THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 257. No, I, Issue of January 10, pp. 485-494, 1982 Printed in U.S.A.

A Unique Class of Compound, Guanosine-Nucleoside Tetraphosphate G(5’)pppp(5’)N, Synthesized during the in Vitro Transcription of Cytoplasmic Polyhedrosis Virus of Bombyx mori STRUCTURAL DETERMINATION AND MECHANISM OF FORMATION*

(Received for publication, May 22, 1981)

Robert E. Smith and Yasuhiro Furuichi From the Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Two structurally different classes of oligonucleotides accumulate in vitro in cytoplasmic polyhedrosis virus (CPV) transcription mixtures in molar excess as com- pared to the completed RNA products. The first class consists of oligonucleotides which correspond to the 5’- terminal sequence of the virus mRNAs (referred to as initiator oligonucleotides). The major species of initia- tor oligonucleotides are (p)ppApG and (p)ppApGpN together with smaller amounts of homologous capped structures (Furuichi, Y. (1981) J. Biol. Chem. 256, 483- 493).

In addition to initiator oligonucleotides, CPV tran- scription mixtures yielded a second new class of com- pounds which were radiolabeled by [~u-~’P]GTP and resistant to phosphatase digestion. Their structures were identified as G(5’)pppp(5’)A, G(5’)pppp(B’)C, G(5’)pppp(5’)G, and G(5’)pppp(5’)U. With the exception of G(5’)pppp(B’)G, these compounds have not been observed previously. The mechanism of synthe$g*of these unique, compounds was elucidated as pppG + pppN -+ GppppN + $fii. The reaction resembles, in principle, a guanylylation reaction which occurs during cap formation in CPV and other eukaryotic mRNA syntheses. It is likely that these compounds are formed in a similar way by a condensation reaction involving a viral guanylyltransferase-pG intermediate complex and ribonucleoside triphosphate.

When the amounts of G(5’)pppp(5’)N were measured, it was found that G(5’)pppp(5’)N reached maximum concentrations (0.4 to 0.7 p ~ ) shortly after the onset of RNA synthesis (1 h) and these levels were maintained or diminished gradually. By contrast, mRNA and (p)ppApG were continuously synthesized. The relative molar ratios of total G(5’)pppp(5’)N and (p)ppApG uer- sus mRNA were comparable (74:24:1 and 30:27:1 dur- ing 1 to 4 h transcription, respectively). The results imply that these unusual compounds G(5’)pppp(5’)N as well as initiator oligonucleotides may be produced re- iteratively during initiation when RNA chain elonga- tion and capping are uncoupled.

CPV,‘ like human reovirus, contains 10 double-stranded

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: CPV, cytoplasmic polyhedrosis virus; AdoMet, S-adenosylmethionine; NTP, nucleoside 5’-triphosphate; NDP, nucleoside 5”diphosphate; NMP, nucleoside 5‘-monophos- phate; EDTA, ethylenediaminetetraacetate; P-enolpyruvate, phos- phoenol pyruvate; M,, molecular weight.

RNA segments (Miura et al., 1969 Fujii-Kawata et al., 1970) and RNA polymerases that transcribe the duplex genome RNA to form mRNA either in the infected animal or in vitro under appropriate conditions (Lewandowski et al., 1969; Shi- motohno and Miura, 1973). The CPV-associated RNA polym- erase, unlike other virus-associated RNA polymerases, is ac- tive without decoating by detergent or proteolytic digestion. This property of CPV permits the in vitro study of virus transcription without disrupting the virus structure. In addi- tion to RNA polymerase, CPV contains other enzymes re- quired for the formation of the complete mRNA with a capped 5’-terminus, m7GpppA”pG- (Furuichi and Miura, 1975). These enzymes are nucleotide phosphohydrolase, mRNA methyl- transferases, and mRNA guanylyltransferase.

Previously, it was shown that the transcription of CPV is unique in that (i) S-adenosylmethionine, a methyl donor, stimulates the initiation of transcription (Furuichi, 1974); (ii) this is due to a lowering of the K,,, for the initiating nucleotide ATP, the effect facilitating RNA polymerase to initiate RNA sythesis (Furuichi, 1981); (iii) the stimulatory effect is me- diated by a cooperative relationship between RNA polymer- ase and methyltransferase in CPV transcription complexes (Wertheimer et al., 1980); the (iv) initiation is perhaps related to the process of capping (Furuichi, 1978). Furthermore, the terminal sequence common to all genome segments has been partially identified (Miura et al., 1975) and the transcription by virus-associated RNA polymerase occurs as:

(+) 5’ m’GpppA”-G-U ___- _-________ c-c 3’ Genome

(-) 3’ U-C-A ___-__________ G-Gp(p) 5’

1 mRNA 5’ m’GpppA”-G-U ------- -____-- C-C 3’

Thus, CPV provides a useful eukaryotic model system for investigation of the mechanisms of transcription initiation and mRNA capping.

Recently, we found in the CPV transcription mixture an accumulation (in molar excess of the completed mRNA) of oligonucleotides. These were classified as “initiator oligonu- cleotides” since they have structures which correspond to the 5”terminus of CPV mRNA. The major species of oligonucle- otides are (p)ppApG and (p)ppApGpN together with small amounts of the homologous capped structures. Incomplete transcription mixtures that support initiation of CPV tran- scription and produce the mRNA 5’-terminal structure m7GpppA”pG yielded more initiator oligonucleotides than complete transcription mixtures. Therefore, it was considered that the oligonucleotides were produced by reiterative initia- tion of the virion RNA polymerase at promoter regions (Fu- ruichi, 1981).

485

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High resolution polyacrylamide gel electrophoresis that sep- arated the oligonucleotides from the completed mRNA and radioactive precursor also resolved a new compound. The compound which was labeled by both [a-32P]GTP and [P-”P] ATP was tentatively referred to as GppppA since it contained a P1 nuclease-phosphatase-resistant -4 charge (Furuichi, 1981). Following these primary observations, we have inves- tigated this new compound of unusual structure synthesized during CPV transcription. Four different species of guanosine- nucleoside tetraphosphates, namely GppppA, GppppC, GppppG, and GppppU, were detected in CPV transcription reaction mixtures. AU have a charge of -4 and differ from the unmethylated mRNA cap precursors GpppN of -3 charge. This is the first description, structural identification, and analysis of the mechanism of biosynthesis of G(5’)pppp(Y)Ns that are formed during eukaryotic virus transcription in vitro. Possible biological functions of GppppN and initiator oligo- nucleotides during initiation of CPV transcription are dis- cussed in relation to mRNA capping.

EXPERIMENTAL PROCEDURES’

In Vitro CPV Transcription-CPV genome transcription in vitro by virus-associated RNA polymerases was carried out under the same conditions as described before (Smith and Furuichi, 1980). The com- plete transcription mixture (10 pl) contained 70 m~ Tris-OAc (pH 8). 100 m~ NaOAc, 10 mM Mg(OAc)a, 1 mM S-adenosylmethionine, 0.2 m g / d of bentonite, 0.4 mM ATP, 0.2 m~ each of CTP, GTP, and UTP, 0.1 mg/ml of proteinase K, 2.5 pCi of a-”P-labeled ribonucleo- side triphosphate, and 3 pg of CPV. In some experiments, 2.5 mM phosphoenol pyruvate and 0.17 mg/d of pyruvate kinase were also added and proteinase K was removed. Incomplete reaction mixtures contain a limited species of ribonucleoside triphosphate, but the concentrations of each nucleotide are same as described in the com- plete mixture. All reactions were carried out by incubation at 31 “C.

The RNA and oligonucleotides were recovered from the reaction mixtures by extraction with water-saturated phenol. The aqueous phases were then extracted with ether. Samples were treated by 0.5 unit/pl of calf intestine alkaline phosphatase for 1 h at 37 “C and lyophilized prior to gel electrophoresis unless otherwise indicated.

RESULTS~

Oligonucleotides Synthesized during CPV Transcription When the products of a complete CPV transcription mix-

ture were analyzed by polyacrylamide gel electrophoresis, several discrete bands (bands D to H in Fig. 1A-a) were detected that migrated between bromphenol blue (BPB) and [ ~ U - ~ ~ P I G D P which was derived from radioactive precursor [cP~’P]GTP. CPV mRNAs synthesized in the mixture stayed at the origin. In an incomplete transcription mixture contain- ing only ATP, [a-”PIGTP, and AdoMet, which are required for formation of the mRNA 5‘-terminus m7GpppAmpG, these bands also occurred at the same position in the gel (Fig. 1B- a) . To eliminate unreacted [w3’P]GTP which may obscure the oligonucleotides during electrophoresis, aliquots of both complete and incomplete reaction products were treated with calf intestine alkaline phosphatase before loading on the gel (Fig. 1 A-b and B-b). As shown in Fig. 1, A-b and B-b, new bands A and B appeared in the position between xylene cyano1 (XC) and bromphenol blue markers. Concomitantly, bands D, G, and H in Fig. 1A-a diminished in intensity. Similar

Portions of this paper (including portions of “Experimental Pro- cedures” and “Results,” and Figs. 2, 4, 6, and 9) 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, Be- thesda, MD 20814. Request Document No. 81M-1225, cite authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

486 In Vitro CPV Transcription and Capping

changes also occurred in the incomplete reaction mixture (Fig. 1, B-a and 4 ) except that band H disappeared after phospha- tase treatment (Fig. 1B-b). The mobility shifts by phosphatase treatment are consistent with the structures of oligonucleo- tides which were identified previously (Furuichi, 1981). For example, band A in bot$ complete and partial reactions con- tains predominantly ApG (* denotes [32P]phosphate). ApG derives from the 5’-phosphorylated precursors pppApG and ppApG which migrate in band H and G, respectively. Simi- larly, band B conpins ApGpN (N is not identified) which derives from ppApGpN in band D by phosphatase treat- ment. Band D, in addition*to pgApGpN, contains 5’pan~I- ylated oligonucleotides GpppApG (85%) and m7GpppApG (15%) which, because of phosphatase resistance, maintain their mobility before and after phosphatase treatment. These structures, in conjunction with the amounts (picomoles) in individual hands, are summarized in Table I.

Guanosine-Nucleoside Tetraphosphate, G(S?pppp(5’)N, Synthesized during CPV mRNA Synthesis

Nucleotide Species Required for the Synthesis of Com- pounds in Bands F, G, and H-In addition to initiator oli- gonucleotides recapitulated above, we found the phosphatase- resistant components in bands F, G, and H. In the complete transcription mixture, three bands F, G, and H were labeled by [LY-~’P]GTP (Fig. 1A-b). By contrast, only two bands F and G occurred in the incompIete reaction mixture in which CTP and UTP were omitted (Fig. 1B-b). To determine the ribo- nucleoside triphosphate requirements for the synthesis of components in each individual band, four sets of incomplete reaction mixtures were tested. These include GTP only, GTP + ATP, GTP + CTP, and GTP + UTP. Each mixture contained [a-32P]GTP as radioactive precursor. After incuba- tion with CPV, the reaction mixtures were extracted by phenol, digested with phosphatase, and the products were analyzed by gel electrophoresis (Fig. 2 A , presented in mini- print). As described in the previous section, the mixture with ATP + GTP forms ApG, (m7)GpppA‘”’pG, and components in bands F and G (lane I). By contrast, the reaction mixture with GTP only forms a single main band F, no band G, a weak band co-migrating with ApG, and faint bands above band F (lane 2). In GTP + CTP (lane 3) or GTP + UTP (lane 4) , two bands F and H appeared. These results indicate that a component in band F is formed solely by GTP. On the other hand, bands G and H contain not only guanosine, but are specific for adenosine and pyrimidine nucleosides, respec- tively.

Next, several other combinations of nucleotides were tested using [~u-~’P]CTP (panel B ) and [cx-~’PIUTP (panel C) as radioactive precursors (Fig. 2) . The results clearly demon- strate that the compound(s) in band H are formed only when GTP is present in addition to CTP (Fig. 2B, lune 3) or UTP (Fig. 2C, lanes 3 and 6) indicating that the reaction is GTP- specific. Moreover, both [a-32P]GMP and [~u-~’P]UMP (or [cx-~’P]CMP) were incorporated into the compounds in band H. Therefore, it was concluded that (i) the synthesis of com- pounds in bands F, G, and H requires GTP, (ii) a compound in band F is formed solely from GTP, (iii) a compound in band G requires both GTP and ATP for its formation, and (iv) compounds in band H require both GTP and CTP or UTP for their synthesis.

It should be noted that from reactions without ATP (lanes 2, 3, and 4 of panel A ) a band was present in the position of ApG but with a lower intensity than that of ApG. The com- ponent in this band contains a -1 charge as determined by DEAE-cellulose-7 M urea column chromatography indicating that its structure is, like ApG, a dinucleoside monophosphate.

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In Vitro CPV Transcription and Capping 487

mRNA-

A-

B-

D- E- F L G- H‘

-xc-

- B PB-

ii&S iii

-A

-B

-C -D - E -F -G - H

FIG. 1. Time course of CPV mRNA and oligonucleotide syn- thesis. The products of both com- plete and incomplete transcription were examined after 0, 1, 2, and 4 h incubation a t 31 “C by 208 polyacryl- amide gel electrophoresis. A-a , a complete reaction mixture; A-b, a complete reaction mixture treated by calf intestine alkaline phosphatase; B-a, an incomplete reaction mixture containing ATP and GTP; B-b, an incomplete reaction mixture contain- ing ATP and GTP treated by alkaline phosphatase. All reactions were car- ried out using [a-:’ZP]GTP as radio- active precursor. Reaction conditions and separation of products are de- scribed under “Experimental Proce- dures.” XC, xylene cyanol; BPB, bromphenol blue.

”-

0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4 REACTION TIME (hr)

TABLE I Oligonucleotides and mRNA synthesized in CPV transcription in oitro

Amounts synthesized“ Digestion products

Bands Species l ~ a ~ ~ ~ Net negative charge 4 NTPs ([a-’2P] ATP + [a-’”P]

GTP) GTP

reaction P I nuclease Pyrophosphatase and phosphatase

pmol Origin mRNA 0.31

A APG 7.3 51 -1 PC APG D (m‘)GpppApG 0.33 6.2 (-3.5), -4 (rn‘)GpppA and pG P, and ApG E GPPPPAPG 0.33 1.1 -5 GPPPPA and PC P, and ApG

GPPPPG 3.6 8.7 -3.8 NCb P, GPPPPA 5.9 11.8 -3.8 NC P, GPPPPPY‘ 14.4 -3.8 NC P,

F G H

Amounts synthesized during 1 h transcription. NC, no change. A mixture of GppppC and GppppU.

Consistently, it was P1 nuclease-sensitive and the nearest neighbor transfer analysis by RNase T 2 digestion revealed that the 5’-proximal nucleoJide was Gp, suggesting that the nucleotide sequence was GpG (data not shown). In ATP + GTP (lane I), the band in the same position contained exclu- sively (98%) ApG. Therefore, it seems likely that CPV RNA polymerase directs the synthesis of an aberrant nucleotide sequence when it lacks the appropriate initiation nucleotide ATP.

Compounds in Bands F, G, a n d H Contain -4 Charge and Are Resistant to P I Nuclease a n d Phosphatase-Each com- ponent in bands F to H from the complete or incomplete

transcription mixture (Fig. 1) was extracted as described under “Experimental Procedures” and analyzed by cellulose thin layer chromatography (Avicel TLC) using isobutyric acid-0.5 M NH,OH (10:6 v/v) as solvent (Fig. 3A). Extracts from band F contain one main component (spot a in lane I; R,G, relative mobility to pG = 0.20) which co-migrates with the authentic marker compound G(S’)pppp(5’)G (P-L Biochemicals). Ex- tracts from G are resolved into two spots (lane 2): a main spot b (RPG = 0.50) and spot a which is probably a contaminant from band F. Extracts from band H of the complete reaction mixture contained two major components (lane 3) and a trace of spot b also probably due to carry-over from band G. These

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488

A

B

c c L

I

m7GpppAm-

m7GpppGm-

GPVPA -

In Vitro CPV Transcription and Capping

-Ap

“c -myGpwCm

2 7 G p p p U m I G P

d -GPW

G~ovvG- GvvvG- -GvvpU

ORIGIN- - -ORIGIN

I 2 3 4 5 6

I ” I

Fractlon

I “ ” t - , ~-

- pu

- PG

I. . I . . .

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 orlgln

FIG. 3. Characterization of oligonucleotides in bands F. G , and H. Components in bands F, G , and H resolved by 206 polyacryl- amide gel electrophoresis were extracted from the gels (Fig. I ) , and their properties were measured by (A ) cellulose thin layer chroma- tography. ( B ) DEAE-cellulose column chromatography, and (C) pa- per electrophoresis in conjunction with alkali and/or various enzyme treatments. Panel A, fane 1, band F from the complete reaction containing [u-”YI’]GTP (Fig. 1); fane 2, band G from the complete reaction (Fig. 1): fane 3 , band H from the complete reaction with [u- .”PJGTP (Fig. 1); fane 4, band H from the incomplete reaction GTP + [a-”’PIUTP (Fig. 2C); fane 5, band H from the incomplete reaction GTP + [a-”P]CTP (Fig. 2B); fane 6, band F containing -3 charge from the incomplete reaction G T P + [a-.”P]CTP (Fig. 2B, fane 3, upper band). Panel B, compound in spot a (panel A, fane I ) was analyzed for negative charge by DEAE-cellulose-7 M urea column chromatography. As a marker, authentic GppppG (P-L Biochemicals) (5 Aw, ,,”, unit) was also included in the chromatography. Similar analyses were carried out for compounds in spots a , h, c, and d. They all contained a negative charge of -3.8. A summary of the charges measured for the other oligonucleotides is shown in Table I. Panel C, [a-’”P]GTP-labeled compounds which migrated in the positions of spots a , b, c, and d were treated with ( b ) PI nuclease and calf intestine alkaline phosphatase, ( c ) alkali (0.2 M NaOH, 37 “C for 6 h), (d ) tobacco acid pyrophosphatase, and ( e ) tobacco acid phyrophsphatase (PyroPase) followed by calf intestine alkaline phosphatase ( ( a ) : no treatment). fane I , compound in spo/ a (presumptive GppppG from band F); fane 2, compound in spot b (presumptive GppppA from band G ) ; fane 3. compound in spot d (presumptive GppppC from band H); fane 4, compound in spo/ c (presumptive GppppU from band H). After treatment, all reaction products were analyzed by paper elec- trophoresis (pH 3.5).

two major components were separated by Whatman No. 3MM paper chromatography in the same solvent, yielding the pu- rified compounds: spot c (lane 4, R,,c = 0.27) and spot d (lane 5, R,G = 0.35). Thus, bands F, G, and H contain four different compounds migrating in the positions of a, b, and c and d , respectively. Each purified compound was analyzed for nega- tive net charge by DEAE-cellulose-7 M urea column chroma- tography with a linear NaCl gradient elution (0.05 to 0.5 M). Fig. 3B represents a typical chromatographic profde that was obtained with the purified compound in spot (I extracted from band F. Nearly identical profdes were obtained for the other three compounds (data not shown). Accurate estimation of the negative charge of the compounds in spots Q to d revealed that they contain in common a -3.8 charge which, for con- venience, we designate as -4. Successive treatment by P1 nuclease and alkaline phosphatase did not change their neg- ative charge as determined by rechromatography under the same conditions. The results clearly indicate that they do not contain a 3’-5’-phosphodiester linkage. Furthermore, com- pound Q extracted from band F co-migrates with authentic G(5’)pppp(5’)G in both thin layer chromatography (Fig. 3A) and DEAE-cellulose column chromatography (Fig. 3B). These results prompted us to conclude that the compound in band F is GppppG and the remaining compounds in bands G and H contain the general structure GppppN. Spot e (lane 6, R,,c; = 0.50), co-migrating with authentic G(5’)ppp(5’)C (P-L Bio- chemicals), was derived from the band (F) migrating above the presumptive GppppC in band H (Fig. 2B, lane 3), and contained a -3 charge.

Identification of Guanosine- Nucleoside Tetraphosphate G(S’)pppp(5’)N”Next, in order to define the nature of the phosphate linkage, purified compounds that were labeled by [a-:”’P]GTP were treated with several different nucleotide hydrolases and/or alkali, and the products analyzed by paper electrophoresis. As shown in Fig. 3C, neither incubation with P1 nuclease and then phosphatase (panel b) , or alkali treat- ment (0.2 M NaOH, 37 “C for 6 h (panel c ) changed the original electrophoretic mobility of the compounds (panel a) . However, when these compounds were treated with tobacco acid pyrophosphatase (abbreviated as PyroPase in Fig. 31, they all were converted to 5’-GMP ( p a n e l d ) . Digestion of the pyrophosphatase-treated samples by alkaline phosphatase rendered the radioactive 5‘-GMP to inorganic phosphate @anel e). These results clearly demonstrate that compounds in bands F, G, and H contain pyrophosphate bond(s) linked to a pG residue. Apparently, they do not contain a phospho- diester linkage (2’-5’ or 3’-5’ bonds). These results, including the negative charge (-4), the presence of a 5’-GMP residue in all the molecules, and differential labeling by other nucleotides as shown in Fig. 2, suggested the structures G(5’)pppp(5’)G, G(5’)pppp(5’)A, and G(5’)pppp(5’)C (spot d in Fig. 3A) and G(5’)pppp(5’)U (spot c in Fig. 3A) for the compounds in bands F, G, and H, respectively.

In order to confirm these structures, partial digestion by tobacco acid pyrophosphatase was carried out. Each com- pound was digested for various incubation times and aliquots of digestion products were analyzed by cellulose thin layer chromatography (Fig. 4, presented in miniprint). In panel A, characterization of the cfmppnent from band F, with pre- sumptive structure G(5’)pppp(5’)G, is shown. Intermediate digestion products pp$G and p$G were released from the stFrting material. As th,e incubation proceeded, pppG and ppG were converted to pG, consistent with the structure of G(5‘)pppp(5‘)G and the mode of hydrolysis by pyrophospha- tase of GplplplpG (Shinshi et al. (1976), arrows indicate the sites of hydrolysis). Similarly, the coppound in band H with the presumptive structure G(5’)pppp(Y)U (prepared as de-

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In Vitro CPV Transcription and Capping 489

scrib$d in Fig. 2, panel C, lane 3) produced ppIfU, pIfU, and then pU as sequential digestion products (panel B). In panels C and D, the compounds labeled by [a-””PICTP and isolated from band H and the upper faint band (Fig. 2, panel B, lane 3) were analyzed. Presumptive G(5’)pppp(5’)C ft;om band*H upon digesiion yielded intermediate products pppC and ppC as well as PC consisient with the proptsed structure (panel C) . By contrast,GpppC produced onlyppC as an intermediate, but it also was converted to PC (panel D). These results are in good agreement with the general structure guanosine-nu- cleoside tetraphosphate G(Y)pppp(5’)N. As for the compound in band G with a putative structure G(5‘)pppp(5’)A, similar lines of evidence are shown in the following section.

P- and y-Phosphates of ATP and GTP are Incorporated into GppppA and GppppG, Respectively-To verify the struc- ture GppppN and obtain some insight into the mechanism of its biosynthesis, we tested [P-:”P]ATP, [ y-’”PIATP, [P-”’P] GTP and [y-”’P]GTP as precursors. The products of incom- plete reactions containing ATP and GTP were analyzed by gel electrophoresis after phosphatase treatment. Each exper- iment contains a control (no incubation) in order to detect any impurities in the radioactive precursors (Fig. 5, lanes I, 3, 5, and 7).

When [P-‘”P]ATP was used, three radiolabeled spots were resolved near the bottom of the gel (Fig. 5, lane 2). Compounds in two of the spots (first and third spots from the top) are (m‘)GpppA‘”’pG and GppppA as shown by their co-migration with authentic markers (Furuichi, 1981). A component in the second spot was characterized as GppppApZ; since Pl-nu- clease digestion of the compound yieldedGppppA.”

In addition to P-phosphate, which is expected to be incor- porated into GppppA (Furuichi, 1981). it was found that y- phosphate of ATP also was incorporated into GppppA (lane 4) . Under the conditions, there is no apparent phosphate exchange between ATP and GTP, as determined by PEI chromatography (data not shown), and between the ,i3 and y position of ATP. The latter possibility is excluded by the finding that y-ATP does not yield the radioactive cap struc- ture m‘GpppApG (lane 4). Incorporation of both P- and y- phosphates of ATP into GppppA was confirmed by partial pyrophosphatatse digestion of GppppA made with either [P- “’PIATP (Gppppfi, Fig. 6, presented in miniprint, panel A ) or [y-”IJATP (GppppA, Fig. 6, panel B). Upnp digestion, GppppA pom [P-’”P]qTP released radio+active ppA in addi- tion to pppG and ppp!, whereas GppppA from [y-’”PI ATP failed to produce ppA, the r p d t expected from the position of labeled phosphate in GppppA. These results not only demonstrate the uptake of both P- and y-phosphate into GppppA, but also confirmed the presence of guanosine, aden- osine, and four phosphate residues comprising three pyro- phosphate linkages.

Similarly, both the P- and y-phosphates of GTP were in- corporated into GppppG (Fig. 5, lanes 6 and 8). Radiolabeled GppppG made from [P-”’P]GTP or [ y-”P]GTP were analyzed by a partial pyrophosphatase digestion. As shown in Fig. 6 (panels Cand D), both GppppG preparations, unlike GppppA, gave rise to an identical digestion profile which included ppG and pppG as intermediate digestion products. This is due to the symmetrical structure of GppppG. From these results we propose that the mode of synthesis of GppppN is as follows:

ppp(5’)G + ;;;(5’)N 4 G(5’)p;Iflf(5’)N + PP,

where 5’-guanosine monophosphate is a common donor and nucleoside triphosphates are acceptors. All three phosphates of the acceptor nucleotide are retained in GppppN.

’’ R. E. Smith and Y. Furuichi, unpublished results.

ORIGIN-

- xc

“ B P B

Irn7)GpppAp G-

GDDPPA-

l ? 3 4 5 6 7 H FIG. 5. Synthesis of gluanosine-nucleoside tetraphosphates

in incomplete reactions with [/3-32P1ATP, [ Y-’?~P]ATP, [p-”P] GTP, or [y-32P]GTP. Guanosine-nucleoside tetraphosphates were produced in incomplete reactions mixtures containing ATP (0.4 mM) and GTP (0.2 mM), and then resolved by 201 polyacrylamide gel electrophoresis. A, GTP + [P-.’”I’]ATI’, lane 1, 0 h (incubation time); lane 2, 4 h. B, GTP + [y-’v21’]ATl’, lane 3. 0 h; lane 4, 4 h. C. [P- ”Pl GTP + ATP, lane 5, 0 h; lane 6, 4 h. D, [y-.”P]GTI’ + ATP, lane 7, 0 h; lane 8, 4 h. I’hosphoenol pyruvate (2.5 mM) and 0.K mg/ml of p.yruvate kinase were present in all reactions. Incubations were carried out at 31 “C. XC, xylene cyanol; B.P.B., bromphenol blue.

R W s for the formation of GppppN

xc- xc

BPB- BPB

(m7)GpppApG- (~’)GPDDAPG

GPPPAL GPDPA

GPPPPA PI Gppppp4= 1

I , i 4 5 t, / t i 1 11, I ’

FIG. 7. Synthesis of guanosine, adenosine pyrophosphate compounds in incomplete reactions using analogs of GTP and ATP. The effect of substituting analogs of GTP and ATP on the formation of guanosine-adenosine tetraphosphate was examined using ( I ) GTP + [P-’”I’]ATP. 0 h (all other reactions were performed by incubation at 31 “C for 2 h); ( 2 ) GTP + [P-.”P]ATP; ( 3 ) GTP + [ p - :’”P]ATP plus phosphoenolpyruvate (2.5 m) and p.mvate kinase (0.17 mg/ml); (4 ) GTP + [P-’”P]ADP (5) GTP + [a-”‘P1AMP; (6)

+ 5 mM sodium pyrophosphate. (8) GTP + [P-,”’P]ATP + 5 mM sodium orthophosphate; (9 ) GDP + [P-:”P]ATP; (IO) n,P-methylene- GTP (ppCH,pG) + [P-’”I’]ATI’; ( I Z ) P,y-imido GTP (pNHppG) + [B-,’”P]ATP. Concentration of analogs of GTP and ATP are 0.2 mM and 0.4 mM, respectively. XC, xylene cyanol; BPB, bromphenol blue.

Requirements for the Formation of GppppN-In order to confirm the mechanism of biosynthesis of these compounds, several reactions containing various nucleotide species and radioactive precursors were analyzed by gel electrophoresis (Fig. 7). Lane I is a control of GTP + [P-”’P]ATP (no

GTP + [P-:”P]ATP + 5 m~ EDTA (-Mg); (7) GTP + [[s”’”P]ATP

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490 In Vitro CPV Transcription and Capping

incubation). Lanes 2 and 3 show the effect of ATP concentra- tion on the formation of GppppA. CPV contains a strong phosphohydrolase activity which preferentially removes the y-phosphate of ATP (Storer et al., 1973). The experiment was, therefore, performed in the absence (lane 2) and the presence (lane 3 ) of a nucleoside triphosphate-regenerating system consisting of phosphoenolpyruvate and pyruvate kinase. [p- 32P]ATP was used as radioactive precursor. In the presence of the triphosphate-regenerating system, (m’)GpppApG and GppppA labeled by [/3-32P]ATP were produced (lane 3 ) . In its absence, GpppA which migrated at the upper part of the spot of GppppA was also synthesized (lane 2). The possible reac- tion between GTP and ADP was investigated using [P-”P] ADP as precursor. As shown in lane 4, GpppA was synthesized from GTP and ADP as determined by co-migration with authentic nucleotide marker (P-L Biochemicals). It should also be noted that GTP and ADP do not support the synthesis of (m’)GpppApG (lane 4 ) . Similar reactions, i.e. pppG + ;pN + G$pN + PPi, seem to occur with the other nucleo- tides, since we also find GpppC and GpppG in the partial reaction mixtures (Fig. 2B, lane 3, band above band H, and Fig. 2 A , lane 2, band above band F, respectively). In contrast to ATP and ADP, 5‘-AMP did not serve as a substrate for the synthesis of GppppA, GpppA, or GppA (lane 5 ) . Mg2+ seems to be required for efficient synthesis of GppppA, although a low level of GppppA was still produced in the absence of Mg2+ and presence of 5 mM EDTA (lane 6). Under these conditions, however, there was no detectable synthesis of GpppA.

In the previous section, we have proposed a reaction scheme for the synthesis of GppppN which included the release of equimolar pyrophosphate. To test whether pyrophosphate inhibits the condensation of two nucleotides, the effects of pyrophosphate and inorganic phosphate on the formation of GppppA from GTP (0.2 m ~ ) and [P-32P]ATP (0.4 m ~ ) were investigated. Pyrophosphate (5 m ~ ) inhibited GppppA for- mation completely (lane 7), while inorganic phosphate at the same concentration did not (lane 8) .

Next, to examine the required structure of the guanylate residue, 5’-GDP, a$-methylene GTP (ppCH2pG) and p,y- imido GTP (pNHppG) were tested in partial reactions with [/3-32P]ATP (lanes 9 to 1 1 ) . Only P,y-imido GTP supported the synthesis of GppppA, although less efficiently than GTP (lane 11 ). Apparently, then GTP is required and is cleaved at the a,p position, with transfer of GMP to the y-phosphate of the acceptor nucleoside triphosphate consistent with the pro- posed reaction scheme. No apparent methylation of GppppA was seen when 32P-GppppA was analyzed by thin layer chro- matography in isobutyric-0.5 M NH40H (10:6 v/v) (data not shown).

Comparison of the Rate of Synthesis of GppppNs, Initiator Oligonucleotides, and mRNA-In order to get some insight into the possible function of GppppN during CPV transcrip- tion, the amounts of individual GppppN, (p)ppApG, (m’)GpppApG, and mRNA synthesized in the mixture were compared in a time course taken for 0, 1, 2, and 4 h at 31 “C. Radioactivities in the bands in Fig. 1A-b (A, D, F, G, and H ) were measured and the amounts (picomoles) of components were calculated at each time point based on the specific radioactivity of [a-”PIGTP and the number of GMP residues in the compounds. As shown in Fig. 8, synthesis of all GppppNs reaches a maximal level (0.4 to 0.7 p ~ ) early in the transcription (1 h). With subsequent incubation, however, the concentrations of GppppNs in the mixtures were either main- tained at the maximum levels (GppppA and GppppG) or decreased gradually (GppppC and GppppU). By contrast, mRNA, (p)ppApG, and (m’)GpppApG were continuously syn- thesized for at least 4 h. The apparent reduction in the rate of

8 -

L O . - n

-I

0 1 2 3 4 Reactlon tme (hr)

FIG. 8. Comparison of the rate of synthesis of GppppNs, initiator oligonucleotides, and mRNA. To compare the rate of synthesis, band F (GppppG), band G (GppppA), band H (GppppC and GppppU), band A (ApG from (p)ppApG), band D ((m’)GpppApG), and the origin portion of the gel (Fig. 1, A-b) were cut out and the radioactivities were determined. The molar amounts (picomoles) of components were calculated based on the number of guanylate residue and the specific radioactivity of [32P]GMP (2,500 cpm/pmol). Molar amount of CPV mRNA was calculated based on the assumption that mRNAs consist, on the average, of 1,600 nucleo- tides (Smith and Furuichi, 1980). A-A, mixture of GppppU and GPPPPC; M, (P)PPAPG; H, GPPPPA; M, GPPPPG; M, CPV mRNA A-A, (m’)GpppA‘””pG.

GppppN synthesis that occurs late in the transcription may be due to inhibition by pyrophosphate which accumu- lates in association with RNA synthesis. Also, the decrease in amounts of GppppU and GppppC may be accounted for by a pyrophosphorolysis reaction which occurs as: GppppN + fiPi --$ fIfIpG + pppN. It should be noted that the molar amounts of the synthesized GppppN and (p)ppApG are always greater than that of mRNA or (m’)GpppApG (the molar ratios for GppppN, (p)ppApG, and (m’)GpppApG versus mRNA are 74, 24, and 1 early (1 h) in transcription, and 30. 27, and 1 late (4 h) in transcription, respectively).

DISCUSSION

In 1963, Finamore and Warner fist reported the occurrence of P’,P4-diguanosine 5’-tetraphosphate, G(5’)pppp(S’)G, in the undeveloped eggs of the brine shrimp Artemia salina (Fina- more and Warner, 1963). It was also found in embryos of other eukaryotic organisms such as Daphnia (Oikawa and Smith, 1966) and Eubranchipus (Warner and McClean, 1968). This compound, referred to as GppppG, is synthesized in large amounts in the shrimp ovarian tissue, and incorporated into yolk platelets in the dormant cyst where it is thought to function as an energy source for generating ATP after hatch- ing (Warner and McClean, 1968). Another dinucleoside tet- raphosphate so far reported, A(5’)pppp(5’)A, was also found in vivo in several mammalian cells at relatively higher con- centrations in rapidly proliferating cells and at low levels in resting cells (Rapaport and Zamecnik, 1976).

A unique class of compounds with a structure similar to G(5’)pppp(5’)G or A(5’)pppp(5’)A but containing guanosine in one position and a variety of nucleosides in the second, G(5’)pppp(5’)N (N: A, C, G, and U), is formed during in vitro transcription by cytoplasmic polyhedrosis virus of Bombyx mori. At least one of these compounds, GppppA as described above, is also synthesized during vaccinia virus in vitro tran- scription (results shown in miniprint). Synthesis of GppppN, therefore, does not seem to be specific for CPV transcription. However, we did not detect GppppN in human reovirus tran- scription mixtures, while chymotrypsin-digested reovirus (cores), like CPV, synthesized viral mRNA and large molar amounts of uncapped oligonucleotides (ppGpC and pp- GpCpU; Yamakawa et al., 1981) and methylated oligonucle-

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In Vitro CPV Transcription and Capping 491

&ides (m7GpppGmpC; Zarbl et al., 1980). The reaction for the synthesis of CPV G(5’)pppp(5’)N is guanosine-specific and the mechanism is elucidated ascIfcG + pppN -G(5‘)6ppp(Y)N + f i f i i . The mechanism is principally the same as that pro- posed previously for the brine shrimp G(S’)pppp(5’)G which is synthesized from 2 GTP residues by cellular GTP:GTP- guanylyltransferase (Warner and Huang, 1974). However, CPV G(5’)pppp(5’)N, although GTP-specific, does not have a strict base specificity for the second nucleotide. Furthermore, our study revealed that ribonucleoside diphosphates, NDP, also can be guanylylated by CPV and give rise to guanosine- nucleoside triphosphate G(Y)ppp(S‘)N. This reaction with NDP, however, does not seem to occur efficiently in the complete transcription mixture because of the low concentra- tion of NDP therein. Little, if any, GpppN were detected especially in the transcription mixtures that contain a nucleo- side triphosphate-regenerating system (Fig. 7, lane 3). These results indicate that the reaction takes place by a guanylyl transfer, perhaps via an intermediate enzyme-pG complex. The guanylate residue in the complex may be transferred to acceptors either NTP or NDP, but not nucleoside monos- phosphate NMP.

A similar reaction but with different acceptors occurs in the process of mRNA cap formation (Furuichi, 1978). mRNA: GTP-guanylyltransferases of CPV, human reovirus (Furuichi et at., 1976), vaccinia virus (Martin and Moss, 1976) and HeLa cell (Wei and Moss, 1977) catalyze the following general reaction: I;I ;~G + p p N p ~ ” + GI;PPN~N” + Pi?. AS postu- lated for vaccinia virus (Shuman and Hurwitz, 1981), an enzyme-pG complex may be a direct precursor for capping as well. The enzyme activity which is responsible for G(5’)pppp(Y)N formation during CPV transcription has not been isolated from virus particles because of difficulties to solubilize the virus without loss of enzyme activity. However, we assume that structures of the type G(S’)pppp(5’)N are derived from a reaction similar to mRNA capping by CPV mRNA:GTP guanylyltransferase, perhaps by the reaction: Enzyme-pG + pppN -+ GppppN + Enz. We have recently found that a CPV structural protein (apparent M, = 124,000) was consistently radiolabeled by incubation of viruses with [cx-~’P]GTP but not with [P-”P]GTP.3 The [32P]GMP residue incorporated into the protein seems to be covalently linked since it is resistant to alkaline phosphatase treatment and is retained on the protein during sodium dodecyl sulfate-poly- acrylamide gel electrophoresis. It remains to be seen whether the protein is a mRNA:GTP guanylyltransferase and capable of transferring pG residues for synthesis of GppppN and

Biological function of GppppG in the undeveloped embryo is still ambiguous except that it is the primary source of all purine-containing compounds required for Artemia develop- ment (Warner, 1979). In Artemia, GppppG, can be methylated by cellular methyltransferase although the methylated struc- ture has not been elucidated (Warner, 1979). I t is noteworthy that the appearance and disappearance of GppppG in Artemia embryos seems to be related with cell dormancy and morpho- genesis, respectively. AppppA exists in many mammalian cells at varying concentrations (0.05 to 5 PM) depending on the doubling time of the cell line or the proliferative state of the cells (Rapaport and Zamecnik, 1976). Grummt has recently shown that the addition of AppppA to permeabilized GI- arrested baby hamster kidney cells resulted in the stimulation of DNA synthesis (Grummt, 1978; 1979).

One of the main differences in the structure of GppppN and CPV cap precursor GpppApG is the number of phosphate residues in the blocked structure. The y-phosphate of ATP in initiator oligonucleotides such as pppApG is excluded from

GPPPAPG.

the cap in completed mRNA (Furuichi and Miura, 1975) but retained in GppppA and GppppApG (Fig. 5, lane 2) . Both blocked structures are made by the same guanylyl transfer mechanism and perhaps by the same enzyme. The prior removal of y-phosphate from pppApG to yield ppApG by the virus-associated nucleotide phosphohydrolase may be impor- tant for subsequent chain elongation and methylation but not for guanylylation.

Clearly, as shown in Fig. 7 (lane 4) , ADP is unable to support the synthesis of capped oligonucleotides which con- tain a phosphodiester linkage, whereas GTP and ADP can form GpppA. Previously, Shimotohno and Miura (1976) have found the occurrence of a small amount of m7GpppA in CPV transcription and proposed a pretranscriptional cap formation in which the m7GpppA serves as primer for RNA synthesis. In the context of present results, however, it is likely that the corresponding m7GpppA may derive from methylation of GpppA which is formed from GTP and ADP by a side reaction of guanylyltransferase. Rather, the occurrence of a large amount of uncapped initiator oligonucleotide ppApG supports the following sequence of reactions for the initiation of CPV transcription and capping.

As shown in Fig. 8, initiator oligonucleotides, like viral mRNA, increase in amount as incubation continues. It seems that these oligonucleotides in the complete reaction mixture are produced by the reiterative initiation of transcription, since they are released from genome t e m ~ l a t e . ~ In regard to the synthesis of initiator oligonucleotides, three points are noteworthy from Table I. First, a greater molar excess (26- fold) of initiator oligonucleotides over mRNA was produced during CPV mRNA synthesis suggesting that a successful chain completion occurred, on the average, every 27 initiation events. Second, molar amounts of these initiation-related oli- gonucleotides in incomplete reactions (after 1-h incubation) are about 20-fold more than the total of oligonucleotides and mRNA (-1,600 nucleotides chain length) in the complete reaction mixture. These results suggest that RNA polymerase, when it is unable to function in chain elongation, continues multiple initiations at the promoter site. Third, the ratio of capped oligonucleotides (mixture of m7GpppApG + GpppApG) uersus uncapped oligonucleotides ApG (originally (p)ppApG) in the complete reaction mixture is 2.5-fold less than in the incomplete reaction mixture (Table I). A lesser amount of capped oligonucleotides in the complete reaction mixture which supports capped mRNA synthesis may imply a positive effect of capped initiator oligonucleotides being more effectively incorporated into the full size transcripts than ppApG. In the sequence of reactions, step 111 is probably the step where nascent and genome-bound ppApG is subject to a decision to “stay” or “release” on/from the genome template. A comparative molar amount of GppppN to that of released ppApG obtained during CPV transcription may im- ply that initiator oligonucleotide synthesis coupled with sub- sequent guanylylation is of primary importance for the elon- gation of ppApG to mRNA. GppppN, thereby, may represent an uncoupled side reaction by guanylyltfansferase during this process. In fact, when %’P-labeled ppApG (0.2 mM) isolated from incomplete CPV transcription mixture was added to the complete transcription mixture, ppApG was incorporated into the 5“termini of CPV full size mRNAs. Moreover, the incor- porated ppApG was guanylylated and methylated in the struc-

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492 In Vitro CPV Transcription and Capping

ture of m7GpppApGp-? These findings indicate that the for- mation of a phosphodiester bond to yield the initiating dinu- cleotide (pppApG) takes place before guanylylation and meth- ylation. A similar sequence of reactions has been reported previously in human reovirus transcription initiation (Furui- chi and Shatkin, 1977).

In eukaryotic transcription directed by cellular RNA polym- erase 11, the data suggesting that the capping site is also the transcription initiation site have been accumulating. These include the absence in adenovirus primary transcripts of se- quences upstream of capping sites (Ziff and Evans, 1978; Manley et aL, 1979) and, more directly, the incorporation of P-phosphate of the initiating nucleotides into caps of SV40- specific transcripts (Gidoni et al., 1981; Contreas and Fiers, 1981). Perhaps, during transcription in these systems, caps are formed through reaction steps I to IV immediately after the first phosphodiester bond formation by capping enzymes as- sociated with RNA polymerase 11. It remains to be seen if reiterative initiation similar to that observed for CPV and human reovirus occurs in cellular transcription directed by RNA polymerase 11.

Acknowledgments-We thank Dr. A. J. Shatkin at Roche Institute for discussion and encouragement. We also thank Drs. M. Miwa and T. Sugimura at the National Cancer Center Research Laboratory (Japan) for purified tobacco acid pyrophosphatase used in the struc- tural determination of GppppN.

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In Vitro CPV Transcription and Capping 493

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R E Smith and Y Furuichiformation.

polyhedrosis virus of Bombyx mori. Structural determination and mechanism ofG(5')pppp(5')N, synthesized during the in vitro transcription of cytoplasmic

A unique class of compound, guanosine-nucleoside tetraphosphate

1982, 257:485-494.J. Biol. Chem. 

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