1 (12). This conclusion is supported by the observation that RNA polymerase I11 activity present in fractions in which nucleic acid was removed by protamine sulfate precipitation or ion filtration on DEAE-Sephadex does bind heparin aga- rose quantitatively.” The amount of RNA polymerase activity lost as a consequence of failure to bind heparin agarose varies from 10-30% of the RNA polymerase I11 activity present in the S35 fraction.

Glycerol gradient centrifugation results in a 10-fold purifi- cation of the polymerases after heparin agarose chromatog- raphy. This step is the most cumbersome in the procedure since the enzymes have to be concentrated and the centrifu- gation is time-consuming. It is likely that the use of vertical rotors at this step would improve yield and significantly shorten the time needed to complete this step. Use of vertical rotors would also improve the procedure for isolations from large quantities of yeast cells. DEAE-Sephadex or DNA-cel- lulose chromatography can be used as a final purification step. The polymerases are 95% pure as judged by SDS-polyacryl- amide gel electrophoresis using either chromatography method.

Multiple forms of each RNA polymerase are resolved after DEAE-Sephadex chromatography but not after DNA-cellu- lose chromatography. The two forms of RNA polymerase I (Ia and Ib) differ by the presence of a 48,000-dalton poly- peptide in lb but not in Ia. The two forms of RNA polymerase I1 differ by the presence of a 24,000-dalton polypeptide asso- ciated with IIb but not IIa. In the case of the Ia and Ib forms, the 48,000-dalton polypeptide has been implicated with effi- cient transcription of double-stranded DNA (11, 15). Since RNA polymerase Ib co-chromatographs with RNA polymer- ase IIIa, we were not able to compare directly the activities of Ia and Ib with denatured and native templates. The 24,000- dalton polypeptides associated with RNA polymerases I and I1 are identical based on fingerprint analysis (23). This poly- peptide appears to be required for RNA polymerase I activity (24). We observe no significant differences in specific activity between the two forms of this enzyme in assays using salmon sperm DNA as template. These data suggest that this poly- peptide is not essential for RNA polymerase I1 activity.

C. I. Hammond and M. J. Holland, unpublished observations.

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3240 Yeast RNA Polymerases

The specific activities of RNA polymerases I and I1 isolated by the procedure reported here are comparable to those determined for enzymes purified by other procedures (8,9,11, 12). Although an accurate determination of the specific activ- ity of RNA polymerase I11 could not be made, it does appear that the specific activity of this form of the enzyme is much higher than observed with other preparations (10, 12). Based on the relative amounts of RNA polymerase I11 subunits analyzed on SDS-polyacrylamide gels, it is clear that the specific activity of RNA polymerase 111 is at least two orders of magnitude higher than RNA polymerases I and 11. The quantity of RNA polymerase I11 protein isolated by the pro- cedure described by Hager et al. (12) is comparable to the amount of RNA polymerase I isolated. Since the specific activity of RNA polymerase 111 isolated by this latter proce- dure is 250 units/mg, it appears likely that heparin agarose chromatography results in preferential retention of active RNA polymerase I11 molecules. This possibility is supported by the fact that 70-90% of the RNA polymerase I11 activity in the S35 fraction is bound to the heparin agarose column.

RNA polymerases I and 11, isolated as described in this report, transcribe supercoiled as well as linear plasmid DNA templates with low efficiency. Utilizing plasmids containing yeast ribosomal DNA and a yeast glyceraldehyde-3-phosphate dehydrogenase structural gene, we did not detect synthesis of discrete transcripts from these templates. RNA polymerases IIIa and IIIb do synthesize discrete transcripts from super- coiled and linear plasmid DNA templates. In the case of restriction endonuclease-digested templates, synthesis of dis- crete transcripts is initiated from the ends of the template and termination occurs at specific sites downstream from the end of the template. The efficiency of synthesis of these discrete transcripts is comparable to or higher than the efficiency of transcription of a mixture of denatured and native salmon sperm DNA depending on the plasmid template used. Effi- cient synthesis of discrete transcripts was detected from the termini of templates cleaved with restriction endonucleases which produce free 5’, free 3’, and blunt termini. These latter data argue against a requirement for single-stranded versus double-stranded termini for efficient initiation. We observed no differences in template efficiency among plasmids which contained no yeast DNA sequences and plasmids containing the yeast 5 S rRNA gene, for example. The transcriptional properties of RNA polymerase 111 after each step in the fractionation procedure are similar to those observed for the purified enzyme.

We observe preferential initiation of transcription from selected termini in the restriction endonuclease-cleaved tem- plate. All of the discrete transcripts synthesized from the plasmid przbl05R after cleavage with Hpa I, for example, are initiated from one end of the template. Greater than 90% of the transcripts synthesized from the 2.4-kb Eco RI fragment of yeast rDNA are also initiated from one end of the molecule. The basis for the preference for certain termini is not clear. Termination of transcription to form discrete transcripts oc- curs at specific sites downstream from the end of the template. In the cases of transcripts synthesized from pribl05R after cleavage with either Hind111 or Hpa I, termination of some of the discrete transcripts synthesized from these two templates occurs at the same sites. We have examined the primary structure of the rDNA portion of this template (20) in the regions containing these termination sites and found no struc- tural similarities among them. Transcripts synthesized from supercoiled templates by RNA polymerase I11 are initiated

from many sites within the template. This conclusion is based on the observation that transcripts synthesized in the presence of sufficient cordycepin triphosphate to produce molecules approximately 100 nucleotides in length hybridize randomly to restriction endonuclease fragments derived from the tem- plate.

Based on the transcriptional properties of purified RNA polymerase 111, it is clear that the enzyme is capable of efficient transcription of defined template DNA. RNA polym- erase 111-dependent selective transcription of yeast 5 S rDNA in crude chromatin preparations has been demonstrated (4). In the latter case, the chromatin template must contain the additional components necessary to support specific initiation. The possibility that efficient initiation of transcription from the ends of template molecules reflects a significant partial reaction for the enzyme remains an open question.

Acknowledgments-We would like to thank Mark Swanson, Janice Holland, and Laura Labieniec for their help in generating some of the experimental data. We also thank Dr. Terrance Leighton for many helpful discussions.

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r l l c c d frm the gel before autoradlqJraphy and Stained 4 t h 0.5 m l m l e t h l d i m b r a i d s ,

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9. IO. 11. 12. is. IO. 15. 16. i7.

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C I Hammond and M J Hollandtemplates.

chromatography. Transcriptional properties of the purified enzymes on defined Purification of yeast RNA polymerases using heparin agarose affinity

1983, 258:3230-3241.J. Biol. Chem. 

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