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Structure and transcription specificity of yeast RNA polymerase A
179
Biochimica et 0 Scientific
Acta, Publishing
395
179-190 Amsterdam
--
in The
BBA 98328
STRUCTURE AND TRANSCRIPTION POLYMERASE A
H. VAN KEULEN, Biochemisch (Received
R.J. PLANTA
Laboratorium, December
24th,
SPECIFICITY
OF YEAST RNA
and J. RETkL
Vrije Universiteit,
Amsterdam
(The Netherlands)
1974)
Summary DNA-dependent RNA polymerase A (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was isolated from whole yeast cells and purified to a nearly homogeneous state. The subunit structure as well as the transcription specificity of the purified enzyme were investigated. Polyacrylamide gel electrophoresis under denaturating conditions revealed that yeast polymerase A is made up of two large subunits having mol. wts of 190 000 and 135 000, and five smaller subunits with mol. wts of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. The molar ratios of all these polypeptides were found to be about unity. The transcription specificity of yeast polymerase A was tested using homologous nuclear DNA as a template. The in vitro synthesized RNA was characterized by determining its degree of self-complementarity and its ability to compete with purified ribosomal RNA in hybridization experiments. It was found that yeast polymerase A is capable of a highly selective transcription in vitro of the rRNA cistrons, provided DNA of high integrity is used as a template. Introduction It is now well-established that mammalian cells contain three different classes of DNA-dependent RNA polymerase (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), designated A (or I), B (or II) and C (or III)., respectively (see for example ref. 1). Previously we have reported that three different RNA polymerase species are present in yeast nuclei as well [2] . These enzymes were found to exhibit properties very similar to those reported for the polymerases from mammalian cells [2-S]. Suggestions about the function of these multiple RNA polymerase species are mainly based on their specific intracellular location. Since polymerase A was found to be localized in the
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nucleolus [g--14], its function is generally accepted to be the synthesis of ribosomal RNA [15-201. Polymerase B is found in the nucleoplasm [9,10,21, 221 and is believed to be responsible for the synthesis of heterogeneous nuclear RNA [ 16-181. The role of the nucleoplasmic polymerase C is even less clear, although recently evidence has been obtained suggesting that this enzyme is probably involved in the synthesis of 5 S ribosomal RNA and tRNA [ 191. One direct way to test the hypothesis that the different types of eukaryotic RNA polymerase transcribe different regions of the genome is to investigate whether preferential transcription can be observed in vitro using purified enzyme, and homologous DNA as a template. In this paper we describe the subunit structure of highly purified yeast polymerase A, isolated from whole cells, as well as the transcription properties of the enzyme on homologous nuclear DNA. The results obtained clearly show that polymerase A preferentially transcribes the rRNA genes, provided DNA of high integrity is used as a template. Materials and Methods Isolation and purification of yeast RNA polymerase A and Escherichia coli RNA polymerase RNA polymerase A was isolated from exponential phase Saccharomyces carlsbergensis cells (Strain S 74) according to the procedure of Buhler et al. [23] with some modifications. All steps of the isolation procedure were performed at 0-4°C in 0.05 M Tris-HCl buffer, pH 7.9, containing 0.1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10% glycerol and (NH4)2S04 as indicated below. Yeast cells (250 g wet wt), suspended in 250 ml of buffer containing 0.3 M (NH4)2 SOa, were disrupted by passing them twice through a Manton Gaulin homogenizer at a pressure of 600 atm. After removal of cell debris and membranes by centrifugation, the (NH,), SO, concentration of the supernatant was reduced to 0.15 M by adding 1 vol. of buffer without (NH4)* S04, and the solution was mixed with phosphocellulose (175 g wet wt). After stirring for 1.5 h, the slurry was poured into a column (25 X 4.5 cm). The column was washed with the buffer containing 0.15 M (NH,),SO, and polymerase A was then eluted with the same buffer containing 0.4 M (NH,), SO,. The fractions containing enzyme activity were pooled and after reducing the (NH,),SO, concentration to 0.05 M, stirred with DEAE-Sephadex (200 g wet wt) for 1.5 h. The slurry was again poured into a column (20 X 2 cm) and the enzyme was eluted with buffer containing 0.3 M (NH,),SO,. RNA polymerase was precipitated by addition of 35 g of solid (NH,),SO,. The precipitate was dissolved in a small amount of buffer containing 0.05 M (NH,),SO, and applied to a Sepharose-6B column (35 X 2 cm). Elution was carried out using the same buffer. The fractions containing polymerase activity were pooled and dialyzed overnight against buffer containing 50% glycerol. At this stage the purity of the enzyme is at least 85% (see Fig. 1) and the final yield is 2 mg protein. The preparation did not contain detectable ribonuclease or deoxyribonuclease activities. The absence of ribonuclease activity was tested gels in by electrophoresis of ‘*P-labeled yeast rRNA on 2.6% polyacrylamide the presence of formamide, after incubation of the RNA with the RNA poly-
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merase A preparation under standard conditions. Similarly the absence of deoxyribonuclease was tested by determination of the double- and single-stranded molecular weight of T,-DNA or nuclear yeast DNA according to Studier [31] after incubation of the DNA with the enzyme preparation. The enzyme can be stored in liquid nitrogen for at least 6 months without loss of activity. E. coli RNA polymerase was isolated according to the procedure of Burgess [ 241 with slight modifications. Assay for RNA polymerase activity The standard incubation mixture (0.2 ml) contained: 0.065 M Tris-HCl, pH 8.0, 5 mM MgCl?, 1.5 mM MnCl*, 20 mM (NH,),SO,, 1 mM each of ATP, GTP and CTP, 0.5 mM UTP, 2 PCi of [ .‘H] UTP (spec. act. 0.02 Ci/mmol), 25 /~g DNA, and enzyme. Assay mixtures were incubated for 15 min at 30” C and were then processed essentially as previously described [ 21 . One unit of activity is defined as the amount of enzyme which incorporates 1 nmol of UMP/h into the acid-insoluble product. Determination of the molecular weight of RNA polymerase A The molecular weight of RNA polymerase A was determined by Sepharose-6B column chromatography according to the method of van ‘t Riet and Planta [25]. The following protein standards were used: thyroglobulin (670 000), urease (480 000), ferritin (460 000) and catalase (240 000); the numbers in brackets indicate the molecular weight. Polyacrylamide gel electrophoresis Electrophoresis under non-denaturing conditions was carried out on 4.7% polyacrylamide gels according to the method of Krakow [26] as modified by Kedinger et al. [27]. Electrophoresis in the presence of dodecyl sulphate was performed on 5%, 7.5% and 10% polyacrylamide gels according to Shapiro et al. [28]. Protein samples were heated for 3 min at 100°C in the presence of 1% dodecyl sulphate before applying them to the gels. Gels were stained with either Coomassie brilliant blue or amido black for 48 h and molecular weights of the various subunits of RNA polymerase A were determined according to Weber and Osborn [ 291. The following protein standards were used: the /3’, p, u and (Y subunits of E. coli RNA polymerase (165 000, 155 000, 96 000 and 39 000, respectively), phosphorylase a (92 500), fructose-6-phosphate kinase (78 000), catalase (57 500), lactate dehydrogenase (35 000) and cytochrome c (13 400); the numbers in brackets indicate the molecular weight of the protein or its subunits. Isolation of DNA and RNA High molecular weight yeast DNA of high integrity was isolated from nuclei as previously described [ 301. Nicked DNA was prepared by mechanical shearing of the high molecular weight DNA. Double- and single-stranded molecular weights were determined from sedimentation velocity measurements in the Spinco analytical ultracentrifuge according to the method of Studier [31]. 3 ’ P-labeled 26 S and 17 S rRNA were prepared from yeast ribosomes as
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described previously [ 321, and purified by sucrose-gradient centrifugation ]331. RNA transcribed in vitro from yeast DNA (cRNA) was obtained by incubating RNA polymerase with DNA (250 pg/ml) in a 1 : 10 ratio in the same reaction mixture as used for the enzyme assay (see above) except that both MnCl, and (NH4 )z SO, were omitted. Incubation time was 40 min at 30” C. The cRNA was isolated in the same way as described by Hecht and Birnstiel [ 341 . cRNA made on a template with a relatively high integrity (double- and single-stranded mol. wts 40 * lo6 and 5-10 * 106, respectively) appears to have a size distribution ranging from approx. 100 to more than 3000 nucleotides; however, the majority of the cRNA has a length ranging from 1600 to 3500 nucleotides, as judged by polyacrylamide gel electrophoresis in the presence of formamide. Assay for symmetric transcription The degree of symmetric transcription was measured by determining the percentage of cRNA capable of self-annealing according to the procedure of Tabak and Borst [ 351. Increasing concentrations of cRNA were incubated in 0.15 M NaC1/0.015 M trisodium citrate, pH 7.0 at 68°C for 20 h and the percentage of RNA resistant to pancreatic plus T, ribonuclease digestion was determined. Competition hybridization The hybridization method used was that of Gillespie and Spiegelman [36] as modified by Birnstiel [37]. Competition hybridization was carried out at 37°C in 0.2 ml of 50% formamide and 0.3 M NaC1/0.03 M trisodium citrate, pH 7.0, for 20 h. Filters loaded with 0.5 pg nuclear yeast DNA were incubated with saturating amounts of purified 3 ’ P-labeled rRNA (at a RNA/DNA input ratio of 0.2) and increasing amounts of cRNA synthesized in vitro.
Results Structural and enzymatic properties of yeast RNA polymerase A The purity of the yeast RNA polymerase A preparation was assessed by polyacrylamide gel electrophoresis under non-denaturating conditions (Fig. 1). It appears that the polymerase migrates as one main band of protein and is largely free of contaminating protein. It was verified by analysis of an unstained gel that the main band contains the enzymatic activity. From quantitative measurements of the distribution of the stained material along the gel the purity of the enzyme was judged to be at least 85%. It is evident that the preparation is devoid of both RNA polymerase B and C, since no other bands could be detected, even when very large amounts of protein were applied to the gel. Moreover, the polymerase activity appeared to be completely insensitive to even very high concentrations (loo--400 pg/ml) of ol-amanitin, indicating the absence of significant amounts of polymerase B and C activities, which in contrast to polymerase A are known to be sensitive to this drug [l] . The molecular weight of the RNA polymerase A was determined by gel
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Fig. 1. Polyacrylamide gel electrophoresis under non-denaturating conditions of purified yeast polymerase A. Electrophoresis was performed on a 4.7% polyacrylamide gel according to Kedinger et al. 1271.
filtration on Sepharose-6B according to the procedure of van ‘t Riet and Planta [25]. A value of 500 000 ? 30 000 was found. In order to investigate its subunit structure, the purified enzyme was subjected to polyacrylamide gel electrophoresis under denaturating conditions. Fig. 2 shows the pattern obtained after electrophoresis in the presence of dodecyl sulphate and extensive staining (48 h) with Coomassie brilliant blue. The results indicate that yeast polymerase A contains two large and five smaller subunits. The molecular weights of the large subunits, determined by gel electrophoresis on 5% polyacrylamide gels according to Weber and Osborn [29], were found to be 190 000 and 135 000, respectively. The five smaller subunits have molecular weights, determined by the same method using 10% polyacrylamide gels, of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. These values are accurate only to within 5-10% due to uncertainties in the values for the molecular weight of some of the marker proteins available. An estimate for the molar ratios of the various subunits present in yeast
2ro-
1 5-
I O-
: Ya 5-
t3-
0
1
234567 Dstance km)
Fig. 2. Subunit pattern of purified yeast polymerase A. The subunit structure was determined by electrophoresis on a 7.5% polyacrylamide gel in the presence of 0.1% dodecyl sulphate according to Shapiro et al. [ 281. After extensive staining with Coomassie brilliant blue, a spectrophotometric tracing of the gel was made, using a Vitatron UFD-100 spectrophotometer.
TABLE
1
THE SUBUNIT
STRUCTURE
OF YEAST
POLYMERASE
A
The molecular weight of the various subunits was determined by polyacrylamide gel electrophoresis in the presence of 0.1% dodecyl sulphate according to Weber and Osborn [291. using the protein standards indicated in Materials and Methods. Determination of the molecular weight of the two large subunits was performed on 5%. and of the five smaller subunits on 10% polyacrylamide gels. After extensive staining for 48 h of the gels with Coomassie brilliant blue or amide black, the molar ratios of the subunits were estimated by quantitative determination of the amount of dye absorbed by them according to Kedinger et al. 1271. ___._ Mol at (XIOJ)
Molar ratio*
19.0 13.5 5.4 4.4 3.5 2.5 1.6
1.0 1.0 0.8-l .O 0.9-1.3 1.0-1.2 0.9-1.1 1.0-1.4
* 5 independent
determinations.
RNA polymerase A was made by quantitative determination of the staining intensity of the various bands obtained after polyacrylamide gel electrophoresis under denaturating conditions. The gels were stained extensively (48 h) with either Coomassie brilliant blue or amido black. As shown in Table I the molar ratios of all subunits, when determined in this way, are about unity. However, even though the same values were obtained using two different dyes, this result should be considered to be tentative for the reasons already pointed out by Kedinger et al. [ 271. The purified yeast polymerase A has largely the same requirements, as far
Enzyme/DNA weight Rotlo Fig. 3. The influence of the enzyme/DNA weight ratio on the amount of RNA synthesized in vitro on yeast DNA. Fixed amounts of yeast DNA (5 pg) of high integrity (double- and single-stranded mol. wts 3+40 10h and ~10. 106, respectively) were incubated with increasing quantities of polymerase A. The reaction mixture (0.2 ml) was the same as used for the enzyme assay (see Materials and Methods) except that both Mn2+ and (NHq)2 SO4 were omitted. Incubation time was 30 min at 30” C. The amount of RNA synthesized was determined by measuring the c3Hl UMP incorporation into acid-insoluble product as described previously [21.
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divalent cations ionic strength concerned, as described for corresponding enzyme, from yeast [2]. Optimal concentration found to 10 mM, Mn” concentration mM, when DNA was as a Maximal activity observed in presence of Mg*+ (5 and Mn*’ mM). However, at a concentration (10 can largely Mn” to the same Increasing the strength by of ( NH4 ) 2 SO4 also results in stimulating of [” H] UMP incorporation. However, the extent of stimulation is rather small; about 20% at a (NH,), SO4 concentration of 20 mM. Higher salt concentrations inhibit the enzyme activity. Fig. 3 shows the effect of increasing the enzyme/DNA ratio on the [ 3 H] UMP incorporation. A linear increase in the amount of RNA synthesized on yeast DNA of high integrity is observed up to an enzyme/DNA ratio of about 1.5. Upon increasing the ratio further the amount of RNA transcribed reaches a maximum at an enzyme/DNA ratio of about 3 (Fig. 3). At low enzyme/DNA ratios (0.1-0.5) ENA synthesis is linear for up to an hour (data not shown). The specific activity of the purified RNA polymerase under optimal conof ditions (i.e. in the presence of Mg*‘, Mn’+ and (NH,), SO, at concentrations 5 mM, 1.5 mM and 20 mM, respectively, and an enzyme/DNA ratio of 3) was found t.o be 1600~-2000 units/mg protein. Transcription
specificity of yeast polymerase A: symmetry of transcription From investigations in both pro- and eukaryotic systems, it has become clear that several factors may play a role in determining the fidelity of transcription on a given DNA template. The most important of these factors are: the molecular structure of the RNA polymerase (see ref. 38), the integrity of the DNA template [34,39-411, the enzyme/DNA ratio [41] and the ionic conditions [39,42--441. In order to gain insight into the influence of the enzyme/DNA ratio as well as the integrity of the DNA template on the fidelity of transcription, we studied first de degree of symmetry of transcription. RNA polymerase A was used to transcribe homologous yeast DNA in vitro in the presence of 5 mM Mg*+ (Mn*’ was omitted) and the degree of symmetry of transcription was determined by measuring the amount of RNA resistant to degradation by pancreatic plus T, ribonuclease after self-annealing. The results are shown in Fig. 4 and Table II. It is clear that the degree of symmetric transcription rises when the enzyme/DNA ratio is increased (Fig. 4). At a ratio of 0.2, which is well below that required for maximal activity (see Fig. l), the degree of symmetry is rather low (lO-15%). However, this value rises to about 35% when the enzyme/DNA ratio is increased to 3 (Fig. 4). In these assays high molecular weight yeast DNA containing a relatively low number of singlestranded breaks was used. Table II shows the influence of the integrity of the DNA template on the amount of symmetric transcription. In accordance with the results of Flint et al. [40] which showed that single-stranded breaks can serve as pseudopromoters, we found that the degree of symmetric transcription increased when DNA templates of low physical integrity were used. The reliability of our measurements can also be judged from Fig. 4, which shows the percentage of self-complementary RNA when heat-denatured yeast
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0
2
4
6 cRNA
6
10
12
(~/ml)
Fig. 4. Influence of the enzyme/DNA weight ratio on the degree of symmetric transcription by yeast RNA pol~merase A. Yeast RNA polymerase A was used forfranscription of 100 pg relatively intact yeast DNA (mol. wts before and after denaturation 40 . 106 and 5-10 . 106, respectively) at enzyme/DNA ratios of 0.2, 1.0 and 3.0, respectively. The yield of RNA at these enzyme/DNA ratios was approx. 7.5, 18 and 35 pg, respectively. E. coli core RNA polymerase was used for transcription of denatured yeast DNA (mol. wt ErlO 106) at an enzyme/DNA ratio of 1.0. The degree of symmetry of transcription was determined by self-annealing increasing concentrations of cRNA in 0.15 M N&1/0.015 M trisodium citrate, PH 7.0. at 65’C, for 29 h and measuring the percentage of RNA resistant to pancreatic plus T1 ribonuclease according to Tabak and Borst [351. 0, RNA made in vitro by yeast RNA polymerase at an enzyme/DNA ratio of 0.2; ~1, at a ratio of 1.0; A, at a ratio of 3.0. a, RNA made in vitro by E. coli core polymerax on denatured yeast DNA.
DNA was transcribed by E. coli core RNA polymerase, i.e. conditions which strongly favor symmetrical transcription [ 451. Under these conditions about 7 5% of the RNA made becomes resistant to ribonuclease after self-annealing. From these experiments it can be concluded that both a low enzyme/DNA ratio and a high physical integrity of the DNA template are required to obtain a high fidelity of transcription. Transcription specificity of yeast polymerase A: selective transcription of the ribosomal RNA genes In order to determine whether yeast polymerase A has any preference for the transcription of the yeast rRNA genes, cRNA was made on total yeast DNA in the presence of 5 mM Mg’+ at a low enzyme/DNA ratio (0.1 or 0.2). The TABLE
II
THE EFFECT OF THE PHYSICAL INTEGRITY OF YEAST TRANSCRIPTION BY HOMOLOGOUS RNA POLYMERASE
DNA ON THE DEGREE A
OF SYMMETRIC
Yeast polymerase A was used to transcribe homologous “intact” DNA (mol. wts before and after denaturation 40 . 106 and 5-10 . 106, respectively) and “nicked” DNA (mol. wt before and after denaturation lo . lo6 and 0.2 . 106, respectively) at a low enzyme/DNA ratio (0.2). The degree of symmetry of transcription was determined by measuring the amount of cRNA, which becomes resistant to pancreatic plus T1 ribonuclease upon self-annealing, as described in the legend to Fig. 4. DNA, used in transcription
Jribonuclease-resistant cRNA after self-annealing
Intact DNA Nicked DNA
13 33
187
Unlabeled
RNA
(pg )
Fig. 5. Sequence horn&&y of RNA, synthesized in vitro by yeast polymerase A on homologous DNA, with purified yeast rRNA. Yeast RNA polymerase A or E. coli polymerase were used for transcription of 200 pg yeast DNA at an enzyme/DNA weight ratio of 0.2. DNA templates of different degrees of integrity were used. The degree of sequence homology of the various cRNA preparations with yeast rRNA was tested by competitive hybridization of fixed saturating amounts of in viva 32P-labeled 26 S plus 17 S rRNA and increasing amounts of cRNA as described in Materials and Methods. 0, purified yeast rRNA; 0. RNA made in vitro (yield a&ox. 16 pg) by yeast RNA polymerase A on yeast DNA with double- and single-stranded mol. wts of 40. 106 and 5-10. 106, respectively: A, RNA made in vitro (yield approx. 50 fig) by yeast RNA polymerase A on yeast DNA with a double- and single-stranded mol. wts of 30. 106 and 1 106, respectively: 0, RNA made in vitro (yield approx. 80 Mg) by yeast RNA polymersse A on yeast DNA with double- and single-stranded mol. wts of 10 . 106 and 0.2 . 106, respectively. l, RNA made in vitro (yield approx. 75 fig) by E. coli RNA polymerase (plus 0) on yeast DNA with double- and single-stranded mol. wts of 40. lo6 and 5-10. 106, respectively.
RNA was subsequently characterized by its ability to compete with yeast rRNA in RNA . DNA hybridization. The results of competitive hybridization of fixed saturating amounts of in vivo 3 * P-labeled 26 S plus 17 S rRNA and increasing amounts of cRNA are presented in Fig. 5. About four times more cRNA, transcribed from a template having a relatively high degree of integrity, than pure yeast rRNA is required to obtain the same degree of competition. It can be concluded, therefore, that about 25% of the RNA synthesized in vitro under these conditions consists of ribosomal sequences. Since the rRNA cistrons comprise only approx. 2% of the total yeast DNA [46], this result shows, that yeast polymerase A has a clear preference for in vitro transcription of the ribosomal genes. It can be calculated that roughly one transcript per ribosomal transcription unit is made in vitro in spite of the fact that the enzyme/DNA molar ratio is less than one. In contrast, E. coli RNA polymerase does not show any selective transcription of the yeast rRNA genes. When tested under the same conditions, only 2% of the cRNA, synthesized in vitro by E. coli RNA polymerase, showed sequence homology with yeast rRNA, as judged from competitive hybridization (Fig. 5). The selective transcription of the rRNA cistrons appeared to be strongly dependent on the integrity of the DNA template. When DNA with a large number of nicks (double- and single-stranded mol. wts 30 * lo6 and 1.0 * 106, respectively) was transcribed by polymerase A, the percentage of rRNA sequences in the in vitro transcript decreased to 8-lo%, whereas in the case of even less intact DNA (double- and single-stranded mol. wts 10 . lo6 and 0.2 * 106,
188
respectively), polymerase A did not show transcription specificity anymore (Fig. 5). From these results we conclude that yeast polymerase A is capable of highly selective transcription of the homologous rRNA cistrons in vitro, providing DNA of a high integrity is used as a template. Discussion The subunit structure of yeast RNA polymerase A, as determined by polyacrylamide gel electrophoresis under denaturating conditions is similar to that described for this enzyme isolated from other eukaryotic organisms [23, 27,47-491. In particular, the occurrence of two large subunits (mol. wts in yeast polymerase A 190 000 and 135 000, respectively) is a common feature of this type of enzyme. However, since there is considerable disagreement concerning the number, molecular weights and molar ratios of the smaller subunits of polymerase A from different organisms, comparison of these subunits is extremely difficult. Although the possibility that the differences observed actually reflect dissimilarities between the enzymes from different sources cannot be excluded, it seems likely that they are, at least partially, due to other factors, such as the isolation procedure, degree of purification or conditions of gel electrophoresis. We have observed, for instance, that both the apparent number and the apparent molar ratios of the small polypeptides could be changed by varying either the amount of protein applied to the gel or the length of the staining period. Only when suitable large quantities of protein were used and when staining was carried out for at least 20 h all five small subunits could be detected and appeared to be present in unimolar amounts. If we assume that all proteins found to be present in yeast polymerase A belong to the enzyme structure, the calculated mol. wt is 499 000, which is in very good agreement with the value of 500 000 + 30 000, determined by Sepharose-6B column chromatography. Recently, Buhler et al. [23] reported the subunit structure of Saccharomyces cerevisiae RNA polymerase A. In addition to two large subunits having molecular weights of 190 000 and 135 000, respectively, they detected one copy each of two smaller subunits (mol. wts 48 000 and 41 000) and two copies each of two other small subunits (mol. wts 29 000 and 16 000). Besides minor differences in the molecular weights of the smaller polypeptides, the subunit structure of the S. cerevisiae polymerase differs in a number of respects with that of the S. carlsbergensis enzyme: one of the small subunits (with a mol. wt of 35 000) found in the enzyme described in this paper is lacking from the enzyme isolabed from S. cereuisiae, and the molar ratios of various small proteins are quite different for the two enzymes. Buhler et al. did detect two additional proteins having mol. wts of 44 000 and 35 000, respectively, in their enzyme preparation. However, they did not consider these proteins as constituents of the enzyme because of the “unsatisfactory” molar ratios of these two polypeptides. In our opinion, this interpretation is not completely justified, also considering the results we have obtained. From the results presented in this paper it can be concluded that the enzyme preparation, isolated by us, is capable of a highly selective transcription
189
of homologous DNA. This conclusion is based on the characteristics of the in vitro RNA product, which, when synthesized under the right conditions, shows a considerable sequence homology with yeast rRNA. In addition, the in vitro transcription appears to be highly asymmetric. The most important prerequisites for this selective transcription appear to be a high physical integrity of the DNA template and a low enzyme/DNA weight ratio. The first requirement can be explained by the observation that singlestranded breaks in the template can act as pseudopromoters which tend to mask the binding of the enzyme to specific promoters [40]. In fact, it was recently shown by Dezelee et al. [50] that yeast polymerase A can be entirely dependent on pseudopromoters, such as single-stranded breaks or regions, using heterologous T7 DNA as a template. A similar conclusion was made by Mandel and Chambon [ 441 using polymerase A from calf thymus and Simian Virus 40 DNA as a template. However, our results demonstrate that yeast polymerase A is able to transcribe preferentially the rRNA cistrons, suggesting that true promoter sites are recognized by the enzyme. The second requirement, that of a low enzyme/DNA ratio, probably can be explained in a similar way. The highly selective transcription at low enzyme/DNA ratios must reflect a strong affinity of the polymerase A for the rRNA promotor sites. At higher enzyme/DNA ratios polymerase A seems to bind to false promoter sites present even in DNA of high integrity which results in a lower fidelity of transcription. The stimulating effect of Mn*’ on yeast polymerase activity (see Results) is probably based on the introduction of unpaired regions [ 441 which may also give rise to false initiation sites. This hypothesis is supported by our observation that the degree of symmetric transcription is strongly enhanced by the addition of Mn” to the incubation mixture (unpublished results). For this reason, characterization of transcription products was carried out using RNA transcribed in the absence of Mn*‘. Our finding that yeast polymerase A in vitro is capable of a highly selective transcription of yeast rRNA genes is in agreement with the recent results of Beebee and Butterworth [ZO] who observed a similar specificity of Xenopus laevis polymerase A. The failure of other investigators [51,52] to obtain selective transcription can be explained in several ways such as use of a template of low physical integrity or lack of essential factors from the enzyme. In this respect it should be noted that both the enzyme used by Beebee and Butterworth [20] and that used in the studies described here was isolated from whole cells. Acknowledgements The present investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). One of us (H.v.K.) is very grateful to Dr J.M. Buhler for making him familiar with the procedure for the isolation of large quantities of RNA polymerase A from whole yeast cells and for his kind help. The authors are indepted to Dr H.A. Raue for helpful critical comment on this manuscript and to Miss E. van der
190
Veer, Mr C.P. Bakker, Mr W.L. Homan, Mr W. Bruggeman Oosten for their skilled technical assistance.
and Mr Chr. van
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Jacob, S.T. (1973) Prog. Nucl. Acid. Res. Mol. Biol. 7. 93-126 Brogt. Th.M. and Planta. R.J. (1972) FEBS Lett. 20, 47-52 Ponta. H., Ponta, U. and Wintersberger, E. (1971) FEBS Lett. 18, 204~~208 Dezt+ie. S., Sentenac, A. and Fromageot, P. (1972) FEBS Lett. 21, l-6 Ponta, H.. Ponta, U. and Wintersberger, E. (1972) Eur. J. Biochem. 29, 110-118 Adman, R., Schulz. L.D. dnd Hall, B.D. (1972) Proc. Natl. Acad. Sci. U.S. 69. 1702. 1706 Da&e, S. and Sentenac. A. (1973) Eur. J. Biochem. 34. 41--52 Sebastian, J.. Bhargava. M.M. and Halvorson, H.D. (1973) J. Bacterial. 114. l-6 Roeder. R.G. and Rutter. W.J. (1969) Nature 224, 234--m237 Roeder. R.G. and Rutter, W.J. (1970) Proc. Natl. Acad. Sci. U.S. 65, 675-682 Jacob. S.T.. Sajdel, EM. and Munro, H.N. (1970) Biochem. Blophys. Res. Commun. 38. 765.--770 Lindell, T.J.. Weinberg:, F., Morris. P.W.. Roeder, R.G. and Rutter, W.J. (1970) Science 170. 447 448 Chesterton, C.J. and Butterworth. P.H.W. (1971) FEBS Lett. 12. 301-308 Chesterton. C.J. nnd Butterworth, P.H.W. (1971) Eur. J. Biochem. 19, 232 -241 Tocchini-Valentini, G.P. and Crippa. N. (1970) Nature 228, 993-995 Blatti, S.P.. lngles, C.J., Lindell. T.J.. Morris, P.W.. Weaver, R.F.. Weinberg. F. and Rutter, W.J. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 649-657 Zylber. E.A. and Penman, S. (1971) Proc. Natl. Acad. Sci. U.S. 68. 2861--2865 Reeder. R.H. and Roeder. R.G. (1972) J. Mol. Biol. 67. 433-441 Weinman. R. and Roeder, R.G. (1974) Proc. Natl. Acad. Sci. U.S. 71. 1790-1794 Beebee. T.J.C. and Butterworth, P.H.W. (1974) Eur. J. Biochem. 45. 395-406 Roeder. R.G. and Rutter, W.J. (1970) Biochemistry 9, 2543-2553 Kedinger, C., Nuret. P. and Chambon, P. (1971) FEBS Lett. 15. 169--174 Buhler, J.M., Sentenac. A. and Fromageot, P. (1974) J. Biol. Chem. 249. 5963-5970 Burgess, R.R. (1969) J. Biol. Chem. 244, 6160 6167 van ‘t Riet, J. and Planta, R.J. (1975) Biochim. Biophys. Acta. 379. 81-94 Krakow, J.S. (1971) Methods Enzymol. 21. 520-528 Kedinger. C., Gissinger, F. and Chambon. P. (1974) Eur. J. Biochem. 44. 421-436 Shapiro, A.L., Vinella. E. and Maizel, J.V. (1967) Biochem. Biop1.w. Res. Commun. 28, 815-820 Weber, K. and Osborn. M. (1969) J. Biol. Chem. 244. 4406-4412 Retel, J. and Planta, R.J. (1972) Biochun. Biophys. Acta 281, 299-309 Studier. F.W. (1965) J. Mol. Biol. 11. 373-390 van den Bos. R.C., Retel, J. and Planta, R.J. (1971) Biochim. Biophys. Acta 232, 494-508 Rettil, J. and Planta, R.J. (1967) Eur. J. Biochem. 3. 248-258 Hecht, R.M. and Birnstiel, M.L. (1972) Eur. J. Biochem. 29, 489-499 Tabak. H.F. and Borst. P. (1970) Biochim. Biophys. Acta 217, 356-364 Gillespie, D. and Spiegelman, S. (1965) J. Mol. Biol. 12. 829-842 Bimstiel, M.L., Sells, B.H. and Purdom, I.F. (1972) .J. Mol. Biol. 63, 21-39 Burgess. R.R. (1971) Ann. Rev. Biochem. 40, 711-740 Dausse. J.P., Sentenac. A. and Fromageot, P. (1972) Eur. J. Biochem. 31, 394-404 Flint, S.J., de Pomerai, D.I., Chesterton, C.J. and Butterworth. P.H.W. (1974) Eur. J. Biochem. 42. 567.-579 Beebee, T.J.C. and Butterworth. P.H.W. (1974) Eur. J. Biochem. 45, 395-406 So, A.G., Davie, E.W.. Epstein. R. and Tissieres, A. (1967) Proc. Natl. Acad. Sci. U.S. 58. 1739-1746 Milletta, R.L. and Trotter, C.D. (1970) Proc. Natl. Acad. Sci. U.S. 66. 701-708 Mandel. J.L. and Chambon. P. (1974) Eur. J. Biochem. 41, 367-378 Mandel, J.L. and Chambon. P. (1974) Eur. J. Biochem. 41, 379-395 Rettil. J. and Planta. R.J. (1968) Biochim. Biophys. Acta 169. 416-429 Burgess. A.rj. and Burgess, R.R. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1174-1177 Gornicki, S.Z.. Vuturo. S.B., West. T.V. and Weaver. R.F. (1974) J. Biol. Chem. 249, 1792- 1798 Schwarz, L.B. and Roeder. R.G. (1974) J. Biol. Chem. 249. 5898-5906 DezBlGe, S.. Srntenac. A. and Fromageot, P. (1974) J. Biol. Chem. 249. 5971-5974 Roeder, R.G., Reeder. R.II. and Brown. D.D. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 727- 735 Ilollenberg. C.P. (1973) Biochemistry 12, 5320 5325
Biochimica et 0 Scientific
Acta, Publishing
395
179-190 Amsterdam
--
in The
BBA 98328
STRUCTURE AND TRANSCRIPTION POLYMERASE A
H. VAN KEULEN, Biochemisch (Received
R.J. PLANTA
Laboratorium, December
24th,
SPECIFICITY
OF YEAST RNA
and J. RETkL
Vrije Universiteit,
Amsterdam
(The Netherlands)
1974)
Summary DNA-dependent RNA polymerase A (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was isolated from whole yeast cells and purified to a nearly homogeneous state. The subunit structure as well as the transcription specificity of the purified enzyme were investigated. Polyacrylamide gel electrophoresis under denaturating conditions revealed that yeast polymerase A is made up of two large subunits having mol. wts of 190 000 and 135 000, and five smaller subunits with mol. wts of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. The molar ratios of all these polypeptides were found to be about unity. The transcription specificity of yeast polymerase A was tested using homologous nuclear DNA as a template. The in vitro synthesized RNA was characterized by determining its degree of self-complementarity and its ability to compete with purified ribosomal RNA in hybridization experiments. It was found that yeast polymerase A is capable of a highly selective transcription in vitro of the rRNA cistrons, provided DNA of high integrity is used as a template. Introduction It is now well-established that mammalian cells contain three different classes of DNA-dependent RNA polymerase (Nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), designated A (or I), B (or II) and C (or III)., respectively (see for example ref. 1). Previously we have reported that three different RNA polymerase species are present in yeast nuclei as well [2] . These enzymes were found to exhibit properties very similar to those reported for the polymerases from mammalian cells [2-S]. Suggestions about the function of these multiple RNA polymerase species are mainly based on their specific intracellular location. Since polymerase A was found to be localized in the
180
nucleolus [g--14], its function is generally accepted to be the synthesis of ribosomal RNA [15-201. Polymerase B is found in the nucleoplasm [9,10,21, 221 and is believed to be responsible for the synthesis of heterogeneous nuclear RNA [ 16-181. The role of the nucleoplasmic polymerase C is even less clear, although recently evidence has been obtained suggesting that this enzyme is probably involved in the synthesis of 5 S ribosomal RNA and tRNA [ 191. One direct way to test the hypothesis that the different types of eukaryotic RNA polymerase transcribe different regions of the genome is to investigate whether preferential transcription can be observed in vitro using purified enzyme, and homologous DNA as a template. In this paper we describe the subunit structure of highly purified yeast polymerase A, isolated from whole cells, as well as the transcription properties of the enzyme on homologous nuclear DNA. The results obtained clearly show that polymerase A preferentially transcribes the rRNA genes, provided DNA of high integrity is used as a template. Materials and Methods Isolation and purification of yeast RNA polymerase A and Escherichia coli RNA polymerase RNA polymerase A was isolated from exponential phase Saccharomyces carlsbergensis cells (Strain S 74) according to the procedure of Buhler et al. [23] with some modifications. All steps of the isolation procedure were performed at 0-4°C in 0.05 M Tris-HCl buffer, pH 7.9, containing 0.1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10% glycerol and (NH4)2S04 as indicated below. Yeast cells (250 g wet wt), suspended in 250 ml of buffer containing 0.3 M (NH4)2 SOa, were disrupted by passing them twice through a Manton Gaulin homogenizer at a pressure of 600 atm. After removal of cell debris and membranes by centrifugation, the (NH,), SO, concentration of the supernatant was reduced to 0.15 M by adding 1 vol. of buffer without (NH4)* S04, and the solution was mixed with phosphocellulose (175 g wet wt). After stirring for 1.5 h, the slurry was poured into a column (25 X 4.5 cm). The column was washed with the buffer containing 0.15 M (NH,),SO, and polymerase A was then eluted with the same buffer containing 0.4 M (NH,), SO,. The fractions containing enzyme activity were pooled and after reducing the (NH,),SO, concentration to 0.05 M, stirred with DEAE-Sephadex (200 g wet wt) for 1.5 h. The slurry was again poured into a column (20 X 2 cm) and the enzyme was eluted with buffer containing 0.3 M (NH,),SO,. RNA polymerase was precipitated by addition of 35 g of solid (NH,),SO,. The precipitate was dissolved in a small amount of buffer containing 0.05 M (NH,),SO, and applied to a Sepharose-6B column (35 X 2 cm). Elution was carried out using the same buffer. The fractions containing polymerase activity were pooled and dialyzed overnight against buffer containing 50% glycerol. At this stage the purity of the enzyme is at least 85% (see Fig. 1) and the final yield is 2 mg protein. The preparation did not contain detectable ribonuclease or deoxyribonuclease activities. The absence of ribonuclease activity was tested gels in by electrophoresis of ‘*P-labeled yeast rRNA on 2.6% polyacrylamide the presence of formamide, after incubation of the RNA with the RNA poly-
181
merase A preparation under standard conditions. Similarly the absence of deoxyribonuclease was tested by determination of the double- and single-stranded molecular weight of T,-DNA or nuclear yeast DNA according to Studier [31] after incubation of the DNA with the enzyme preparation. The enzyme can be stored in liquid nitrogen for at least 6 months without loss of activity. E. coli RNA polymerase was isolated according to the procedure of Burgess [ 241 with slight modifications. Assay for RNA polymerase activity The standard incubation mixture (0.2 ml) contained: 0.065 M Tris-HCl, pH 8.0, 5 mM MgCl?, 1.5 mM MnCl*, 20 mM (NH,),SO,, 1 mM each of ATP, GTP and CTP, 0.5 mM UTP, 2 PCi of [ .‘H] UTP (spec. act. 0.02 Ci/mmol), 25 /~g DNA, and enzyme. Assay mixtures were incubated for 15 min at 30” C and were then processed essentially as previously described [ 21 . One unit of activity is defined as the amount of enzyme which incorporates 1 nmol of UMP/h into the acid-insoluble product. Determination of the molecular weight of RNA polymerase A The molecular weight of RNA polymerase A was determined by Sepharose-6B column chromatography according to the method of van ‘t Riet and Planta [25]. The following protein standards were used: thyroglobulin (670 000), urease (480 000), ferritin (460 000) and catalase (240 000); the numbers in brackets indicate the molecular weight. Polyacrylamide gel electrophoresis Electrophoresis under non-denaturing conditions was carried out on 4.7% polyacrylamide gels according to the method of Krakow [26] as modified by Kedinger et al. [27]. Electrophoresis in the presence of dodecyl sulphate was performed on 5%, 7.5% and 10% polyacrylamide gels according to Shapiro et al. [28]. Protein samples were heated for 3 min at 100°C in the presence of 1% dodecyl sulphate before applying them to the gels. Gels were stained with either Coomassie brilliant blue or amido black for 48 h and molecular weights of the various subunits of RNA polymerase A were determined according to Weber and Osborn [ 291. The following protein standards were used: the /3’, p, u and (Y subunits of E. coli RNA polymerase (165 000, 155 000, 96 000 and 39 000, respectively), phosphorylase a (92 500), fructose-6-phosphate kinase (78 000), catalase (57 500), lactate dehydrogenase (35 000) and cytochrome c (13 400); the numbers in brackets indicate the molecular weight of the protein or its subunits. Isolation of DNA and RNA High molecular weight yeast DNA of high integrity was isolated from nuclei as previously described [ 301. Nicked DNA was prepared by mechanical shearing of the high molecular weight DNA. Double- and single-stranded molecular weights were determined from sedimentation velocity measurements in the Spinco analytical ultracentrifuge according to the method of Studier [31]. 3 ’ P-labeled 26 S and 17 S rRNA were prepared from yeast ribosomes as
182
described previously [ 321, and purified by sucrose-gradient centrifugation ]331. RNA transcribed in vitro from yeast DNA (cRNA) was obtained by incubating RNA polymerase with DNA (250 pg/ml) in a 1 : 10 ratio in the same reaction mixture as used for the enzyme assay (see above) except that both MnCl, and (NH4 )z SO, were omitted. Incubation time was 40 min at 30” C. The cRNA was isolated in the same way as described by Hecht and Birnstiel [ 341 . cRNA made on a template with a relatively high integrity (double- and single-stranded mol. wts 40 * lo6 and 5-10 * 106, respectively) appears to have a size distribution ranging from approx. 100 to more than 3000 nucleotides; however, the majority of the cRNA has a length ranging from 1600 to 3500 nucleotides, as judged by polyacrylamide gel electrophoresis in the presence of formamide. Assay for symmetric transcription The degree of symmetric transcription was measured by determining the percentage of cRNA capable of self-annealing according to the procedure of Tabak and Borst [ 351. Increasing concentrations of cRNA were incubated in 0.15 M NaC1/0.015 M trisodium citrate, pH 7.0 at 68°C for 20 h and the percentage of RNA resistant to pancreatic plus T, ribonuclease digestion was determined. Competition hybridization The hybridization method used was that of Gillespie and Spiegelman [36] as modified by Birnstiel [37]. Competition hybridization was carried out at 37°C in 0.2 ml of 50% formamide and 0.3 M NaC1/0.03 M trisodium citrate, pH 7.0, for 20 h. Filters loaded with 0.5 pg nuclear yeast DNA were incubated with saturating amounts of purified 3 ’ P-labeled rRNA (at a RNA/DNA input ratio of 0.2) and increasing amounts of cRNA synthesized in vitro.
Results Structural and enzymatic properties of yeast RNA polymerase A The purity of the yeast RNA polymerase A preparation was assessed by polyacrylamide gel electrophoresis under non-denaturating conditions (Fig. 1). It appears that the polymerase migrates as one main band of protein and is largely free of contaminating protein. It was verified by analysis of an unstained gel that the main band contains the enzymatic activity. From quantitative measurements of the distribution of the stained material along the gel the purity of the enzyme was judged to be at least 85%. It is evident that the preparation is devoid of both RNA polymerase B and C, since no other bands could be detected, even when very large amounts of protein were applied to the gel. Moreover, the polymerase activity appeared to be completely insensitive to even very high concentrations (loo--400 pg/ml) of ol-amanitin, indicating the absence of significant amounts of polymerase B and C activities, which in contrast to polymerase A are known to be sensitive to this drug [l] . The molecular weight of the RNA polymerase A was determined by gel
183
Fig. 1. Polyacrylamide gel electrophoresis under non-denaturating conditions of purified yeast polymerase A. Electrophoresis was performed on a 4.7% polyacrylamide gel according to Kedinger et al. 1271.
filtration on Sepharose-6B according to the procedure of van ‘t Riet and Planta [25]. A value of 500 000 ? 30 000 was found. In order to investigate its subunit structure, the purified enzyme was subjected to polyacrylamide gel electrophoresis under denaturating conditions. Fig. 2 shows the pattern obtained after electrophoresis in the presence of dodecyl sulphate and extensive staining (48 h) with Coomassie brilliant blue. The results indicate that yeast polymerase A contains two large and five smaller subunits. The molecular weights of the large subunits, determined by gel electrophoresis on 5% polyacrylamide gels according to Weber and Osborn [29], were found to be 190 000 and 135 000, respectively. The five smaller subunits have molecular weights, determined by the same method using 10% polyacrylamide gels, of 54 000, 44 000, 35 000, 25 000 and 16 000, respectively. These values are accurate only to within 5-10% due to uncertainties in the values for the molecular weight of some of the marker proteins available. An estimate for the molar ratios of the various subunits present in yeast
2ro-
1 5-
I O-
: Ya 5-
t3-
0
1
234567 Dstance km)
Fig. 2. Subunit pattern of purified yeast polymerase A. The subunit structure was determined by electrophoresis on a 7.5% polyacrylamide gel in the presence of 0.1% dodecyl sulphate according to Shapiro et al. [ 281. After extensive staining with Coomassie brilliant blue, a spectrophotometric tracing of the gel was made, using a Vitatron UFD-100 spectrophotometer.
TABLE
1
THE SUBUNIT
STRUCTURE
OF YEAST
POLYMERASE
A
The molecular weight of the various subunits was determined by polyacrylamide gel electrophoresis in the presence of 0.1% dodecyl sulphate according to Weber and Osborn [291. using the protein standards indicated in Materials and Methods. Determination of the molecular weight of the two large subunits was performed on 5%. and of the five smaller subunits on 10% polyacrylamide gels. After extensive staining for 48 h of the gels with Coomassie brilliant blue or amide black, the molar ratios of the subunits were estimated by quantitative determination of the amount of dye absorbed by them according to Kedinger et al. 1271. ___._ Mol at (XIOJ)
Molar ratio*
19.0 13.5 5.4 4.4 3.5 2.5 1.6
1.0 1.0 0.8-l .O 0.9-1.3 1.0-1.2 0.9-1.1 1.0-1.4
* 5 independent
determinations.
RNA polymerase A was made by quantitative determination of the staining intensity of the various bands obtained after polyacrylamide gel electrophoresis under denaturating conditions. The gels were stained extensively (48 h) with either Coomassie brilliant blue or amido black. As shown in Table I the molar ratios of all subunits, when determined in this way, are about unity. However, even though the same values were obtained using two different dyes, this result should be considered to be tentative for the reasons already pointed out by Kedinger et al. [ 271. The purified yeast polymerase A has largely the same requirements, as far
Enzyme/DNA weight Rotlo Fig. 3. The influence of the enzyme/DNA weight ratio on the amount of RNA synthesized in vitro on yeast DNA. Fixed amounts of yeast DNA (5 pg) of high integrity (double- and single-stranded mol. wts 3+40 10h and ~10. 106, respectively) were incubated with increasing quantities of polymerase A. The reaction mixture (0.2 ml) was the same as used for the enzyme assay (see Materials and Methods) except that both Mn2+ and (NHq)2 SO4 were omitted. Incubation time was 30 min at 30” C. The amount of RNA synthesized was determined by measuring the c3Hl UMP incorporation into acid-insoluble product as described previously [21.
185
divalent cations ionic strength concerned, as described for corresponding enzyme, from yeast [2]. Optimal concentration found to 10 mM, Mn” concentration mM, when DNA was as a Maximal activity observed in presence of Mg*+ (5 and Mn*’ mM). However, at a concentration (10 can largely Mn” to the same Increasing the strength by of ( NH4 ) 2 SO4 also results in stimulating of [” H] UMP incorporation. However, the extent of stimulation is rather small; about 20% at a (NH,), SO4 concentration of 20 mM. Higher salt concentrations inhibit the enzyme activity. Fig. 3 shows the effect of increasing the enzyme/DNA ratio on the [ 3 H] UMP incorporation. A linear increase in the amount of RNA synthesized on yeast DNA of high integrity is observed up to an enzyme/DNA ratio of about 1.5. Upon increasing the ratio further the amount of RNA transcribed reaches a maximum at an enzyme/DNA ratio of about 3 (Fig. 3). At low enzyme/DNA ratios (0.1-0.5) ENA synthesis is linear for up to an hour (data not shown). The specific activity of the purified RNA polymerase under optimal conof ditions (i.e. in the presence of Mg*‘, Mn’+ and (NH,), SO, at concentrations 5 mM, 1.5 mM and 20 mM, respectively, and an enzyme/DNA ratio of 3) was found t.o be 1600~-2000 units/mg protein. Transcription
specificity of yeast polymerase A: symmetry of transcription From investigations in both pro- and eukaryotic systems, it has become clear that several factors may play a role in determining the fidelity of transcription on a given DNA template. The most important of these factors are: the molecular structure of the RNA polymerase (see ref. 38), the integrity of the DNA template [34,39-411, the enzyme/DNA ratio [41] and the ionic conditions [39,42--441. In order to gain insight into the influence of the enzyme/DNA ratio as well as the integrity of the DNA template on the fidelity of transcription, we studied first de degree of symmetry of transcription. RNA polymerase A was used to transcribe homologous yeast DNA in vitro in the presence of 5 mM Mg*+ (Mn*’ was omitted) and the degree of symmetry of transcription was determined by measuring the amount of RNA resistant to degradation by pancreatic plus T, ribonuclease after self-annealing. The results are shown in Fig. 4 and Table II. It is clear that the degree of symmetric transcription rises when the enzyme/DNA ratio is increased (Fig. 4). At a ratio of 0.2, which is well below that required for maximal activity (see Fig. l), the degree of symmetry is rather low (lO-15%). However, this value rises to about 35% when the enzyme/DNA ratio is increased to 3 (Fig. 4). In these assays high molecular weight yeast DNA containing a relatively low number of singlestranded breaks was used. Table II shows the influence of the integrity of the DNA template on the amount of symmetric transcription. In accordance with the results of Flint et al. [40] which showed that single-stranded breaks can serve as pseudopromoters, we found that the degree of symmetric transcription increased when DNA templates of low physical integrity were used. The reliability of our measurements can also be judged from Fig. 4, which shows the percentage of self-complementary RNA when heat-denatured yeast
186
0
2
4
6 cRNA
6
10
12
(~/ml)
Fig. 4. Influence of the enzyme/DNA weight ratio on the degree of symmetric transcription by yeast RNA pol~merase A. Yeast RNA polymerase A was used forfranscription of 100 pg relatively intact yeast DNA (mol. wts before and after denaturation 40 . 106 and 5-10 . 106, respectively) at enzyme/DNA ratios of 0.2, 1.0 and 3.0, respectively. The yield of RNA at these enzyme/DNA ratios was approx. 7.5, 18 and 35 pg, respectively. E. coli core RNA polymerase was used for transcription of denatured yeast DNA (mol. wt ErlO 106) at an enzyme/DNA ratio of 1.0. The degree of symmetry of transcription was determined by self-annealing increasing concentrations of cRNA in 0.15 M N&1/0.015 M trisodium citrate, PH 7.0. at 65’C, for 29 h and measuring the percentage of RNA resistant to pancreatic plus T1 ribonuclease according to Tabak and Borst [351. 0, RNA made in vitro by yeast RNA polymerase at an enzyme/DNA ratio of 0.2; ~1, at a ratio of 1.0; A, at a ratio of 3.0. a, RNA made in vitro by E. coli core polymerax on denatured yeast DNA.
DNA was transcribed by E. coli core RNA polymerase, i.e. conditions which strongly favor symmetrical transcription [ 451. Under these conditions about 7 5% of the RNA made becomes resistant to ribonuclease after self-annealing. From these experiments it can be concluded that both a low enzyme/DNA ratio and a high physical integrity of the DNA template are required to obtain a high fidelity of transcription. Transcription specificity of yeast polymerase A: selective transcription of the ribosomal RNA genes In order to determine whether yeast polymerase A has any preference for the transcription of the yeast rRNA genes, cRNA was made on total yeast DNA in the presence of 5 mM Mg’+ at a low enzyme/DNA ratio (0.1 or 0.2). The TABLE
II
THE EFFECT OF THE PHYSICAL INTEGRITY OF YEAST TRANSCRIPTION BY HOMOLOGOUS RNA POLYMERASE
DNA ON THE DEGREE A
OF SYMMETRIC
Yeast polymerase A was used to transcribe homologous “intact” DNA (mol. wts before and after denaturation 40 . 106 and 5-10 . 106, respectively) and “nicked” DNA (mol. wt before and after denaturation lo . lo6 and 0.2 . 106, respectively) at a low enzyme/DNA ratio (0.2). The degree of symmetry of transcription was determined by measuring the amount of cRNA, which becomes resistant to pancreatic plus T1 ribonuclease upon self-annealing, as described in the legend to Fig. 4. DNA, used in transcription
Jribonuclease-resistant cRNA after self-annealing
Intact DNA Nicked DNA
13 33
187
Unlabeled
RNA
(pg )
Fig. 5. Sequence horn&&y of RNA, synthesized in vitro by yeast polymerase A on homologous DNA, with purified yeast rRNA. Yeast RNA polymerase A or E. coli polymerase were used for transcription of 200 pg yeast DNA at an enzyme/DNA weight ratio of 0.2. DNA templates of different degrees of integrity were used. The degree of sequence homology of the various cRNA preparations with yeast rRNA was tested by competitive hybridization of fixed saturating amounts of in viva 32P-labeled 26 S plus 17 S rRNA and increasing amounts of cRNA as described in Materials and Methods. 0, purified yeast rRNA; 0. RNA made in vitro (yield a&ox. 16 pg) by yeast RNA polymerase A on yeast DNA with double- and single-stranded mol. wts of 40. 106 and 5-10. 106, respectively: A, RNA made in vitro (yield approx. 50 fig) by yeast RNA polymerase A on yeast DNA with a double- and single-stranded mol. wts of 30. 106 and 1 106, respectively: 0, RNA made in vitro (yield approx. 80 Mg) by yeast RNA polymersse A on yeast DNA with double- and single-stranded mol. wts of 10 . 106 and 0.2 . 106, respectively. l, RNA made in vitro (yield approx. 75 fig) by E. coli RNA polymerase (plus 0) on yeast DNA with double- and single-stranded mol. wts of 40. lo6 and 5-10. 106, respectively.
RNA was subsequently characterized by its ability to compete with yeast rRNA in RNA . DNA hybridization. The results of competitive hybridization of fixed saturating amounts of in vivo 3 * P-labeled 26 S plus 17 S rRNA and increasing amounts of cRNA are presented in Fig. 5. About four times more cRNA, transcribed from a template having a relatively high degree of integrity, than pure yeast rRNA is required to obtain the same degree of competition. It can be concluded, therefore, that about 25% of the RNA synthesized in vitro under these conditions consists of ribosomal sequences. Since the rRNA cistrons comprise only approx. 2% of the total yeast DNA [46], this result shows, that yeast polymerase A has a clear preference for in vitro transcription of the ribosomal genes. It can be calculated that roughly one transcript per ribosomal transcription unit is made in vitro in spite of the fact that the enzyme/DNA molar ratio is less than one. In contrast, E. coli RNA polymerase does not show any selective transcription of the yeast rRNA genes. When tested under the same conditions, only 2% of the cRNA, synthesized in vitro by E. coli RNA polymerase, showed sequence homology with yeast rRNA, as judged from competitive hybridization (Fig. 5). The selective transcription of the rRNA cistrons appeared to be strongly dependent on the integrity of the DNA template. When DNA with a large number of nicks (double- and single-stranded mol. wts 30 * lo6 and 1.0 * 106, respectively) was transcribed by polymerase A, the percentage of rRNA sequences in the in vitro transcript decreased to 8-lo%, whereas in the case of even less intact DNA (double- and single-stranded mol. wts 10 . lo6 and 0.2 * 106,
188
respectively), polymerase A did not show transcription specificity anymore (Fig. 5). From these results we conclude that yeast polymerase A is capable of highly selective transcription of the homologous rRNA cistrons in vitro, providing DNA of a high integrity is used as a template. Discussion The subunit structure of yeast RNA polymerase A, as determined by polyacrylamide gel electrophoresis under denaturating conditions is similar to that described for this enzyme isolated from other eukaryotic organisms [23, 27,47-491. In particular, the occurrence of two large subunits (mol. wts in yeast polymerase A 190 000 and 135 000, respectively) is a common feature of this type of enzyme. However, since there is considerable disagreement concerning the number, molecular weights and molar ratios of the smaller subunits of polymerase A from different organisms, comparison of these subunits is extremely difficult. Although the possibility that the differences observed actually reflect dissimilarities between the enzymes from different sources cannot be excluded, it seems likely that they are, at least partially, due to other factors, such as the isolation procedure, degree of purification or conditions of gel electrophoresis. We have observed, for instance, that both the apparent number and the apparent molar ratios of the small polypeptides could be changed by varying either the amount of protein applied to the gel or the length of the staining period. Only when suitable large quantities of protein were used and when staining was carried out for at least 20 h all five small subunits could be detected and appeared to be present in unimolar amounts. If we assume that all proteins found to be present in yeast polymerase A belong to the enzyme structure, the calculated mol. wt is 499 000, which is in very good agreement with the value of 500 000 + 30 000, determined by Sepharose-6B column chromatography. Recently, Buhler et al. [23] reported the subunit structure of Saccharomyces cerevisiae RNA polymerase A. In addition to two large subunits having molecular weights of 190 000 and 135 000, respectively, they detected one copy each of two smaller subunits (mol. wts 48 000 and 41 000) and two copies each of two other small subunits (mol. wts 29 000 and 16 000). Besides minor differences in the molecular weights of the smaller polypeptides, the subunit structure of the S. cerevisiae polymerase differs in a number of respects with that of the S. carlsbergensis enzyme: one of the small subunits (with a mol. wt of 35 000) found in the enzyme described in this paper is lacking from the enzyme isolabed from S. cereuisiae, and the molar ratios of various small proteins are quite different for the two enzymes. Buhler et al. did detect two additional proteins having mol. wts of 44 000 and 35 000, respectively, in their enzyme preparation. However, they did not consider these proteins as constituents of the enzyme because of the “unsatisfactory” molar ratios of these two polypeptides. In our opinion, this interpretation is not completely justified, also considering the results we have obtained. From the results presented in this paper it can be concluded that the enzyme preparation, isolated by us, is capable of a highly selective transcription
189
of homologous DNA. This conclusion is based on the characteristics of the in vitro RNA product, which, when synthesized under the right conditions, shows a considerable sequence homology with yeast rRNA. In addition, the in vitro transcription appears to be highly asymmetric. The most important prerequisites for this selective transcription appear to be a high physical integrity of the DNA template and a low enzyme/DNA weight ratio. The first requirement can be explained by the observation that singlestranded breaks in the template can act as pseudopromoters which tend to mask the binding of the enzyme to specific promoters [40]. In fact, it was recently shown by Dezelee et al. [50] that yeast polymerase A can be entirely dependent on pseudopromoters, such as single-stranded breaks or regions, using heterologous T7 DNA as a template. A similar conclusion was made by Mandel and Chambon [ 441 using polymerase A from calf thymus and Simian Virus 40 DNA as a template. However, our results demonstrate that yeast polymerase A is able to transcribe preferentially the rRNA cistrons, suggesting that true promoter sites are recognized by the enzyme. The second requirement, that of a low enzyme/DNA ratio, probably can be explained in a similar way. The highly selective transcription at low enzyme/DNA ratios must reflect a strong affinity of the polymerase A for the rRNA promotor sites. At higher enzyme/DNA ratios polymerase A seems to bind to false promoter sites present even in DNA of high integrity which results in a lower fidelity of transcription. The stimulating effect of Mn*’ on yeast polymerase activity (see Results) is probably based on the introduction of unpaired regions [ 441 which may also give rise to false initiation sites. This hypothesis is supported by our observation that the degree of symmetric transcription is strongly enhanced by the addition of Mn” to the incubation mixture (unpublished results). For this reason, characterization of transcription products was carried out using RNA transcribed in the absence of Mn*‘. Our finding that yeast polymerase A in vitro is capable of a highly selective transcription of yeast rRNA genes is in agreement with the recent results of Beebee and Butterworth [ZO] who observed a similar specificity of Xenopus laevis polymerase A. The failure of other investigators [51,52] to obtain selective transcription can be explained in several ways such as use of a template of low physical integrity or lack of essential factors from the enzyme. In this respect it should be noted that both the enzyme used by Beebee and Butterworth [20] and that used in the studies described here was isolated from whole cells. Acknowledgements The present investigations were supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). One of us (H.v.K.) is very grateful to Dr J.M. Buhler for making him familiar with the procedure for the isolation of large quantities of RNA polymerase A from whole yeast cells and for his kind help. The authors are indepted to Dr H.A. Raue for helpful critical comment on this manuscript and to Miss E. van der
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Veer, Mr C.P. Bakker, Mr W.L. Homan, Mr W. Bruggeman Oosten for their skilled technical assistance.
and Mr Chr. van
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