the journal of val. 23, issue of december 10, pp. 11588 ... · omission of mgcl2 completely...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Val. 255, No. 23, Issue of December 10, pp. 11588-11598, 1980

Purification and Characterization of a GTP-Pyrophosphate Exchange Activity from Vaccinia Virions ASSOCIATION OF THE GTP-PYROPHOSPHATE EXCHANGE ACTIVITY WITH VACCINIA mRNA GUANYLYLTRANSFERASE *RNA (GUANINE-7-)METHYLTRANSFERASE COMPLEX (CAPPING ENZYME) *

(Received for publication, May 22, 1980)

Stewart Shuman, Martin Surks$, Henry Furneaux, and Jerard Hurwitz From the Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgC12 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP.

The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through succes- sive chromatography steps on DEAE-cellulose, DNA- cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPI exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described.

The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.

Purified vaccinia virions contain a variety of enzymatic activities involved in the metabolism of nucleic acids and nucleotides. Several of these, including RNA guanylyltransferase and 7-methyltransferase (1-3), 2-0-methyltransferase (4), poly(A) polymerase (5), and DNA-dependent RNA polymerase (6, 7) are involved in mRNA synthesis and process- ing. Other enzymes, whose roles in transcription remain un- clear, include DNA topoisomerase (8), 5’-phosphate polynucleotide kinase (9), protein kinase (lo), single strand specific DNase ( l l ) , RNA triphosphatase (12), and two nucleic acid- dependent nucleoside triphosphatases (13, 14).

We have reported recently the purification of the DNA- dependent RNA polymerase present in vaccinia virus cores.

* This work was supported by Grant 5R01 GM13344-15 from the National Institutes of Health, Grant 5R01 CA21622-04 from the National Cancer Institute, and Grant NP 89L from the American Cancer Society. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

tabolism, Department of Medicine, Montefiore Hospital and Medical 4 A visiting scientist from the Division of Endocrinology and Me-

Center and the Albert Einstein College of Medicine, Bronx, N.Y.

The enzyme was characterized as a rifampicin- and a-amani- tin-resistant polymerase capable of transcribing single- stranded DNAs in the presence of Mn2+ and the four rNTPs (6). During further studies on the RNA polymerase, we observed a novel nucleoside triphosphate-PPi exchange reaction catalyzed by partially purified RNA polymerase preparations. This exchange reaction was unusual in that it was independent of a DNA template, was insensitive to nucleases, and required only GTP for activity. Furthermore, the activity was not evident in more highly purified RNA polymerase preparations, indicating that the exchange reaction was catalyzed by an enzyme other than RNA polymerase. This exchange activity, previously undetected in vaccinia virus, was observed readily in permeabilized virions and in isolated viral cores.

The occurrence of a virion-associated PPi displacement reaction specific for GTP suggested some relationship between this new exchange activity and the vaccinia virus guanylyltransferase reaction (RNA capping), which has been shown to involve a reversible transfer of GMP from GTP to RNA, with elimination of PPi (2). Recently, Mizumoto and Lipmann have reported that a GTP-PPi exchange activity was associated with partially purified capping enzyme from calf thymus (15). In order to address this question in the case of vaccinia, the properties of the GTP-PP, exchange reaction catalyzed by vaccinia virions have been studied and the enzyme has been solubilized and purified from viral cores. The results presented herein indicate that the GTP-PPi exchange reaction is catalyzed by the vaccinia virus mRNA capping enzyme.

EXPERIMENTAL PROCEDURES’

RESULTS

PP, Exchange Reaction in Purified Vaccinia Virwns- Incubation of permeabilized vaccinia virions with [”P]PPi in the presence of MgCL and GTP resulted in the incorporation of 32P into acid-soluble, Norit-adsorbable material. (This material, the product of a PPi exchange, will later be identified as guanosine triphosphate.) As shown in Table I, 32P incorporation catalyzed by vaccinia virus is contingent upon the addition of GTP. Neither UTP, CTP, or ATP will replace GTP in this reaction. Inclusion of ATP, CTP, and UTP along

’ Portions of this paper (including “Experimental Procedures,” Figs. 3 to 6, and Tables VI11 to X) 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. 20014. Request Document No. 8OM-1031, cite author(s), and include a check or money order for $2.40 per set of photocopies. Full sized photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

11588

GTP-Pyrophosphate Exchange Activity Associated with Vaccinia Capping Enzyme 11589

with GTP had only a slight effect on 32P incorporation, while the omission of GTP from the reaction containing the other rNTPs resulted in a drastic reduction in activity. Of the guanosine nucleotides, only GTP supported the exchange reaction; GDP and GMP were inert, as was ITP. dGTP did support 32P incorporation, although less efficiently (-10%) than GTP.

Characterization of the Reaction Product-The 32P-labeled product synthesized by vaccinia virions in the presence of GTP and [32P]PPi was eluted from Norit with ethanolic ammonia and analyzed by thin layer chromatography on polyethyleneimine cellulose developed in 1.6 M LiC1. Under these conditions, the radioactive material migrated as two discrete species with mobilities which were identical with those of the GTP and GDP markers (Fig. 1). No radioactivity could be detected in a position corresponding to GMP or PPi (the latter migrates slightly slower than GTP using these chromatographic conditions).

As shown in Fig. 1, 80% of the radioactive products was GTP and 20% was GDP. We propose that the reaction we are monitoring proceeds as follows: G-P-PP + PPi e G-P-PP to generate GTP labeled at the fl- and y-phosphate residues. Radioactive GDP presumably arises via the action of the GTPase activity present in the vaccinia virion. The 32Pi gen- erated by the GTPase would not be detected by our assay procedure and would not be expected to appear in the product analysis in Fig. 1.

Properties of the GTP-PP, Exchange Reaction-The incorporation of 32P into Norit-adsorbable material catalyzed by penneabilized virions was linear with time up to 40 min, and continued at a lesser rate up to 60 min. The activity required Mg2+ as a cofactor (Fig. 2b) . Omission of MgCl2 completely abolished activity; optimal exchange occurred between 3 to 7 l ~ l ~ MgClz with a gradual decline in activity at concentrations in excess of 10 m. The dependence of the exchange reaction on PPi concentration is shown in Fig. 2c. The reaction velocity was proportional to PPi concentration up to 1 mM PPi, pla- teaued between 1 to 1.4 m, and decreased at higher PPi concentrations; activity at 5 m PPi was 24% of the optimal rate. The reaction displayed a hyperbolic dependence on GTP concentration, as indicated in Fig. 2a, with optimal activity at

TABLE I NTP-PP, exchange reaction catalyzed by permeabilized vaccinia

virions Reaction mixtures (50 p i ) contained 60 mM Tris.HC1, pH 8.4, 10

mhf dithiothreitol, 5 mM MgC12, 1 mhf [32P]PPi, 0.05% NP-40, 0.2 AZm unit of purified vaccinia virus, and nucleotides as indicated at a final concentration of 1 m. dGTP was freed of ribonucleotide contaminants by periodate treatment followed by re-purification on DEAE- Sephadex. Reactions were incubated for 30 min and processed as described under “Experimental Procedures.”

* * * *

Nucleotides added Norit-adsorbable 32P P W l

None GTP

7 871

CTP 18 UTP ATP

12 10

ATP, GTP, CTP, UTP 550 ATP, CTP, UTP dCTP

43 100

ITP 12

None GTP

22 1114

GDP 25 GMP <I

Experiment 1

Experiment 2

FIG. 1. Characterization of the PPi exchange reaction product synthesized by vaccinia virions. Reaction conditions were as described in the legend to Table I with the following modifications; reaction volume was increased to 0.25 ml and contained 1 mM GTP and 1 A260 unit of purified virus. After 30 min at 37”C, the reaction was halted as described under “Experimental Procedures.” The acid- soluble material was bound to Norit and centrifuged to remove [32P]PPi. The charcoal was resuspended in 5 mM HCI and recentrifuged. This step was repeated three times until all nonadsorbed radioactivity was eliminated from the reaction product. The charcoal was washed with distilled water, recentrifuged, and eluted with a solution containing 50% ethanol and 2% NH4OH. The eluted material was lyophilized, resuspended in water, and chromatographed on PEI- cellulose plates developed with 1.6 M LiCl. The chromatograms were cut into I-cm slices and assayed for 32P by Cerenkov counting. The positions of UV marker nucleotides are indicated by the arrows.

3 1 5 2 0

- 0 1 2 3 4 5

FIG. 2. Properties of the GTP-PPi exchange reaction catalyzed by vaccinia virions. Reaction mixtures containing 0.14 A ~ w unit of vaccinia virus were prepared as described in the legend to Table I and were incubated for 30 min at 37°C. The concentrations of individual reaction components were varied as indicated in a to d. In the experiment shown in c, blank values of Norit-adsorbable 32P were determined for each concentration of [32]PP, and were subtracted from the incorporation obtained in the presence of virus. In the experiment illustrated in d, reaction mixtures contained 60 mM Tris . HCl buffer at the indicated pH values.

1 m GTP. Enzymatic activity was dependent on alkaline pH, with optimum exchange between pH 8.1 to 8.5 (Fig. 2 4 . Activities at pH 7.1 and pH 8.9 were reduced by 95% and 35%, respectively.

To determine whether the GTP-PPi exchange activity was associated with viral cores, the outer envelope was removed

11590 GTP-Pyrophosphate Exchange Activity Associated with Vaccinia Capping Enzyme

from purified vaccinia virions with 0.5% NP-40 and 50 mM dithiothreitol using a modification (13) of the procedure of Easterbrook (19); the viral cores were isolated by centrifugation as described (6). This treatment removed approximately one-third of the virus protein (Table 11), yet all of the GTP- dependent PPi exchange activity was recovered in the viral cores. In order to study the exchange reaction in detail, we have purified the enzyme from viral cores. It was of particular interest to establish the relationship of the exchange activity to two previously described vaccinia enzymes: (a) the DNA- dependent RNA polymerase, which was noted to contain a GTP-PPi exchange activity during the initial stage of purification, and (b ) the RNA guanylyltransferase - 7-methyltransferase complex (capping enzyme), which shares certain properties with the exchange activity, i.e. a PPi displacement specific for GTP. The purification (along with Figs. 3 to 6) is presented in the miniprint supplement.

Summary of Purification-The results of the purification of the GTP-PPi exchange enzyme from viral cores indicate that the exchange reaction is catalyzed by the vaccinia virus mRNA capping enzyme. As shown in Figs. 4 and 5, co-chromatography of GTP-PPi exchange activity with RNA (guanine-7-)methyltransferase and/or RNA-guanylyltransferase as well as with a newly described nucleic acid-independent ATPase was observed on columns of DNA-cellulose and phosphocellulose that were developed with linear salt gradients. A summary of the enzyme purification through the phosphocellulose stage (Table 111) indicates that the ratios of GTP-PPi

TABLE I1 Association of GTP-PP, exchange with vaccinia cores

Fractions (NP-40 pellet and NP-40 supernatant) were prepared from 280 AZm units of purified vaccinia virus as described in the text. Protein was determined by the method of Bradford (27). Reaction mixtures were as described in the legend to Table I and contained 1 m~ GTP where indicated. Either virus (13.5 pg of protein), viral cores (9.8 pg of protein), or envelope fraction (4.4 pg of protein) were assayed for PPi exchange activity. NP-40 was omitted from the reactions containing NP-40 pellet or NP-40 supernatant fractions. Incubation was for 30 min in each case.

Fraction Norit-adsorbable “P

+GTP -GTP Total protein

pmol w virus 667 5 27.0 Cores (NP-40 pellet) 673 4 19.5 Envelope (NP-40 supernatant) 9 9 10.3

exchange, 7-methyltransferase, and nucleic acid-independent ATPase activities were relatively constant, particularly during the latter purification steps. In the case of the ATPase, the lower yield of enzyme at the DNA-cellulose step may be attributed to the elimination of the nucleic acid-independent ATPase activity contributed by phosphohydrolase I1 (see Fig. 4). Physical association of GTP-PPi exchange with capping enzyme and nucleic acid-independent ATPase was maintained even through glycerol gradient sedimentation in 1 M NaC1. The observed sedimentation coefficient of 6.5 S (Fig. 6) is in agreement with the results of Martin et al. (1) who obtained a value of 6 S for the purified capping enzyme complex using 5 to 20% sucrose gradients in 0.25 M NaC1, and with the glycerol gradient sedimentation profiles for capping enzyme reported by Monroy et al. (3) and Venkatesan et al. (20). Our data strongly suggest that the guanylylation, methylation,

- 80

w N V

-60 -.

0

CL - I

a-“

- 4 0 1 - 20

V I I 1 1 I I 0 IO x) 30 40 50 60

Tame (min)

FIG. 7. Time course of y-phosphate cleavage and RNA capping. A 50-4 reaction mixture containing 50 mM Tris. HC1, pH 7.5,5 mM dithiothreitol, 1.25 m~ MgC12, 17 BM [3H]GTP, approximately 80 pmol of triphosphate termini of [y-32P]poly(A), and enzyme was incubated at 37°C. At the indicated times, 7 . 5 ~ 1 aliquota were with- drawn and acid-insoluble material was collected on glass fiber filters. Cleavage of the y-phosphate of [y-32P]triphosphate-terminated poly(A) was assayed by the loss of acid-insoluble 32P (determined by Cerenkov counting) and is expressed as the percentage of triphosphate termini hydrolyzed. Cap formation was assessed by tritium counting of the same filters in Econofluor scintillation fluid.

TABLE I11 Purification of GTP-PP, exchange enzyme and associated activities

GTP-PPi exchange was assayed as described in the legend to Fig. [3H]SadenosylmethioNne to GTP in 30 min at 37°C. Reaction mix- 3. One unit of exchange enzyme directed the incorporation of 1 m o l tures for assay of nucleic acid-independent ATPase contained ( 5 0 4) of 32P from [32]PPi into acid-soluble, Norit-adsorbable material in 30 40 mM Tris. HCl, pH 8.3, 2 mM dithiothreitol, 6 mM MgClz, 1 mM [y- min at 37OC. RNA (guanine-7-)methyltransferase was assayed as 32P]ATP, and enzyme. One unit of ATPase released 1 m o l of 32Pi described under “Experimental Procedures.” One unit of 7-methyl- from [y3’P]ATP in 30 min at 3 7 T . Protein was determined by the transferase catalyzed the transfer of 1 pmol of [3H]methyl group from method of Bradford (27).

Fraction GTP-PPi exchange RNA (guanine-7-)methyltransferase independent Nucleic acid-

ATPase unitslml units % yield units %yield units mg

Virions 331 1986 100 4800 100 22.0 NP-40 pellet (cores) 175 1050 53 6Ooo 125 Deoxycholate supernatant 240 1776 5328 3.6 Deoxycholate pellet 148 888 2760 5.9

Triton supernatant 70 595 2550 Triton pellet 60 180 300

DEAE I 115 2300 116 3680 77 17,600 5.0 DEAE I1 unbound 18.5 1332 67 4608 96 13,840 DEAE I1 bound 30 300 960 2,780 DNA-cellulose 140 1148 58 3411 71 6,382 Phosphocellulose 65 877 44 3240 67 6,413


-I - 2

- 3

o b c d e f FIG. 8. Purification of GTP-PPI exchange enzyme shown by

sodium dodecyl sulfate polyacrylamide gel electrophoresis. Samples of GTP-PPi exchange enzyme at various stages of purification were analyzed by gel electrophoresis as described under “Exper- imental Procedures.” The polypeptide composition of vaccinia virions, viral cores (NP-40 pellet), the DEAE-cellulose I enzyme preparation, the flow through from DEAE-cellulose 11, DNA cellulose column Fraction 24, and the pooled phosphocellulose enzyme preparation is shown in Lanes a through j , respectively. The approximate amount of enzyme applied to each lane was: Lane a, 1 pl of purified vaccinia virions (0.33 unit of enzyme); Lane b, 1 pl of viral cores (0.18 unit of enzyme); Lane c, 8 pl of DEAE-I enzyme (0.92 unit); Lane d, 20 pl of DEAE-I1 flow through (0.37 unit); Lane e, 20 pl of DNA-cellulose column Fraction 24 (-3.2 units); Lane f, 20 pl of phosphocellulose enzyme (1.3 units). The positions of marker proteins run in the same gel are indicated by the arrows. The markers are I) /3 and p’ subunits of E. coli RNA polymerase (-M, = 160,000); 2) @galactosidase (-Mr = 130,000); 3) large subunit of y-globulin (-M, = 53,000); 4) small subunit of y-globulin (-Mr = 23,000); and 5) cytochrome c (-Mr = 12,000). The molecular weights of the major polypeptides in the purified enzyme preparations (discussed in the text) were determined from a semilogarithmic plot of molecular weight uersus migration of marker proteins of known molecular weight.

GTP-PPi exchange, and ATPase reactions are catalyzed by a multifunctional capping enzyme complex.

RNA Triphosphatase Associated with GTP-PPi Exchange and Capping Enzyme Complex-During assays of guanylyltransferase in the presence of a [y-32P]triphosphate terminated poly(A) acceptor, it was noted that a loss of acid-insoluble 32P radioactivity (ascertained by Cerenkov counting) occurred in large excess over the amount of ends capped, and that this loss of acid-insoluble 32P was independent of GTP. Venkatesan et al. (20) have recently reported that an RNA triphosphatase activity is associated with the vaccinia capping enzyme, and that conversion of triphosphate-terminated RNA to diphosphate-terminated RNA precedes guanylylation. Fig. 6 shows that our preparation of GTP-PPi exchange enzyme does indeed contain an RNA triphosphatase which co-sediments with capping enzyme and nucleic acid-independent ATPase. The results of Fig. 7 support their conclusion that terminal cleavage precedes capping.

Sodium Dodecyl Sulfate Gel Electrophoresis-Due to the minute amounts of protein present in the more purified enzyme fractions, we have assessed qualitatively the purity of the GTP-PPi exchange enzyme preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subse- quent visualization of the protein bands using the highly sensitive silver staining procedure (25). Fig. 8 shows the polypeptide composition of virions, cores, DEAE-cellulose I, DEAE-cellulose 11, DNA-cellulose, and phosphocellulose fractions, revealing an extensive purification via this protocol. The phosphocellulose enzyme contains two major polypeptides of

molecular weights 95,800 f 1,600 and 26,400 f 800. These values are the average of four separate molecular weight determinations using sodium dodecyl sulfate gels and have average deviations as indicated. The presence of these same two polypeptides paralleled the GTP-PPi exchange activity and associated activities across the glycerol gradient (Fig. 9). Martin et al. (1) reported that their capping enzyme preparations contained two major polypeptides of 95,000 and 31,400 molecular weight.

Characterization of the PPi-Exchange Reaction Product Synthesized by the Purified Enzyme-The product synthe- sued by the purified PPi exchange enzyme in the presence of GTP and [32P]PPi was eluted from Norit and analyzed by thin layer chromatography on PEI-cellulose developed with 1.6 M LiCI. As shown in Fig. loa, virtually all the radioactivity co- migrated with the GTP marker; in contrast to the results with permeabilized virions, no peak of radioactive GDP was observed, suggesting that GTPase activity may have been eliminated during enzyme purification (see below). Since this chromatography system served only to resolve nucleoside

PC 17 18 19 20 21 22 FIG. 9. Polypeptide composition of GTP-PPI exchange en-

zyme sedimented in a glycerol gradient. Aliquots (20 pl) of the phosphocellulose enzyme preparation and of Fractions 17 through 22 from the glycerol gradient sedimentation step (Fig. 6) were analyzed by gel electrophoresis as described under “Experimental Procedures.” From left to right, the lanes contain phosphocellulose enzyme (PO and glycerol gradient fractions indicated by number.

I f , ’ ~ , J 0 8 16 24 32 8 16 24 32

Frochon Number

FIG. 10. Analysis of the PPI exchange reaction product synthesized by purified PPI exchange enzyme. A reaction mixture (0.1 ml) containing 60 mM Tris-HC1, pH 8.4, 10 m~ dithiothreitol, 5 m~ MgC12, 1 mM [32P]PPi, 0.2 mM GTP, and the phosphocellulose enzyme preparation was incubated for 30 min at 37°C. The reaction product was isolated as described in the legend to Fig. 1 and chromatographed on PEI-cellulose plates developed with either 1.6 M LiCl ( a ) or 0.75 M (NH,)*O, (b) . The chromatograms were cut into slices and assayed for 32P by Cerenkov counting. The positions of UV marker nucleotides are indicated by solid bars.


mono-, di-, and triphosphates, the reaction product was also analyzed using PEI-cellulose developed with 0.75 M (NH4)2S04, a system capable of resolving the four NTPs. Fig. 10b indicates that only GTP was formed in the PPi-exchange reaction; no labeled ATP, CTP, or UTP were detected as contaminants of GTP.

Fate of GTP during PP, exchange-In view of the presence of an ATPase activity in the purified PPi exchange enzyme preparation, it was of interest to determine whether the failure to observe 32P-labeled GDP as a reaction product (Fig. 1 uersus Fig. 10) was due to an inherent inability of the ATPase to hydrolyze GTP, or to a lack of GTPase activity under the reaction conditions employed for PPi exchange. This issue was addressed by following the fate of [3H]GTP when incubated with purified enzyme. Fig. 11 shows that incubation of [3H]- GTP with enzyme in the absence of PPi results in significant cleavage of GTP to GDP. GDP formation was linear for 30 min, and continued at a lesser rate up to at least 60 min of incubation. No formation of [3H]GMP was detected at any time (not shown). Clearly, the nucleic acid-independent ATP- ase is not restricted in its NTP specificity, and is more appro- priately termed a nucleoside triphosphate phosphohydrolase. When 1 m~ PPi was included in the reaction mixture, a dramatically different result was obtained (Fig. 11); under conditions which supported GTP-PP, exchange, the cleavage of GTP to GDP was negligible. Thus, the absence of a GDP product is explained by an inhibition of phosphohydrolase activity by PPi. It was noteworthy that in the presence of 1 m~ PPi no formation of [3H]GMP could be detected at any time; this was the case even when (as depicted in Fig. 11) unlabeled GMP was included in the reaction mixture to “trap” any free [3H]GMP that might arise as a reaction intermediate during PPi exchange. Further evidence that free GMP is not an intermediate in PPi exchange catalyzed by capping enzyme came from studies of [3H]GMP-GTP exchange, an approach which affords far greater sensitivity than GMP “trapping.” As indicated in Table IV, [3H]GMP did not exchange with GTP under conditions permissive for GTP-PPi exchange. Further

z 0”- -1 500

30 45 60 Tlme(mln)

FIG. 11. Metabolism of GTP by purified PPi exchange enzyme. A reaction mixture (50 p l ) containing 60 m~ Tris. HC1, pH 8.3, 4 m~ MgClz, 0.2 mM [3H]GTP, 0.2 mM GMP, and purified enzyme was incubated at 37OC, as was the second reaction mixture containing the same components plus 1 mM PPi. At the indicated time, 6-4 aliquots were removed and spotted on PEI-cellulose plates along with GTP, GDP, and GMP markers. The plates were developed with 1.5 M Lick GTP, GDP, and GMP spots were located by UV illumination,

sion of [3H]GTP to [3H]GDP is plotted as a function of time for the cut out, and counted in toluene-based scintillation fluid. The conver-

reaction containing 1 m~ PPi (M) and for the reaction in which PP, was omitted ( O ” - O ) . No formation of C3H]GMP was detected in either case. A third reaction mixture was prepared and incubated as above but contained [32P]PPi and unlabeled GTP. Aliquots of this reaction were removed at indicated times and assayed for formation of Norit-adsorbable 3zP (U - -0) as described under “Experimental Procedures.” Similar results were obtained when assays were performed without the inclusion of GMP in the reaction mixtures.

TABLE IV Inability to t3H]GMP to exchange with GTP during GTP-PP,

exchange reaction The complete reaction mixture (50 1.1) contained 60 mM Tris. HCl,

pH 8.3,4 mM MgCL, 1 m~ sodium PPi, 0.2 m~ GTP, 19 p~ C3H]GMP (6.3 Cihmol), and enzyme. After 30 min at 37°C. a 6-9 aliquot was taken from each reaction and applied to PEI-cellulose plates along with GTP, GDP, and GMP markers. The plates were developed with 1.5 M LiC1. After drying, the marker spots were cut out and counted in toluene-based scintillation fluid. A parallel incubation containing 1 mM [32P]PPi and enzyme resulted in 503 pmol of 32P incorporation into Norit-adsorbable material in 30 min. The reaction indicated in Line 4 contained 60 mM Tris.HC1, pH 8.3, 4 mM MgClz, 13 p~ [3H]GMP, 1 mM ATP, and 6 pg of GMP kinase from hog brain.

Distribution of [3H]Guanosine nucleotide

GTP GDP GMP % % %

Reaction conditions

1. Omit enzyme 0.2 0.6 99.2 2. Omit PPi 0.2 0.6 99.2 3. Complete 0.2 0.5 99.3 4. Omit enzyme, add GMP 18.7 78.8 2.5

kinase

TABLE V Divalent cation specificity of GTP-PP, exchange

Reaction mixtures contained 60 mM Tris.HC1, pH 8.3, 1 mM [“2P]sodium pyrophosphate, 0.2 mM GTP, and divalent cations as indicated. All divalent cations except zinc were added as the chloride salt; zinc was added as the sulfate salt, Mg‘+ was present at 4 mM concentration; all other cations were at 1 m~ concentration.

Divalent cation added Norit-adsorbable ,r’P pmol/40 rnin

Mg‘+ 566 Ca2+ 4 Ca2+ + Mg2+ 396 CO” < l CO’+ + Mg2’ 410 cu2+ <I Cu” + Mg” 102 Mn” <I Mn2+ + Mg’+ 79 Znz+ <1 Zn” + Mg’+ 122

discussion of possible exchange reaction intermediates is included below.

Characteristics of the PPi Exchange Reaction Catalyzed by Purified Capping Enzyme Complex-The ability of vaccinia guanylyltransferase to catalyze a [32P]PPi exchange with GTP would seem to afford a sensitive and convenient assay for capping enzyme function. In the interest of facilitating such an assay, the optimal conditions for GTP-PPi exchange by purified enzyme were determined.

Divalent Cation Requirement-PPi exchange required a divalent cation in the form of Mg2+ (Table V). The dependence of PPi exchange on MgClz concentration was similar to that described for the exchange in permeabilized virions; optimal activity occurred from 3 to 7 m M MgCh. Of the various divalent cations tested, only Mg2+ supported exchange activity, calcium and cobalt did not support PPi exchange but did not significantly inhibit the reaction in the presence of magnesium. Manganese, copper, and zinc also failed to support activity, but did inhibit PP, exchange by approximately 80% when included with magnesium (Table V).

pH Dependence-Optimal GTP-PPi exchange occurred from pH 7.9 to 8.5 (60 m~ Tris.HC1 buffer). Activity at pH 7.3 was 37% of the activity at pH 8.3.

PP, Dependence-In the presence of 0.2 mM GTP, PPi exchange readily occurred from 0.6 to 2 mM PPi. Use of lower


or higher PPi concentrations resulted in a reduction of activity as shown in Fig. 12.

Nucleotide Dependence-The rate of PPi exchange increased with increasing GTP concentration up to 0.3 mM GTP. Activity decreased at higher GTP concentrations as indicated in Fig. 12. The apparent K,,, for GTP of the purified enzyme was lower than that of permeabilized virions (see Fig. 2b); this may be due to the elimination of GTPase activity during enzyme purification.

Of the various ribonucleoside triphosphates, only GTP was able to participate in the PPi exchange reaction catalyzed by purified capping enzyme complex (Table VI). Among the guanine nucleotides, GDP and GMP were inactive while dGTP did support PPi exchange, albeit far less efficiently than GTP. It is noteworthy that 7-methyl-GTP did not participate in PPi exchange; Martin and Moss have previously shown that 7-methyl-GTP cannot serve as a cap donor in RNA guanylylation (21).

Effect of Salts and Inhibitors The GTP-PPi exchange reaction was unaffected by the omission of dithiothreitol, yet was almost completely inhibited by the sulfhydryl antagonist, p-hydroxymercuribenzoate. This inhibition was reversible by the addition of dithiothreitol (Table VII). The effect of ionic strength on enzyme activity is shown in Table VII. GTP-PPi exchange was unaffected at NaCl concentrations up to 0.1 M and decreased gradually as the ionic strength was increased from 0.1 to 0.7 M NaC1. The PPi exchange activity was not inhibited quantitatively by NaCl concentrations as high as 1 M.

As indicated in Table VII, neither Pi nor inorganic sulfate were inhibitory to GTP-PPi exchange up to 40 m~ concentrations; Pi in particular had a stimulatory effect on enzyme activity.

Properties of the RNA Triphosphatase and Nucleic Acid- independent NTP Phosphohydrolase Associated with Cap-

1 0 0 / = y 1 I , I 1 - E CII c c

0 0.2 0.4 0.6 0 8 1.0 c) GTP (mM)

FIG. 12. Dependence of GTP-PPI exchange on GTP and PPi concentration. Reaction mixtures (50 p l ) contained 60 mM Tris. HCl, pH 8.3, 10 mM dithiothreitol, 4 m~ MgC12, 1 mM [32P]PPi (in experiments in which GTP was varied), and 0.2 mM GTP (in experiments in which E3'P]PPi was varied). Incubations were for 30 min a t 37°C.

TABLE VI Nucleotide specificity ofpurified PP, exchange enzyme

Reaction conditions were as described in the legend to Table V except that nucleotides were added at 0.2 m~ concentration as indicated.

Nucleotide added Norit-adsorbable "P pmol

Experiment 1 None GTP ATP CTP UTP GTP, ATP, CTP, UTP ATP, CTP, UTP dGTP ITP GDP GMP

GTP None

7-Methyl-GTP

Experiment 2

430 <I

<I <I

322 <I

6 27

<I 5

<I

<l 304 <I

TABLE VI1 Influence of various additions on GTP-PP, exchange activity

Complete reactions contained 60 mM Tris.HC1, pH 8.3, 4 mM MgC12, 1 mM [32P]PPi, 0.2 mM GTP, and enzyme. Incubations were for 40 min at 37OC.

Additions Norit-adsorbable a 2 p

Experiment 1 Complete Add 3 mM p-hydroxymercuribenzoate Add 3 mM p-hydroxymercuribenzoate plus

8 m~ dithiothreitol Experiment 2

Complete Add 5 mM Pi

Add 20 mM Pi Add 40 mM P, Add 5 mM Na2S04 Add 10 mM Na~S04 Add 20 mM NazSO4 Add 40 mM Na2S04

Experiment 3 Complete Add 0.1 M NaCl Add 0.3 M NaCl Add 0.5 M NaCl Add 1.0 M NaCl

Add 10 mM Pi

pmof

477 17

462

517 540 635 688 561 577 552 531 394

490 485 250 132 39

ping Enzyme-RNA triphosphatase, an activity which cleaves only the y-phosphate of triphosphate-terminated RNA was described initially in extracts of Escherichia coli by Maitra and Hurwitz (23). Two RNA triphosphatases were purified from E. coli. One enzyme hydrolyzed the y-phosphate of ATP- terminated RNAs as well as the terminal phosphate of ATP and dATP. A second activity hydrolyzed the terminal phosphate of RNAs without regard to the nucleotide at the 5'-end; this enzyme also cleaved the y-phosphate of all four ribonucleoside triphosphates. Tutas and Paoletti (12) have purified an RNA triphosphatase from vaccinia virus cores. The vaccinia enzyme was reported to cleave the y-phosphate of 5'- ATP- and 5'-GTP-terminated polyribonucleotides, but was reported not to cleave the y-phosphates of ATP or GTP. These authors proposed that the function of this enzyme is to generate diphosphate-terminated RNAs which may in turn be capped by the guanylyltransferase - 7 methyltransferase complex. Evidence in support of this model was provided by


Venkatesan et al. (20). Our own data support their conclusion that an RNA triphosphatase is a component of the capping enzyme complex; however, we found that the cleavage of y- phosphate by the capping enzyme was not restricted to RNA termini but occurred with ATP and GTP as well. Studies of hydrolysis of y-phosphate by purified capping enzyme are presented in the miniprint supplement.

Further Studies of RNA Guanylylation and Methylation- The capping enzyme activity obtained using the present purification procedure catalyzed both the guanylylation and methylation of the 5‘-ends of triphosphate-terminated poly(A). The transguanylylation reaction showed an absolute requirement for MgC12 and a polynucleotide cap acceptor (ppp-terminated poly(A)). Activity was extremely sensitive to inhibition by PPi (97% inhibition at 100 PM PPi) but was

n

10 X) 30 40 50 60 Fraction Number

FIG. 13. Phosphocellulose chromatography of RNA polymerase. DEAE-cellulose I1 Fractions 10 to 15 were pooled and chromatographed on phosphocellulose as described in the text. Aliquots (5 p1) of the fractions were assayed for RNA polymerase activity (shown in c) . GTP-PP, exchange was assayed as indicated in the legend to Fig. 3. ATPase was assayed as described in the legend to Fig. 3 in the presence (M) and absence ( o ” 0 ) of 0.014 AXXI unit of +X174 DNA (Panel a) . 7-Methyltransferase activity was determined as decribed under “Experimental Procedures.” Capping enzyme activity was assayed as described in the legend to Fig. 6, except that incubations were for 30 min. The activity profiles shown in Panels a to c are from the same column run but are illustrated separately for the sake of clarity.

relatively insensitive to Pi (no inhibition up to 20 mM Pi; 55% inhibition at 40 mM Pi). The donor specificity of the capping reaction was examined indirectly, e.g. by assessing the ability of various nucleotides to substitute for GTP in the GTP- dependent methylation of triphosphate-terminated poly(A) (see “Experimental Procedures”). Of the nucleotides tested, only GTP, dGTP, and GTPyS supported the methylation reaction. ATP, CTP, and UTP did not support methylation.

Association of GTP-PPi Exchange Activity and Capping Enzyme with DNA-dependent RNA Polymerase-The studies presented above were all performed with the GTP-PPi exchange activity purified from the DEAE-cellulose 11 unbound fraction. As indicated in Fig. 3 and Table 111, a small but significant portion of the GTP-PPi exchange and RNA (guanine-7-)methyltransferase activities were retained on DEAE-cellulose (DEAE 11) and eluted in parallel with DNA- dependent RNA polymerase (and with ATPase assayed in the presence of +X174 DNA). For further purification, column Fractions 10 to 15 were combined and the pooled DEAE- cellulose 11-bound fraction was applied to a 6-ml column of phosphocellulose which had been equilibrated with 50 mM NaCl in Buffer A. The column was developed with a 60-ml linear gradient of 0.15 to 0.5 M NaCl in Buffer A. Fractions (-1 ml) were collected and assayed as indicated in Fig. 13. RNA polymerase was retained completely on this column and eluted as a peak at 0.35 M NaC1; here too, the GTP-PPi exchange and capping enzyme activities eluted in parallel with the RNA polymerase, as did nucleic acid-independent ATPase activity. ATPase activity in the presence of a DNA cofactor chromatographed as two (perhaps three) components; the major activity did not co-elute with RNA polymerase, GTP- PPi exchange enzyme, or capping enzyme. The relationship of these ATPase activities to the three activities resolved in Fig. 4 has not been determined.

The chromatograms in Figs. 3 and 13 explain the initial observation of GTP-PPi exchange activity in partially purified RNA polymerase preparations as well as the failure to detect incorporation of [y-32P]ATP into RNA synthesized using the phosphocellulose RNA polymerase preparation.2 The data (Figs. 3 and 13) are consistent with the concept that capping enzyme and RNA polymerase may exist as a complex in the vaccinia virion and that this complex may be dissociated during disruption of the virus particle. Preliminary evidence indicates that the RNA polymerase obtained after glycerol gradient centrifugation (6) does not possess GTP-PPi exchange activity although the fate of the GTP-PPi exchange enzyme during centrifugation was not evaluated. Studies of the presumptive complex between capping enzyme and RNA polymerase are in progress.

DISCUSSION

The vaccinia virion-catalyzed NTP-PPi exchange reaction described above is distinguished by its specific requirement for GTP as the nucleoside triphosphate substrate (Table I); this reaction clearly differs from the PPi exchange reactions mediated by aminoacyl-tRNA synthetases, T4 RNA ligase, and some DNA ligases insofar as these enzymes utilize an ATP substrate. Moreover, the vaccinia virus-catalyzed PPi exchange reaction contrasts with the NTP-PPi exchange activities associated with DNA and RNA polymerase in its independence of a polynucleotide template and its nuclease insensitivity.

Among those vaccinia enzymes which have been studied in detail, the RNA-guanylyltransferase (capping enzyme) shares common features with the GTP-PPi exchange activity. The

* S. Shuman, unpublished work.


capping enzyme, which catalyzes the reaction

Gp;; + (p)ppRNA + GpppRNA + $f’i + (Pi)

specifically requires GTP as a cap donor (21). Several groups have shown that the vaccinia guanylylation reaction involves a PPi displacement in GTP, and that the reaction is reversible by pyrophosphorolysis of the GpppX structure to yield GTP (1-3). A GTP-PPi exchange activity in the absence of a cap acceptor has not been reported previously for the vaccinia capping enzyme, but such an activity has been shown to be associated with guanylyltransferase from rat liver nuclei (15). By the criterion of co-purification, we have established that the vaccinia GTP-PP, exchange reaction is indeed catalyzed by vaccinia virus guanylyltransferase; at no point in purification were the exchange activity and the capping enzyme resolved. The exchange reaction thus affords a simple, inex- pensive, and sensitive assay for the presence of vaccinia capping enzyme.

The fact that guanylyltransferase catalyzes GTP-PPi exchange in the absence of a cap acceptor has interesting impli- cations for the mechanism of transguanylylation. We propose (by analogy to the mechanism described for the DNA ligase reaction (22)) that the initial step in cap formation is the generation of an enzyme - guanylate complex with concomitant release of PPi. The enzyme guanylate complex would then transfer its GMP residue to the 5’-terminus of a diphosphate- terminated RNA to generate the G(5’)ppp(5’)X cap structure. In the presence of GTP and [32P]PPi, rapid formation of the enzyme. guanylate complex and reversal of this reaction would constitute the basis of the GTP-PPi exchange described herein. Again, by analogy to DNA ligase, the capping enzyme. guanylate complex may involve a covalent nucleotide-protein linkage. This is consistent with our failure to detect free GMP as a reaction intermediate during PPi exchange and with the lack of [3H]GMP-GTP exchange.

The analogy between the transguanylylation and DNA ligation reactions is not factitious (22). E. coli DNA ligase forms a covalent enzyme-adenylate complex which transfers the AMP moiety to the 5’-end of 5’-phosphate-terminated DNA. The resulting intermediate A(5’)pp(5’)X is quite similar in general structure to the unmethylated RNA cap; Moreover, the capping enzyme, like DNA ligase, catalyzes nucleotide- PPi exchange in the absence of a polynucleotide acceptor. Direct evidence for the proposed transguanylylation scheme, i.e. the demonstration of a covalent enzyme-guanylate intermediate, has indeed been obtained and will be presented el~ewhere.~

In addition to the guanylyltransferase, 7-methyltransferase, and GTP-PPi exchange activities, the capping enzyme contains an RNA triphosphatase and a nucleic acid-independent NTP phosphohydrolase, all of which are associated in a 6.5 S complex containing two major polypeptides of 96,000 and 26,000 molecular weight. Using a similar purification procedure, Venkatesen et al. (20) have demonstrated an association of capping enzyme with RNA triphosphatase in a complex previously reported (1) to contain major polypeptides of 95,000 and 31,400 molecular weight. Tutas and Paoletti (12) have purified an RNA triphosphatase which does not contain guanylyl- or methyltransferase activities, but, interestingly, this enzyme sediments at 6.2 S on sucrose gradients and consists of major polypeptides of 90,000 and 26,000 molecular weight.

S. Shuman, manuscript in preparation.

On the other hand, Monroy et al. (3) have isolated a guanylyltransferase - methyltransferase complex which lacks an RNA triphosphatase component; this enzyme consisted of polypeptides of 95,000,28,000, and 59,000 molecular weight at an analogous stage of purification. At this time, none of these activities (GTP-PPi exchange, guanylyltransferase, 7-methyltransferase, RNA triphosphatase, or nucleic acid-independent nucleoside triphosphatase) have been assigned with certainty to any of the polypeptides found in the enzymes obtained by various investigators. It is conceivable that the observation, or failure to observe association between the activities may stem from selective inactivation or dissociation during enzyme extraction and purification. However, comparison of the various purification protocols has not yet been performed.

The observation that vaccinia capping enzyme is associated with the virus DNA-dependent RNA polymerase through two column chromatography steps is provocative. These data suggest a pre-existing complex between the two activities in the virus particle. Such a complex would facilitate the capping of nascent RNAs soon after initiation. Although it has not yet been demonstrated, the postulation of a complex between guanylyltransferase and RNA polymerase I1 in the cell nu- cleus offers one explanation of the preponderence of RNA capping of RNA polymerase I1 products (24).

REFERENCES 1. Martin, S. A,, Paoletti, E., and Moss, B. (1975) J. Biol. Chem.

2. Martin, S. A., and Moss, B. (1975) J. Biol. Chem. 250,9330-9335 3. Monroy, G., Spencer, E., and Hurwitz, J. (1978) J. B i d . Chem.

4. Barbosa, E., and Moss, B. (1977) J. Biol. Chem. 253, 7692-7697 5. Moss, B., Rosenblum, E. N., and Gershowitz, A. (1975) J. B i d .

6. Spencer, E., Shuman, S., and Hurwitz, J. (1980) J. B b l . Chem.

7. Baroudy, B. M., and Moss, B. (1980) J. Biol. Chem. 255, 4372-

8. Bauer, W., Ressner, E., Kates, and J., and Patzke, J. (1977) Proc.

9. Spencer, E., Loring, D., Hunvitz, J., and Monroy, G. (1978) Proc.

250,9322-9329

253,4481-4489

Chem. 250,4722-4729

255, 5388-5395

4380

Natl. Acad. Sei. U. S. A . 74, 1841-1845

Natl. Acad. Sei. U. S. A . 75,4793-4797 10. Kleiman, J., and Moss, B. (1975) J. B i d . Chem. 250,2420-2429 11. Rosemond-Hornbeak, H., Paoletti, E., and Moss, B. (1974) J.

12. Tutas, D. J., and Paoletti, E. (1977) J. Biol. Chem. 252, 3092-

13. Paoletti, E., Rosemond-Hornbeak, H., and Moss, B. (1974) J.

14. Paoletti, E., and Moss, B. (1974) J. Biol. Chem. 249, 3281-3286 15. Mizumoto, K., and Lipmann, F. (1979) Proc. Natl. Acad. Sci. U.

16. Joklik, W. (1962) Bzochim. Biophys. Acta 61,290-301 17. Shuman, S., Spencer, E., Furneaux, H. and Hurwitz, J. (1980) J.

18. Alberts, B., and Herrick, G. (1971) Methods Enzymol. 22, 198-

19. Easterbrook, K. B. (1966) J. Ultrastruct. Res. 14,484-496 20. Venkatesan, S., Gershowitz, A., and Moss, B. (1980) J. Biol.

21. Martin, S., and Moss, B. (1976) J. Bwl. Chem. 251,7313-7321 22. Lehman, I. R. (1974) Science 186, 790-797 23. Maitra, U., and Hurwitz, J. (1973) J. B i d . Chem. 248, 3893-3903 24. Shatkin, A. (1976) Cell 9, 645-653 25. Oakley, B., Kirsch, D., and Morris, R. (1980) Anal. Biochem. 105,

26. Switzer, R., Merril, C., and Shifiin, S. (1979) Anal. Biochem. 98,

27. Bradford, M. (1976) Anal. Biochem. 72, 248 28. Laemmli, U. K. (1970) Nature 227, 680-685

Biol. Chem. 249,3287-3291

3098

Biol. Chem. 249, 3273-3280

S. A . 76,4961-4965

Biol. Chem. 255, 5396-5403

217

Chem. 255,903-908

361-363

231-237


EXPERIMENTAL P R O C E D G

3 C m p l e t e Add 0x174 DNA Add p o l y ( r l )

1 Complete

Add 3 mM PP; Add 1 mM PP

Add 5 mN PP Add 5 mM IN:! SO Add 20 mM INdf2884

5 Complete Add 1 . mM PI Add 2 5 nt4 PI Add 5 dA P1 A d d 10 mM 6 ,

6

"

EXPt.

E m .

E m t .

2.

3.

c m p i e t e

Add 0.3 M Nacl Add 0.1 M NaCl

Add 0.5 M NaC1

0 0 7 1.65

1.98 1 15

4 22 3 11 I 30

3 4 8 2 93 3 19

2.66 0 46 0 12 0 02

0 .50 1.46

2.12 1.75 1 00 0.55 0.14

a. 15 3.19 1.16 0.74 0.30

GTP-Pyrophosphate Exchange Ac RESULTS

Pur,flcat?an of the GTP-PPi Exchange A c t i v i t y f m Vaccinia Ylrimnr

- VaCClnla Extracts

Extracts of vacclma v i r i m r were prepared by a d i f i c a t i m of the procedure used 3" the P u r i f i c a t i m Of DU-dependent W polyrrase (6). Yaccima Cores were cbtained by incubat im o f 280 "2 uni ts Of p u r i f i e d v i rus ~n 7.2 m1 o f a m l u t l m c m t a i n i n g 5O .II T r i s a h , pH 8.4. 5O 1111 d7- t h i o t h r e i t o l . and 0.51 NPW f o r MI .in a t 37' wi th In termi t tent shak ing. Corer were i so la ted by c c n t r i f u g a t i m and resuspended i n 6 ml o f a r o l v t i m

The r u r p e n r i m was ude 0.11 i n sod im deoycho la te and placed i n i c e f o r c m t a i n i n g 0.3 M'Tril.HC1 pn 8.1, 50 m d i t h i o t h r e i t o l . and 0.25 M Nacl.

M min. A f te r cen tn fuga t im to m v e i n s o l u b l e M t e r i a l , t h e deoycho-

EDTA i n d f i n a l "01- Of 7 . 1 . I . .The s w l e was i m d i a t c l y applied t o a late supematant was adjusted t o 1 M g l y c e m l . 0.11 Tr i ton X 1W and 1 .II

5 m1 c o l m of &&E cel lu lose *lid had been equ i l i b ra ted w i th 0.2 M UaC1 rn buffer A (50 dl TriS-HCI. pH 8.4. 1 M EOTL2.5 .* d i t h i o t h r e i t w l 0.1% T r i t o n X 100. 1 M g l y c e r o l ) . The c o l u m was washed wi th the s ~ l c buff& and f m c - tlms h i c h c m t a i n e d h e PPi exchange a c t i v i t y . r k i c h were not re ta ined by the res in were placed m lu f o r s e v e n 1 h w m .

The insoluble material cbtalned after deoxycholate treatment con- ta lned s igni f icant PPI exchange I c t l v l t y . Therefore, th is mater ia l was re- extracted. The p e l l e t vas resuspended ~n 6 m l o f buf fer A c m t a l m n g 0.2 M

quently vortexed. The r u r p e n r i m was centnfuged for 10 min a t 10,OW rpn NaCl. and placed m rce f o r 90 m n . Curing this period the tube was f r e -

NaCl i n buffer A. The centr r fugat ion was repeated and the supernatant frac- ~n a Sorval l 55-34 m t o r and the p e l l e t was re-extracted wi th 3 m l O f 0.2 W

t l m s were cmbined ( 8 . 5 m l ) and der lgnated Tr i ton rummatant. The p e l l e t was suspended tn 3 m1 O f 0.2 M Hac1 I " buf fer A (Tntm P e l l e t ) and stored at 4'. This procedure effcct7vely Solubilized the PP, elchange a c t i v i t y which had not been released frm the v i ra l cores dunng treatment with deOXyChollte (see Jab& 111). Ye have &served tha t t he e f f i c i ency o f t he deoycha la te s l t rac t lm vdrles frm m e preparat>an t o the next . not only

polymrale'as well. Re-extraction of the deoxycholate pel let as described for the PP exchange act lv l ty, but for the phmph3ydmlaseL and the RNA

above overcrmer t h l s difficulty and c m t r t b u t e r t o I h7gher enzyme y i e l d . The Tmtm supernatant vas Chrmatographed m OEM cellulose as described

enzyme cbtalned frm step 1. The pwled preparat ion (20 m l ) was designated for the deoxycholate fraction and the unbound enzyme was conbined with the

DEAE-I.

- DEAE ce l l u lose Chrmtwraphy I 1

a 6 m l c o l m Of DEAE cel lu lose which had been equl l ibrated wi th 50 nll NaCl The OEAE- I enzynr was d7luted with 49 ml of buffer A and applled t o

0.35 M NaCl ~n buffer A. nqp??x?aatcly 83% of the PP, exchange a c t l r i t y was ~n buffer A and the s c t i w t y was eluted wi th a 50 "1 gradient of 0.05 W - recoueed i n the unbovnd fract lon (MAE-11). almg r l t h the w o n t y O f the phosphohydmlare and capping a c t l v l t l e s . As prev ious ly Rpor ted (6) a11 o f the RNA p o l m e r a r e a c t i v i t y was re ta ined m t h i s r o l - and was e lu ted a t 0.15 M NaCl. as r h m i n F i g . 3. A >ma11 f r a c t i m of the PP. exchange a c t i - v i t y . ATPase activity and the 7 m t h y l transferase x t l r i t y idserbcd t o t h e c o l m and was e l u t e d i n p a r a l l e l w i t h the RNA po lmrase (F ig . 3 ) . The f u r - t h e r p w i f i c a t i m of the MA ~ o l m ~ w e and i t s I s 1 o c i a t e d a c t i v i t i e s i s d l l -

- Pharphoccellulorc Chrwtography

OW1 cellulose fractions 21-28 we- pooled (8.2 n l ) and applied t o a 5 nl c o l m Of phorphacellulore rt l ich had h e n q w l i b r a t e d w i t h 50 .II *IC1 ~n buf fer A. The col- was washed with buf fer A cmrain ing 0.15 M *IC1 and e lu ted w i th a 60 nl l inear gradient of 0.15 M - 0.4 M NaCl i n k f f c r A. Fract imr (1 .1 m l ) were Collected md assayed as indrcatcd i n Fig. 5. The PP. exchange activity r1ut.Q .I a s ing le peak a t 0.22 M NaCI' thc e l u t i m prb f i le o f the exchrnpl a c t i v i t y was coincident r ich that sf 'capping e n w

ere p w l e d and r w l d be stored m ice f o r several r t k s wi th no s i g n i f i c a n t and the nucleic acid-indcpndent ATPase. PhmphoceI lu lose f ract ims 211-39

loss o f d c t i v l t y Unless O U v n i r e n o m . the phormocellulore m- prepa- r a t i o n was used for a11 s tud ies o f GTP-PP, exchange and a r r m i a M a c t i v i t i e s .

- Glyccml Grad ien t Cmtr i fugat im

A %-le of the phmphocellulme enzyme preparat im was made 1 W w t h respect t o NaCl and a 1 1 1 al iquot us layered mto an 11.5 "1 l inear gva- dicnt O f 15-351 g l y c e r o l i n b u f f e r A cant l in ing 1 M uC1. T b gradient 1.1

nl) were col lected frm the b o t t m O f the tu& and arrayed as i n d i c a t e d i n centnfuged for 62 h m a t 41 .WO - i n 1 Spinco Y 4 1 rotbr. F r a c t i m s (0.3

F ig. 6. The GTP-PP exchange a c t i v i t y s e d i m t e d IS a s i n g l e p a k O f 6.5 S and c o - r e d i m t c d r ! t h t h e g u a r y l y l t r a n s f e r a s e . 7 - r t h y l t m r f e n r c a c t i - v i t y and the nucleic acid-independent ATPase. No lass of PPI exchange a c t i - n t y occurred during the g lyc lml g rad ien t step.

:tivity Associated with Vaccinia Capping Enzyme 11597

The c h l n e d DEAE-I f r a c t i m was chrmatmraohed M DEAE-Cellulose

Effect of OH

The ATPase displayed wtlml aCt iv l tY under a lka l i ne Cmd i t ims , be-

duced by 70X. Ween pH 8 and pH 8.9 (40 .II T r l l . H C I b u f f e r ) . A c t i r i v a t p* 6.9 was re-

11 1598 GTP-Pyrophosphate Exchange A Effect of Nucleic Acids

The a d d i t i m of either single-stranded Iw l Ix174 v i ra l I**) or rw Yllf). These features CleI r ly d is t inguish the phosphohydmlare a c t i v i t y (Poly r(1)) t o the ATPase r e a c t i m had l i t t l e effect m ATP cleavage (r.ble

a s s a i a u d r l t h capping en- c o q l a x f r a the two p r w i w s l y described nYc1eic acid-6.pndent phosphmydmlasrrs present i n vaccinia (13). ATP

ther DX171 Illl 07 poly(r11 1171.

Effect vf ATP Concentration

hyperbolrc; the apparent Un f o r ATP was 8 x 10-pM (not r h m ) .

Effect of Salts and Inh ib i tors

The w l a t ? m r h i p between reaction Veloci and ATP Cmcentrat im was

was i n h i b i t e d 83; i n the presence of p-hydraxylrrruribenzaate; t h i s i n h i b l - t i a n 111 mvened by the addition Of d i t h i o t h n i t o l (Table V I I I ) . The effect of i m i c strength m ATPase a c t i v i t y i s s h a n i n Table VIlI. The a c t i v i t y was unaffected by NaCl up t o 0.1 M concentration but declined IS the cmcen- t r a t i o n of NaCl was increased f m 0.1 t o 1 W . The effect Of var iow anions m enzpe a c t i v i t y i s s h a m in Table Y I I I ; Of these, PP i was the mnt effec- t i ve i nh ib i t o r o f ATP h y d m l y r i r f o l l a d i n Potency by P i and inorganic 1u1- fate.

SpCci f ic i ty of r-phosphate C ~ ~ ~ Y I D C

ATP hydrolysis was unaffected by the m i s l i m of d i t h i o t h n i t o l . b u t

e n z m c m l e x catalyzed the cmverrim Of ,%]ATP t o I ' H I M P . NO f o - t i m The nucleic acid-independent phosphoh dro la re ac t i v i t y of the O w i n g

of nuclcmlde nmophorphste yas dCtected nor d i d the en- Catalyze W

hydrolysis Of GTP. dGTP and dATP t o t h e i r n r p e c t i r r diphosphates [Table X ) . f a m a t i o n i n the pm3en.x of I HINIP. Thc phosphohydrolare also Catalyzed

CTP and UTP ere less cxtrnrively hydrolyzed, and d l l P cleavage was not detected.

Pmpcr t ier O f the lbyh Triphosphatase Associated r i t h Capping En-

p u r i f j c d capping en- required the pmsmce O f MgCl . Act iv i t y d id no t y a w s i g n i f i c a n t l y b e h w q , ~ m nd y+m 1lpc12; I-? cmcentvatirns were not ermined. k i t h e r Ca Co2' t u or ZnZ+ cwld sat is fy the d iva lent cation r q u i m n t . Ihz+ .;I abl; t o a c t i a t e the RIU triphosphatase. t h w g h m l y 12) as e 1 1 as @. Cd+ C& Lnd Z d t *7+pOtent inhibitors of the e n z m i n the presence o f mgcl,: while mz+ and t a were p a r t i a l l y i n h i b i t o r y i n the pmrence (If MgC12. RIU triphosphatase 11s not affected by mirrim of d i th io th re i ta l bu t wss i n h i b i t e d by 791 i n the presence (If 2 .5 nW p - h y d r s r m r c u r i b m r ~ a t e . T h i s l n h i b i t i m was reversed by d i t h i o - t h m t o l (no t rhan l . PP. "as a potent inhibitor of the R M triphosphatase; 2 .5 nW PPI decrcared a c t i i i t y by 981. P i inh ib i ted the enzyme at h igher cmcentrat imr (50; i n h i b i t i o n a t 20 nW P., 94% i n h i b i t i o n a t 40 .* P . ) . The Im Of the RNA t r i p h sphatlse far 5' trlphozphetc terminated poly d l wes w- proximately 6 x 10-9W r i t h respect t o 5' ends.

The cleavagc of the Y-phosphate Of t r iphosphate-teninated wly(A) by

r i t h the capping enzyme c w l e x . i t resenbles the e n z m described by Total To the extent that re hare exmined the lDyl triphosphatase associated

and Paolet t i (12). Our r r r u l t s by no mans c m s t i t u t e d e f i n i t i v e c w w i s o n of the two c n z m r , nor have we ru led wt the possibi l i ty that vaccinia v i - rions may contain mom than m m- capable O f hydrolyzing they -phosphate I n RNA.

lase act iv i t ies described above nay be Catalyzed by the IMT funct ional uni t The RNA tr iQhOrphatart and the nucleic acid-independent phoSphOhydrC-

wi th in the cappin e n z m c a p l e i . The fact that the M for tr iphorphate- terminated ~ o l ~ ( B . 4 i s three orders cf maclnitude 1-7 than the M for ATP suggests that ih7r m 2 m 1 1 pr imar i ly concerned with the mtabol i fm of 5' RNA t c n i n i and not with the cleavage of free nucleotides. Highel aff ini ty of RM triphosphatase for RNA termini than for free NTP has k e n f w n d i n the case Of the E . c o l i RNA triphosphatase as wel l (23) . Though not tested d i rect ly . we i n f G ?i?6 the resul ts of Table X that the lllu triphosphatase LA" act on RNAs terminated r i t h nucleotides other than ATP.

xtivity Associated with Vaccinia Capping Enzyme

Froctm Numbcr

Fig. 5. Phosphocellulosc chmatognphx.

was chnnutcqaphed'm phosph-llulose as described i n the text. GTP-PP. The GTP-PP. exchange en- obtained frm the Du-cel lu lose step

exchange, ATPase ( i n the absence of DX174 MA) and 7 - r t h y l t n n r f c r a r c we& GTP-dewdent n r t h y l a t i m Of t r lmosphate-terminated ~oly(A1) was arrayed arrayed as described i n the legend t o Fig. 3. '"Capping en-" ( i .e. the

i n the presence of 8 pol Of triphosphate t e n i n i o f poly(A1 a% described i n Experimental Pmecdunr.

Fig. 6 , Glycerol Gradient Ccntnfuclation

The GTP-PP- exchange enzyme was Sedimntcd i n a glycerol gradient as described i n t h e ' t e r t GTP-PPI exchange assay mixtures mtaining 60 nll Tnr'HC1. pH 8.3. 10 111 i i t h i o t h n i t o l . 4 nll P!gClz, 1 mU [3rPlPPi, 0 . 2 nll GTP and enzyme were incubated fov 40 min a t 37.. Nucleic acid-independent ATPase a $ x y mirtu containing 40 rW Trlr4lC1. pH 8 . 3 . 2 M d>th iothre i to l . 6 111 MgClp, 1 nll ly!FPIATP and en- ere incubated for 15 m n a t 37'. "Cappin m z m " reactim mixtures contained 4-1 O f tr iphosphate-temini of poly?A) and other c n p o n n t r as indicated i n E i p e r i m t a l Procedures. lncubationr ere f o r 40 min a t 37.. 7 e t h y l transferase act ivi ty was dc- temined as described i n Experlmntal Pmceduns. Guanylyl tr lnl ferdfe assays rrc P r f O m d as described i n Experimental Procedures i n the pRsence

were f o r 40 min a t 37'. R M triphosphatase assay mixturns w e n as lndlcated of 10 YM [~HlGTP and 9 m o l of triphosphate termini O f paly(A). lncubatrmr

i n EIperinontal Pmcedums and Cmtained 4 p o l of tripharphate tFm8nl Of p o l y l l ) . The redinentation Q m f i l C I i n panels a-c a n fro7 the lme gva- d ient and we pmsented separately for the sake of c l a r i t y . Ildl((er pvo-

were r e d i n n t e d i n a para l le l gradient . The positions Of the mavkcn are t e ~ n s (catalase. aldolase. baCteFia1 a lka l ine phosphatase and c y t a c h r m C)

indicated by the a r m s .

the journal of val. 23, issue of december 10, pp. 11588 ... · omission of mgcl2 completely...

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