71, 74-84
VIROLOGY
Enzymatic R. SALOMON, Department
(1976)
Acylation I. SELA,’
of Biochemistry, Agriculture,
The Virus
of Histidine
to Tobacco
H. SOREQ, Weizmann Laboratory, Accepted
D. GIVEON,
Mosaic Virus RNA AND
U. Z. LITTAUER
Institute of Science, Rehovot, Israel; The Hebrew University, Rehovot, November
and ’ The Faculty Israel
of
24, 1975
A method for the purification of rat liver histidyl-tRNA synthetase was developed. The enzyme, freed of trace nuclease contamination by affinity chromatography on DNASepharose, could catalyze the aminoacylation of tobacco mosaic virus (TMV) RNA with histidine. The aminoacylation reaction of TMV RNA was inhibited by KCl, while that of rat liver tRNA exhibited a maximum rate at 140 mM KCl. TMV N-acetyl PHlhistidylRNA was prepared in order to stabilize the TMV histidyl-RNA ester bond. The TMV Nacetyl [3Hlhistidyl-RNA showed a migration identical to that of unacylated TMV RNA upon electrophoresis in polyacrylamide agarose gels. Removal of 5 to 10 nucleoside residues from the 3’-terminus using Escherichia coli polynucleotide phosphorylase or oxidation of the 3’-terminal ribose with periodate (to produce TMV RNA,,,,) eliminated the aminoacylation capacity of TMV RNA as well as its infectivity in tobacco leaves. It is concluded that the aminoacylation site is located at the 3’-terminal adenosine of TMV RNA. Fragmentation of TMV RN&,, with a crude rat liver enzyme did not expose additional sites for acylation with histidine, nor did it produce acylation capacity for other amino acids. The reduction of TMV RN&. with sodium borohydride converted the terminal dialdehyde group to a dialcohol-lacking covalent bond between the C2’ and CB’ of the terminal adenosine, but did not restore its ability to accept histidine or its infectivity. Similar reduction of rat liver tRN&,, to tRNA,,,,,,,, however, did restore its ability to accept phenylalanine while activity for histidine was not regained.
et al ., 1972) the precise site of aminoacylation of plant viral-RNA molecules has not been determined. Moreover, turnip yellow mosaic virus RNA is cleaved by the tRNA maturation enzyme to yield a 3’-terminal fragment of about 111 nucleosides, which retains its valine-acceptor capacity (Prochiantz and Haenni, 1973). In addition, as will be shown in this study, partially purified aminoacyl-tRNA synthetase preparations still contain enough endonucleolytic activity to fragment TMV RNA during the histidine acylation reaction. Thus, the possibility that enzymatic cleavage of TMV RNA is required prior to the aminoacylation reaction cannot be ruled out. It seemed important, therefore, to determine whether the original 3’-terminal adenosine of TMV RNA is the site for histidine attachment, or whether this site resides internally in the molecule and must be exposed to enzymatic cleavage before ami-
INTRODUCTION
RNA obtained from several plant viruses (Pinck et al., 1970, 1972; Yot et al., 1970; Oberg and Philipson, 1972: Hall et al., 1972; Carriquiry and Litvak, 1974; Salomon and Littauer, 19741, as well as from mengovirus (Salomon and Littauer, 19741, can be enzymatically aminoacylated with eukaryotic aminoacyl-tRNA synthetase preparations. The similarity of these aminoacylation reactions to that exhibited by tRNA, and the ability of plant virus RNA to interact with several other tRNA specific enzymes (Yet et al., 1970; Litvak et al., 1970, 1973; Haenni et al., 1973; Prochiantz and Haenni, 1973) may indicate that a tRNA-like structure is covalently linked to the genomic RNA. Although the esterification reaction appears to occur with a relatively intact RNA molecule (Pinck et al., 1970; Yot et al., 1970; Oberg and Philipson, 1972; Hall 74 Copyright All rights
6 1976 by Academic F’ress, Inc. of reproduction in any form reserved.
AMINOACYLATION
noacylation can take place. In the present report, we examine the acylation reaction of TMV RNA with histidine. As TMV RNA is more sensitive to nuclease attack than tRNA, it was of vital importance to acquire a nuclease-free histidyl-tRNA synthetase preparation. Such an enzyme was prepared, allowing us to demonstrate that no enzymatic modification of TMV RNA is necessary prior to its acylation with histidine. We also found a correlation between the histidine acylation capacity of TMV RNA and its infectivity. MATERIALS
AND
METHODS
TMV (strain vulgare) was grown in tobacco plants (N: tubacum cv Samsun). The virus particles were isolated by repeated centrifugation cycles and purified by banding in a CsCl gradient (Salomon and Littauer, 1974). The RNA was then isolated from the virus suspension by phenol-chloroform extraction (Littauer, 1971). The integrity of the TMV RNA chain was checked by polyacrylamide-agarose gel electrophoresis. Nuclease-free polynucleotide phosphorylase was purified as previously described (Soreq et al., 1974) and exhibited a sp act of 250 unitsimg of protein. Carrier-free and polyphosphate-free [32Plorthophosphate (100 mCi/ml) was purchased from the Nuclear Research Center, Negev, Israel. L3Hlhistidine (58 Ci/mmole) and [14C]histidine (342 mCi/mmole) were obtained from Radiochemical Center, Amersham, England. Preparation of DNA-Sepharose. The binding of DNA to Sepharose 4B was accomplished through a modification of the method of N. D. H. Rosa, J. Kallos, and P. B. Sigler (personal communication). Salmon sperm DNA (highly polymerized sodium salt, ICN, Cleveland, Ohio) was dissolved in 0.02 M Tris-HCl, pH 7.5, and precipitated with 2 vol of ethanol. The precipitate was redissolved in 80% N,N’-dimethylformamide (DMF) solution (BDH, Poole, England), containing 0.1 M NaHCO,, 0.02 M Tris-HCl, pH 7.5, to a final concentration of 1.0 mg/ml, and the DNA was denatured by heating to 50” for 5 min. Sepharose 4B (Pharmacia) was washed with 10 vol of cold water, collected
OF
TMV
RNA
75
on a sintered glass filter, and suspended in 2 vol of cold water, the pH of the solution was adjusted to 11 by the addition of 10 N NaOH while stirring at 0”. Cyanogen bromide (20 g) was dissolved in 20 ml of DMF and was added to 200 ml of the Sepharose suspension. The suspension was constantly stirred, and the pH was maintained at 11 by continuous dropwise addition of 10 N NaOH. The whole procedure was performed in an ice bath, and the temperature was kept under 6”. When the decrease in pH ceased, the activated Sepharose was immediately washed with 6 vol of cold water and 5 vol of 0.1 M NaHCO,. Four volumes of denatured DNA solution (1 mg/ml) in 80% DMF containing 0.1 M NaHCO, were added to the ’ activated Sepharose suspension, and the mixture was slowly stirred overnight at 2”. The DNA-Sepharose was washed with 5 vol of 5 M NaCI, and the amount of DNA fixed was calculated by subtraction of the eluted material from the amount of DNA used. With this procedure, between 2 to 5 mg of DNA were fixed per milliliter of Sepharose. The DNA-Sepharose column was washed with 5 M NaCl until no further A,,, could be eluted. The free active groups left on the Sepharose were blocked by washing with 3 vol of 0.05 M ethanolamine, and the suspension was kept until use in 0.02 M Tris-HCl buffer, pH 7.5, containing 2 mM sodium azide. After use the column was regenerated with 2 vol of 0.1 N NaOH and equilibrated with 5 vol of elution buffer. Optimum conditions for enzyme adsorption by the DNA-Sepharose column were obtained when the DNA was first heated for 5 min at 50” and then rapidly cooled at a concentration of 1 mg/ml. Denaturation of the DNA in aqueous solutions or the use of DNA solutions at concentrations higher than 1 mg/ml considerably decreased the amount of DNA that could be fixed to Sepharose. When the average DNA chainlength was reduced to 4 x lo4 daltons by ultrasonication (as measured by polyacrylamide-agarose gel electrophoresis), the enzyme was not adsorbed on the DNASepharose column, although the amount of DNA bound to the Sepharose was markedly high.
76
SALOMON
Preparation of histidyl-tRNA synthetase. All steps were performed at 4”. Mouse
or rat liver was washed thoroughly with a solution containing 10 n&f Tris-HCl, pH 7.5, 10 mM MgC12, 60 mM NH&l, 10 miV reduced glutathione, and 10% glycerol. The livers were homogenized for 15 set in an Ultra Turrax (Ika Werk, Breisgau, Germany) high-speed homogenizer with 3 vol of the same buffer solution. The homogenate was centrifuged for 10 min at 13,000g and then for 3 hr at 100,OOOg. The supernatant solution (S-100) was subjected to phase partition according to the procedure of Babinet (1967). The S-100 solution (600 ml) was mixed with 150 ml of a solution containing 10.6 g of dextran T500 (Pharmacia), 42.6 g of polyethylene glycol 6000 (Uniroyal), and 7.5 ml of 1.0 M TrisHCl buffer, pH 7.5, previously dissolved by boiling. The mixture was stirred for 40 min, centrifuged for 10 min at 13,000 g, and the supernatant was designated Phase I and was subsequently discarded. The pellet was suspended in a 70-ml solution containing 46 ml of 13.8% polyethylene glycol 6000, 1.4 ml of 1.0 M Tris-HCl, pH 7.5, 3.4 ml of 0.2 M reduced glutathione, and 19.2 ml of water. Sixteen grams of NaCl was then added to bring the salt concentration to 4 M, and the suspension was stirred for 30 min. After centrifugation for 10 min at 13,000 g, the supernatant (Phase II) was dialyzed for 3 hr against three changes of 1 liter of a solution containing 10 n-&f TrisHCl, pH 7.5; 1 miV reduced glutathione, and 10% glycerol, and the reduced glutathione concentration was adjusted to 50 mM. The dialyzed Phase II solution (120 ml) was diluted with 1 vol of a solution containing 10 m&f potassium phosphate buffer, pH 7.5, 1 m&f MgC&, 0.1 m&f EDTA, 10 mM reduced glutathione, and 5% glycerol, and applied on a DEAE-cellulose column (3 x 10 cm) pre-equilibrated with the same buffer. The column was washed with 100 ml of the same buffer, and the enzyme was eluted from the column with a 0.01 - 0.36 M (50-ml each) linear gradient of potassium phosphate buffer, pH 7.5, containing 1 mM MgC12, 0.1 n-& EDTA, 10 n&f reduced glutathione, and 5% glycerol, and 2-ml fractions were
ET AL.
collected. Histidyl-tRNA synthetase activity was recovered between 0.15-0.25 M, and the enzyme fraction was dialyzed for 1 hr against 2 liters of 10 mM potassium phosphate buffer, pH 6.8, containing 1 n&f reduced glutathione and 10% glycerol. The reduced glutathione and glycerol were adjusted to final concentrations of 10 mM and 50%, respectively, and the enzyme fraction (87.5 ml) was kept at -20”. Ten milliliters of the above enzyme preparation was diluted with 1 vol of 10 n&f potassium phosphate buffer, pH 6.8, containing 0.1 n-&f DlT and 10% glycerol, and the solution was applied to 2 x 4-cm hydroxylapatite column (Bio-Rad), pre-equilibrated with the same buffer. The column was washed with 12 ml of the application buffer, and then eluted stepwise with 5-ml each of 0.1 M, 0.15 M, 0.20 M phosphate buffer containing DTT and glycerol. The enzyme activity was eluted with 0.25 M phosphate buffer containing D’M’ and glycerol. The histidyl-tRNA synthetase was purified about 145-fold and showed a final sp act of 77 units/mg of protein (Table 1). To remove trace amounts of nucleases, the enzyme preparation (12 ml) was diluted fourfold with 36 ml of 10 m&f TrisHCl buffer, pH 7.7, containing 0.1 mM EDTA, 0.1 n-&f DTT, and 5% glycerol. It was immediately applied to a 2 x 7.5-cm column of DNA-Sepharose. The column was washed with 20 ml of the above buffer. Histidyl-tRNA synthetase activity was eluted between 1.0-1.5 M NaCl, with a 50ml linear salt gradient of O-2 M NaCl in 0.01 M Tris-HCl buffer, pH 9.0, 0.1 miV DTT, 0.1 n&f EDTA, and 5% glycerol. The very dilute enzyme preparation obtained was extremely labile and was stabilized by TABLE PURIFICATIONOF
1
HwrmyL-tRN.4
SYNTHETASE
Fractions
Proteins hnghnl)
Total mtivity (units)
Specific activity (units/ mg of pmtein)
s-100 Phase II DEAE-cellulose Hydroxylapatite DNA-Sepharose
7.4 1.8 0.35 0.055 0.0055
2330 1100 550 372 151
0.5 5.1 18 71 69
AMINOACYLATION
the addition of iodoacetate-treated bovine serum albumin (Zimmerman and Sandeen 1966) and glycerol to a final concentration of 0.5 mg/ml and 50%, respectively. The iodoacetate treatment of serum albumin was necessary to inactivate its nuclease contamination. Enzyme assay. The reaction rate was measured in an incubation mixture (0.05 ml) containing 40 m&f Tris-HCl, pH 7.5, 2 mM ATP, 10 mM MgC12, 3 mM reduced glutathione, 140 n&f KCl, 0.5 &i of 13Hlhistidine (58 Ci/mmole) or 0.1 &i of [14Clhistidine (342 mCi/mmole), 30 pg of tRNA, and 0.002-0.01 units of enzyme. Incubation was carried out at 30” for 10 min, and the reaction was stopped by immersing the tubes in an ice bath; 0.5 ml of bovine serum albumin solution (0.25 mg) and 7 ml of cold 5% trichloroacetic acid were then added. The precipitate was filtered through glass fiber filters (Whatman GF/C), washed three times with cold 5% trichloroacetic acid, and counted in a scintillation counter with toluene-based scintillation fluid. One unit of enzyme is defined as the amount which incorporates 1 nmole of histidine in 60 min, and the specific activity is expressed as enzyme units per milligram of protein. When the extent of the aminoacylation was assayed, the incubation mixture contained 5-15 pg of tRNA and 0.01-0.06 units of enzyme, and the reaction was carried out for 20 min at 30”. The histidine acceptance capacity of TMV RNA was assayed in the absence of KC1 in the reaction mixture. N-acetylation
of TMV
histidyl-RNA.
The ester bond of TMV histidyl-RNA was stabilized by treatment with N-hydroxysuccinimide acetylester to form TMV Nacetylhistidyl-RNA as previously described (Daniel et al., 1970; Salomon and Littauer, 1974). Protein determination. Protein was determined by the method of Lowry et al. (19511, with crystalline bovine serum albumin as the standard. Dilute protein solutions were concentrated prior to protein determination by the addition of 2 vol of ethanol and precipitation overnight at -20”. The precipitate was collected by cen-
OF
TMV
77
RNA
trifugation and dissolved in 0.01 ml of 0.1 N NaOH by incubating for 30 min at 37”. Preparation
of RNA,,
and RNA,,,.
One milliliter of TMV RNA or tRNA solution (2.2 mg/ml) was supplemented with 0.15 ml of 1 M sodium acetate buffer, pH 5.0, and then mixed with 0.35 ml of freshly prepared 0.1 M NaIO,. The solution was kept in the dark for 30 min at room temperature and precipitated with 0.3 ml of 5 M NaCl followed by 3.0 ml of ethanol. Excess periodate in the ethanolic supernatant was titrated with ethylene glycol (Tal et al., 1972). Oxidized TMV-RNA and tRNA were reduced by the addition of 1 M NaBH, to a final concentration of 0.07 A4 and 0.25 M Tris-HCl, pH 8.0. This mixture was incubated at room temperature until bubbles ceased to emerge (about 90 min), and the RNA was precipitated with ethanol. Phosphorolysis assay of TMV RNA. The reaction mixture contained 20 pg of TMV RNA, 30 m&f Tris-HCl buffer, pH 8.0, 1 mM Na,EDTA, 10 mM MgCl*, 20 n-&f potassium phosphate buffer (pH 8.0), containing carrier-free [32P]orthophosphate (about lo* cpm/pmole), 5 pg of purified polynucleotide phosphorylase (1.25 units), and 5 mM dCDP, in a final volume of 30 ~1. The mixture was incubated at o”, and at various time intervals 5-~1 aliquots were removed and applied onto DEAE-cellulose paper sheets (Whatman DE 811, and the 32P-labeled nucleoside diphosphates were chromatographed as previously described (Soreq et al., 1974). Preparation of phosphorolyzed TMV RNA. The reaction mixture was as de-
scribed above except for the omission of ls2Plorthophosphate and the addition of 160 pg of TMV RNA, and 20 pg of purified polynucleotide phosphorylase (5 units), in a final volume of 270 ~1. The mixture was incubated at 0”. At various time intervals, 40-~1 aliquots were removed, and 2 vol of 95% ethanol were added to precipitate the RNA. Infectivity assay of TMV RNA. Serial dilutions of intact TMV RNA were applied to half leaves of Nicotiana glutinosa L. Five microliters of the corresponding dilutions of different treated RNA samples
78
SALOMON
were applied to the opposite half leaves. In addition, samples of each preparation were incubated at 37” with a solution containing 0.15 M NaCl, 0.015 M trisodium citrate, and 10 pg/ml of pancreatic-RNAse extracted with phenol and then applied to another set of half leaves. This was done to rule out the possibility that positive infectivity could be due to the presence of residual whole TMV molecules in the RNA preparation. Polyacrylamide-agarose resis. Gel electrophoresis
ET AL.
Mgz+ concentration required for acylation of tRNA and TMV RNA with histidine. On the other hand, KC1 inhibited the reaction with TMV RNA. Figure 2 demonstrates that at 20 mM KCl, the aminoacylation of
gel electropho-
was performed according to Peacock and Dingman (1968), and the gels were stained with “stains all” (Dahlberg et al., 1969). RESULTS
Properties of purified enzyme. TMV RNA was shown to be aminoacylated with histidine by extracts from either yeast or KB cells @berg and Philipson, 1972; Carriquiry and Litvak, 1974). In comparable tests carried out in this laboratory, rat and mouse liver extracts were found to contain aminoacylation activity while E. coli MRE 600 extract was totally inactive (Salomon and Littauer, 1974). However, since the histidyl-tRNA synthetase preparation did contain endonucleotic activity (see below), it was impossible to dissociate the aminoacylation reaction of TMV RNA with histidine from the fragmentation reaction. A prerequisite for the separation of these two processes is the acquisition of nucleasefree histidyl-tRNA synthetase. Such an enzyme was purified from rat liver extracts employing phase partition, DEAEcellulose, and hydroxylapatite chromatography, as well as affinity chromatography, on a DNA-Sepharose column (Table 1). Although the affinity chromatography step did not enrich the enzyme preparation, it was found to be essential for final removal of trace amounts of nucleases. With this enzyme preparation, the requirements for the TMV RNA aminoacylation reaction were characterized and compared with those for tRNA. The optimal pH range for histidine incorporation with both types of RNA was found to be broad between 7.5-8.5. As shown in Fig. 1, there was no essential difference in the optimal
FIG. 1. Effect of MgCl, on acylation to TMV RNA and mixtures contained 35 wg of purified enzyme, the indicated tions, and were incubated for
the rate of histidine tRNA. The reaction RNA, 0.003 units of MgCl, concentra10 min at 30”.
FIG. 2. Effect of KC1 on the rate of histidine acylation to TMV RNA and tRNA. The reaction mixture contained 35 pg of RNA, 0.003 units of purified enzyme, the indicated KC1 concentrations, and was incubated for 10 min at 30”.
AMINOACYLATION
TMV RNA was inhibited to 50%, whereas reaction with tRNA was stimulated by KC1 with an optimal concentration at 140 mM. Figure 3 shows a time curve for TMV RNA aminoacylation using the purified enzyme preparation. The extent of histidine acylation of TMV RNA was about 0.3 mole/mole of RNA, which is similar to previously reported values (Carriquiry and Litvak, 19741, which were attained with crude yeast enzyme preparations. The extent of histidine acylation could not be increased by lowering the temperature or pH of the reaction mixture. The reason for incomplete acylation of TMV RNA with histidine has been recently shown to be due to a heterogeneity of the TMV RNA population, some of the RNA chains being defective at the 3’-terminus (Littauer et al., 1975). Presence of nuclease contamination in the purified enzyme preparation. In pre-
vious studies,
the size of the aminoacylI
I
I
.
TMVRNP
TMV
I
RNAor
,./--Al, I IO
I Time
I
I 20
(min)
FIG. 3. The effect of periodate oxidation on the aminoacylation capacity of TMV RNA. 0 - 0, Untreated TMV RNA; A-A, TMV RNA,,,; 0- - -0, a 1:l mixture ,of TMV RNA and TMV RNA,. The extent of reaction was measured with 15 fig of RNA and 0.06 units of purified enzyme. In the latter case, the attachment of histidine was expressed as Wlhistidine, cpm/Fg of unoxidized TMV RNA present in the above mixture.
OF
TMV
RNA
79
FIG. 4. Polyacrylamide-agarose gel electrophoresis of TMV A’-acetyl [3H]histidyl-RNA prepared with a partially purified histidyl-tRNA synthetase. TMV RNA was aminoacylated with 13H]histidine with partially purified rat liver enzyme (DEAEcellulose fraction). The resulting [3H]histidyl-RNA was reacted with N-hydroxysuccinimide acetyl ester to yield N-acetyl [3H]histidyl-RNA. About 10 pg of N-acetyl 13H1histidyl-RNA was applied to 1.7% polyacrylamide-0.5% agarose slab gel, and electrophoresis was carried out for 2.5 hr at 0” at 11.4 V/cm. Radioactivity was measured on l-mm slices as previously described (Salomon and Littauer, 1974). In adjacent slots untreated TMV RNA, E. coli rRNA and tRNA markers were run simultaneously.
ated RNA molecule has been estimated by sucrose gradient centrifugation or gel IXtration (Oberg and Philipson, 1972; Litvak et al., 1973). In our work we preferred to use acrylamide gel electrophoresis. For electrophoresis, the ester bond of TMV histidyl-RNA was stabilized by treatment with N-hydroxysuccinimide acetylester to form TMV N-acetyl-histidyl-RNA (Salomon and Littauer, 1974). Using ZV-acetyl [3Hlhistidyl-RNA molecules, we have been able to estimate their size by monitoring the radioactivity of the sliced gel. Figure 4 shows the electrophoretic migration of TMV RNA which was aminoacylated with 13Hlhistidine using a partially purified enzyme preparation (DEAE-cellulose fraction). The labeled product migrated as a broad band with an apparent molecular weight between 0.4 x lo5 to 1.8 x 10”. It is obvious that considerable degradation of the TMV RNA chains took place during the aminoacylation reaction. On the other hand, when the purified enzyme preparation (DNA-Sepharose fraction) was employed for the aminoacylation of TMV RNA, the labeled N-acetylhistidyl-RNA corn&rated with the marker TMV RNA as demonstrated by its staining
80
SALOMON
profile (Fig. 5). Thus, no fragmentation of TMV RNA occurred during the aminoacylation with the purified enzyme preparation. Oxidation and reduction of TMV RNA. Although size determinations by polyacrylamide gel electrophoresis are very accurate (Peacock and Dingman, 1968), they do not exclude the possibility that a few nucleoside residues are cleaved, at or near the 3’-terminus. In order to determine whether the 3’-terminus of TMV RNA is indeed the site for histidine aminoacylation, we employed a chemical approach, in which the RNA was oxidized with sodium periodate. The 3’4erminal sequence of TMV RNA has been determined to be GCCCA-OH (Steinschnieder and FrankelConrat, 1966a). Therefore, periodate will oxidize TMV RNA at the 3’-adenosine, the expected site of histidine attachment, to form the dialdehyde derivative (TMV RN&,). Figure 3 shows that oxidation with periodate abolished the aminoacylation capacity of TMV RNA as well as that of rat liver tRNA, which served as a control. Addition of TMV RN&, had no effect on the aminoacylation of untreated TMV RNA, thus showing that no excess periodate remained attached to the isolated TMV RNA,,. An aliquot of the oxidized TMV RNA was analyzed by polyacryl-
FIG. 5. Polyacrylamide-agarose gel electrophoresis of TMV N-acetyl [3H]histidyl-RNA prepared with a nuclease-free histidyl-tRNA synthetase. TMV RNA was aminoacylated with [$H]histidine by a nuclease-free rat liver histidyl-tRNA synthetase (DNA-Sepharose fraction) and treated as described in the legend to Fig. 4. The smooth line represents the optical density at 570 nm of the stained RNA (“stains all”), and the dotted line shows the radioactivity distribution.
ET AL.
Time(min)
FIG. 6. The effect of cleavage of TMV RNA and TMV RN&,, on histidine aminoacylation capacity. The reaction mixture contained 350 pg of untreated TMV RNA (O-O], or 320 Fg TMV RNA, (A-A), and 40 n&f Tris-HCl, pH 7.5, 2 n-&f ATP, 10 mM MgCl,, 3 u&f reduced glutathione, and 10 ~1 of Phase II extract. After incubation for 30 min at 37” the RNA was extracted with phenol-chloroform mixture and precipitated with 2 vol of ethanol in the presence of 1 M NaCl. The RNA precipitates were dissolved in water and aliquots were removed for the assay. The extent of reaction was measured with 15 pg of RNA and 0.06 units of purified enzyme.
amide-agarose gel electrophoresis to ascertain that the viral RNA was not degraded during the oxidation process, and this was found to be the case. Thus, the results of these experiments would support the notion that the aminoacylation site is located at the 3’-terminal adenosine. We have also attempted to determine whether fragmentation of TMV RNA with a crude rat liver RNase would increase the number of sites available for acylation with histidine or any other amino acids. We argued that if such sites exist, cleavage of oxidized TMV RNA, which itself is inactive, would expose these sites for aminoacylation. TMV RN&, and nonoxidized TMV RNA were incubated with a crude rat liver extract (Phase II fraction). Analysis by gel electrophoresis showed that both preparations were cleaved and migrated as a broad band between the 16 S RNA and the tRNA markers. Figure 6 shows a time
AMINOACYLATION
OF
curve for histidine acylation of cleaved TMV RNA,,, as compared with cleaved, nonoxidized TMV RNA. It is clear from this figure, as compared with Fig. 3, that in both RNA preparations no new sites for aminoacylation are exposed by the cleavage treatment. The TMV RN&, remained as inactive as before treatment, and the histidine-acceptor capacity of the nonoxidized TMV RNA was unaltered. In addition, cleaved TMV RNA did not show acceptor activity for amino acids other than histidine (data not shown). Reduction of tRN&, with sodium borohydride converts the terminal dialdehyde group to a dialcohol lacking a covalent bond between the CZ’ and Cs’ of the terminal adenosine. It is known (Tal et al., 1972) that some, although not all, of the isoaccepting tRN&, species regain their aminoacylation capacity upon reduction. It was therefore of interest to examine whether reduction of TMV RNA,, would restore its histidine-acceptance capacity. Figures 7A and B show that both TMV RNbweci and rat liver tRNAXTed were unable to accept histidine, whereas reduction of tRNA,,, did restore most of its phenylalanine-acceptor activity. Oxidized TMV RNA was previously
5
IO
15
5
IO
15
5
IO
15
Ttmehn)
FIG. 7. The effect of oxidation and reduction of TMV RNA on the histidine aminoacylation capacity. TMV RNA and rat liver tRNA were oxidized with NaIO,. A portion of the oxidized material was then reduced with NaBH, as described in Materials and Methods. (A) Aminoaclyation of TMV RNA with histidine. (B) Aminoacylation of rat liver tRNA with histidine. (C) Aminoacylation of rat liver tRNA with phenylalanine. 0 - 0, Untreated RNA, A - A, oxidized RNA; A-A, oxidized and reduced RNA. The extent of reaction was measured with 15 pg of RNA and 0.06 units of purified enzyme.
TMV
60
81
RNA I
I
I
I
FIG. 8. The effect of oxidation and reduction on the infectivity of TMV RNA. Five microliters of the indicated RNA solutions were applied to the half leaves.
found to be noninfective (Steinschnieder and Frankel-Conrat, 196613). These results were confirmed by us, as is shown in Fig. 8. In addition, reduction of TMV RNA,,, did not restore its infectivity to tobacco leaves. Limited
phosphorolysis
of TMV
RNA.
To rule out the possibility that periodate oxidation affected parts of the RNA molecule other than the 3’-terminus, we employed an enzymatic approach, in which a few nucleoside residues are removed from the 3’-terminus. The method is based on the ability of polynucleotide phosphorylase to phosphorolyse long polynucleotides starting at the 3’-end by a processive mechanism. Using a 1:l molar ratio of enzyme to substrate, a synchronous mode of phosphorolysis can be established. Inclusion of 13*Plorthophosphate in the phosphorolysis medium leads to an incorporation of the label into the p-phosphate moieties of the generated nucleoside diphosphates, the quantities of which enable the extent of phosphorolysis to be followed (Soreq et al., 1974). When TMV RNA was incubated with polynucleotide phosphorylase for 30 set at O”, between 5 to 10 nucleoside residues were removed from the 3’terminus. This treatment eliminated the infectivity of TMV RNA (Fig. 9) and also reduced its 13Hlhistidine-acceptor capacity to a value of 500 cpm/lO c(g of RNA, as compared with 27,000 cpm/lO ,ug for the untreated molecules.
82
SALOMON
FIG. 9. The abolition of infectivity by limited phosphorolysis of TMV RNA with polynucleotide phosphorylase. Phosphorolyzed TMV RNA was prepared by incubating TMV RNA with polynucleotide phosphorylase for 30 set at 0” as described under Materials and Methods. Five microliters of the indicated RNA solutions were applied to the half leaves. DISCUSSION
The overall requirements for histidine charging of both TMV RNA and tRNA were found to be similar, except that the presence of KC1 was shown to stimulate aminoacylation of tRNA, but strongly inhibited that of TMV RNA (Fig. 1). This may suggest that the binding between the TMV RNA and the enzyme is weak and, therefore, readily dissociated by increasing salt concentrations, perhaps due to a difference in the secondary and/or tertiary structure of the aminoacylation region, or a masking of this region by other parts of the TMV RNA molecule in the presence of salt. The high molecular weight TMV RNA molecules appear to contain regions which are highly resistant to nuclease degradation. During incubation with various rat liver enzyme preparations, these regions survive degradation, and the viral RNA is cleaved into long fragments. Depending on the enzyme fraction and incubation conditions, the size of the fragments may vary between 3 x lo4 to 1.8 x 10” daltons. Prolonged incubation with the crude rat liver extract (Phase II fraction) resulted in fragments with a discrete size range that migrated on gel electrophoresis in a range between the tRNA and 5 S RNA markers;
ET AL.
smaller oligonucleotides, however, were absent. Moreover, fragmented viral RNA has the same histidine-acceptance capacity as intact TMV RNA (Figs. 3 and 6). This indicates that the aminoacylation region of TMV RNA, like tRNA, is relatively resistant to nuclease action. It also suggests that no new sites for aminoacylation are exposed during the incubation period. The latter suggestion was reaffirmed by the absence of amino acid acceptance of fragmented TMV RN&, (Fig. 6). The situation may be different when TMV RNA is fragmented with a crude tobacco cell-free extract (Sela, 1972). The presence of nuclease contamination in aminoacyl-tRNA synthetase preparations made it impossible to distinguish between the cleavage and charging reaction, and no answer could be given as to the precise location and number of the aminoacylation site(s). For this reason, the above described preparation of a nucleasefree histidyl-tRNA synthetase became essential. Using the nuclease-free enzyme, we were able to show by gel electrophoresis that the aminoacylated TMV RNA corn&grates with intact TMV RNA, suggesting that the histidine-acceptor site is located at or near the 3’-terminus. To demonstrate more conclusively that the 3’-terminus of TMV RNA is indeed the only site for aminoacylation, periodate oxidation of the 3’-ribose was performed. It was found that periodate oxidation entirely abolished the aminoacylation capacity of TMV RNA. It should be noted that when a crude aminoacyl-tRNA synthetase preparation was employed, TMV RN&, molecules could partially regain their acceptor capacity. Apparently, under these conditions, replacement of the oxidized terminal adenosine does occur, thereby restoring some of the RNA chains to their original form. Thus, the examination of the amino-acid-acceptor capacity of periodate-oxidized RNA molecules can only be performed with relatively purified enzyme prhparations. The possibility that periodate can affect parts of the molecule other than the 3’terminus cannot be excluded a priori. This is particularly relevant since in many
AMINOACYLATION
viral RNAs a modified structure has been detected at the 5’-terminus, which is susceptible to periodate oxidation (cf., Muthukrishnan et al., 1975; Zimmern, 1975). To rule out such a possibility, 5 to 10 nucleoside residues were removed from the 3’terminus of TMV RNA by limited phosphorolysis with E. coli polynucleotide phosphorylase. This treatment also eliminated the histidine-acceptor capacity of TMV RNA. The specificity of the highly purified exonuclease used in this experiment excludes any possibility of damage to other parts of the viral-RNA molecule and enabled us to determine the precise location of the aminoacylated site, which is the 3’-terminus of the TMV RNA. In contrast to the phenylalanine-acceptor capacity of tRN&,,d, both TMV RN&-m and tRN&x+.ed were unable to accept histidine (Fig. 7). It would be interesting to examine whether such a parallelism in response to the oxidation-reduction reactions would also hold for other plant virus RNAs, in which the corresponding isoaccepting tRN&, regains its aminoacylation capacity upon reduction. A case in point would be brome mosaic virus RNA. It has already been shown that BMV RN&, has lost its tyrosine-acceptor ability (Shih et al., 1974), but whether it will be regained upon reduction, similar to the situation in E. coli tRNA,,, (Tal et al., 1972), remains to be determined. A complete analogy between the loss of infectivity of TMV RNA,, TMV RN&X+.ed, and phosphorolyzed TMV RNA, and the reduction in their aminoacylation capacity was observed. These observations show that the same site is essential for maintaining both aminoacylation capacity and infectivity of the viral RNA, and may indicate that the viral aminoacyl-RNA is involved in the recognition site of viralRNA replicase. ACKNOWLEDGMENT This National 332220.
work was supported, Cancer Institute,
in part, Contract
by the U.S. No. NOICP
REFERENCES BABINET, C. (1967). A new tion of RNA polymerase.
method for the puriticaBiochem. Biophys. Res.
OF
TMV
RNA
83
Commun. 26, 639-644. CARRIQUIRY, E., and LITVAK, S. (1974). Further atudies on the enzymatic aminoacylation of TMV RNA by histidine. FEBS Lett. 38, 287-291. DAHLBERG, A. E., DINGMAN, C. W., and PEACOCK, A. C. (1969). Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels. J. Mol. Btil. 41, 139-147. DANIEL, V., SARID, S., and LITTAUER, U. Z. (19701. Bacteriophage induced tRNA in E. coli. Science 167, 1682-1688. HAENNI, A.-L., PROCHIANTZ, A., BERNARD, O., and CHAPEVILLE, F. (1973). TYMV valyl-RNA as an aminoacid donor in protein biosynthesis. Nature New Biol. 241, 166-168. HALL, T. C., SHIH, D. S., and KAESBERG, P. (1972). Enzyme mediated binding of tyrosine to BMV RNA. Biochem. J. 129, 969-976. LITTAUER, U. Z. (1971). The synthesis of peptidylaminoacyl-tRNA and its use as a method for the purification of tRNA. In “Methods in Enzymology” (K. Moldave and L. Grossman, eds.), Vol. 20, p. 70. Academic Press, New York. LITTAUER, U. Z., SALOMON, R., SOREQ, H., FLEISCHER, G. and SELA, I. (1975). Characterization of the aminoacylated region at the 3’-terminus of TMV RNA. Proceedings of the 10th FEBS Meeting, 133-139. LITVAK, S., CARRE, D. S., and CHAPEVILLE, F. (19701. TYMV RNA as a substrate of the tRNA nucleotidyltransferase. FEBS Lett. 11, 316-319. LITVAK, S., TARRAGO, A., TARRAGO-LITVAK, L., and ALLENDE, J. E. (1973). Elongation factor-viral genome interaction dependent on the aminoacylation of TYMV and TMV RNA’s. Nature New Biol. 241, 88-90. LOWRY, 0. H., RO~EBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MUTHUKRISHNAN, S., BOTH, G. W., FURUICHI, Y., and SHATKIN, A. J. (19751. 5’ Terminal ‘I-methylguanosine in eukaryotic mRNA is required for translation. Nature (London) 255, 33-37. OBERG, B., and PHILIPEON, L. (1972). Binding of histidine to TMV RNA. Biochem. Biophys. Res. Commun. 48, 927-932. PEACOCK, A. C., and DINGMAN, C. W. (1968). Molecular weight estimation and separation of RNA by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7, 668-674. PINCK, M., YOT, P., CHAPEVILLE, F., and DURANTON, H. M. (1970). Enzymatic binding of valine to the 3’ end of TYMV RNA. Nature (London) 226, 954956. PINCK, M., CHAN, S. K., GENEVAUX, M., HIRTH, L., and DURANTON, H. (1972). Valine specific tRNAlike structure in RNA’s of two viruses of TYMV groups. Biochimie 54, 1093-1094.
a4
SALOMON
A., and HAENNI, A-L. (1973). TYMV RNA as a substrate of the tRNA maturation endonuclease. Nature New Biol. 241, 168-170. SALOMON, R., and LITTAUEB, U. Z. (1974). Enzymatic acylation of histidine to mengovirus RNA. Nature PROCHIANTZ,
(London) SELA, I.
249, 32-34.
(1972). Tobacco enzyme-cleaved fragments of TMV RNA specifically accepting serine and methionine. Virology 49, 90-94. SHIH, D. S., KAEBBERG, P., and HALL, T. C. (1974). Messenger and aminoacylation functions of BMV RNA after chemical modification of 3’-terminus. Nature
(London)
249, 353-355.
SOREQ,H., NUDEL, Il., SALOMON, R., REVEL, M., and LIT~AUER, U. Z. (1974). In vitro translation of poly(A)-free rabbit globin mRNA. J. Mol. Biol. 88, 233-245. STEINSCHNIEDER, A., and FRAENKEL-CONRAT, H. (1966a). Studies of nucleotide sequences in TMV RNA III. Periodate oxidation and semicarbazone
ET
AL.
formation. Biochemistry STEINSCHNIEDER, A.,
5, 2729-2734.
and FRAENKEL-CONRAT, H. (1966b). Studies of nucleotide sequences in TMV RNA. IV. Use of aniline in stepwise degradation.
Biochemistry 5, 2735-2743. TAL, J., DEUTSCHER, M. P.,
and LITTAUER, U. Z. (1972). Biological activity ofE. coli tRNAPhe modified in its C-C-A-terminus. Eur. J. Biochem. 28, 478-491. YOT, P., PINCK, M., HAENNI, A-L., DURANTON, H. M., and CHAPEVILLE, F. (19701. Valine specific tRNA-like structure in TYMV RNA. PFOC. Nat. Acad. Sci. USA 67, 1345-1352. ZIMMERMAN, S. B., and SANDEEN, G. (1966). The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal. B&hem. 14, 269-277. ZIMMERN, D. (1975). The 5’ end group of tobacco mosaic virus RNA is m7GS1,,,5’G,. Nucleic Acid Res. 2. 1169-1201.
VIROLOGY
Enzymatic R. SALOMON, Department
(1976)
Acylation I. SELA,’
of Biochemistry, Agriculture,
The Virus
of Histidine
to Tobacco
H. SOREQ, Weizmann Laboratory, Accepted
D. GIVEON,
Mosaic Virus RNA AND
U. Z. LITTAUER
Institute of Science, Rehovot, Israel; The Hebrew University, Rehovot, November
and ’ The Faculty Israel
of
24, 1975
A method for the purification of rat liver histidyl-tRNA synthetase was developed. The enzyme, freed of trace nuclease contamination by affinity chromatography on DNASepharose, could catalyze the aminoacylation of tobacco mosaic virus (TMV) RNA with histidine. The aminoacylation reaction of TMV RNA was inhibited by KCl, while that of rat liver tRNA exhibited a maximum rate at 140 mM KCl. TMV N-acetyl PHlhistidylRNA was prepared in order to stabilize the TMV histidyl-RNA ester bond. The TMV Nacetyl [3Hlhistidyl-RNA showed a migration identical to that of unacylated TMV RNA upon electrophoresis in polyacrylamide agarose gels. Removal of 5 to 10 nucleoside residues from the 3’-terminus using Escherichia coli polynucleotide phosphorylase or oxidation of the 3’-terminal ribose with periodate (to produce TMV RNA,,,,) eliminated the aminoacylation capacity of TMV RNA as well as its infectivity in tobacco leaves. It is concluded that the aminoacylation site is located at the 3’-terminal adenosine of TMV RNA. Fragmentation of TMV RN&,, with a crude rat liver enzyme did not expose additional sites for acylation with histidine, nor did it produce acylation capacity for other amino acids. The reduction of TMV RN&. with sodium borohydride converted the terminal dialdehyde group to a dialcohol-lacking covalent bond between the C2’ and CB’ of the terminal adenosine, but did not restore its ability to accept histidine or its infectivity. Similar reduction of rat liver tRN&,, to tRNA,,,,,,,, however, did restore its ability to accept phenylalanine while activity for histidine was not regained.
et al ., 1972) the precise site of aminoacylation of plant viral-RNA molecules has not been determined. Moreover, turnip yellow mosaic virus RNA is cleaved by the tRNA maturation enzyme to yield a 3’-terminal fragment of about 111 nucleosides, which retains its valine-acceptor capacity (Prochiantz and Haenni, 1973). In addition, as will be shown in this study, partially purified aminoacyl-tRNA synthetase preparations still contain enough endonucleolytic activity to fragment TMV RNA during the histidine acylation reaction. Thus, the possibility that enzymatic cleavage of TMV RNA is required prior to the aminoacylation reaction cannot be ruled out. It seemed important, therefore, to determine whether the original 3’-terminal adenosine of TMV RNA is the site for histidine attachment, or whether this site resides internally in the molecule and must be exposed to enzymatic cleavage before ami-
INTRODUCTION
RNA obtained from several plant viruses (Pinck et al., 1970, 1972; Yot et al., 1970; Oberg and Philipson, 1972: Hall et al., 1972; Carriquiry and Litvak, 1974; Salomon and Littauer, 19741, as well as from mengovirus (Salomon and Littauer, 19741, can be enzymatically aminoacylated with eukaryotic aminoacyl-tRNA synthetase preparations. The similarity of these aminoacylation reactions to that exhibited by tRNA, and the ability of plant virus RNA to interact with several other tRNA specific enzymes (Yet et al., 1970; Litvak et al., 1970, 1973; Haenni et al., 1973; Prochiantz and Haenni, 1973) may indicate that a tRNA-like structure is covalently linked to the genomic RNA. Although the esterification reaction appears to occur with a relatively intact RNA molecule (Pinck et al., 1970; Yot et al., 1970; Oberg and Philipson, 1972; Hall 74 Copyright All rights
6 1976 by Academic F’ress, Inc. of reproduction in any form reserved.
AMINOACYLATION
noacylation can take place. In the present report, we examine the acylation reaction of TMV RNA with histidine. As TMV RNA is more sensitive to nuclease attack than tRNA, it was of vital importance to acquire a nuclease-free histidyl-tRNA synthetase preparation. Such an enzyme was prepared, allowing us to demonstrate that no enzymatic modification of TMV RNA is necessary prior to its acylation with histidine. We also found a correlation between the histidine acylation capacity of TMV RNA and its infectivity. MATERIALS
AND
METHODS
TMV (strain vulgare) was grown in tobacco plants (N: tubacum cv Samsun). The virus particles were isolated by repeated centrifugation cycles and purified by banding in a CsCl gradient (Salomon and Littauer, 1974). The RNA was then isolated from the virus suspension by phenol-chloroform extraction (Littauer, 1971). The integrity of the TMV RNA chain was checked by polyacrylamide-agarose gel electrophoresis. Nuclease-free polynucleotide phosphorylase was purified as previously described (Soreq et al., 1974) and exhibited a sp act of 250 unitsimg of protein. Carrier-free and polyphosphate-free [32Plorthophosphate (100 mCi/ml) was purchased from the Nuclear Research Center, Negev, Israel. L3Hlhistidine (58 Ci/mmole) and [14C]histidine (342 mCi/mmole) were obtained from Radiochemical Center, Amersham, England. Preparation of DNA-Sepharose. The binding of DNA to Sepharose 4B was accomplished through a modification of the method of N. D. H. Rosa, J. Kallos, and P. B. Sigler (personal communication). Salmon sperm DNA (highly polymerized sodium salt, ICN, Cleveland, Ohio) was dissolved in 0.02 M Tris-HCl, pH 7.5, and precipitated with 2 vol of ethanol. The precipitate was redissolved in 80% N,N’-dimethylformamide (DMF) solution (BDH, Poole, England), containing 0.1 M NaHCO,, 0.02 M Tris-HCl, pH 7.5, to a final concentration of 1.0 mg/ml, and the DNA was denatured by heating to 50” for 5 min. Sepharose 4B (Pharmacia) was washed with 10 vol of cold water, collected
OF
TMV
RNA
75
on a sintered glass filter, and suspended in 2 vol of cold water, the pH of the solution was adjusted to 11 by the addition of 10 N NaOH while stirring at 0”. Cyanogen bromide (20 g) was dissolved in 20 ml of DMF and was added to 200 ml of the Sepharose suspension. The suspension was constantly stirred, and the pH was maintained at 11 by continuous dropwise addition of 10 N NaOH. The whole procedure was performed in an ice bath, and the temperature was kept under 6”. When the decrease in pH ceased, the activated Sepharose was immediately washed with 6 vol of cold water and 5 vol of 0.1 M NaHCO,. Four volumes of denatured DNA solution (1 mg/ml) in 80% DMF containing 0.1 M NaHCO, were added to the ’ activated Sepharose suspension, and the mixture was slowly stirred overnight at 2”. The DNA-Sepharose was washed with 5 vol of 5 M NaCI, and the amount of DNA fixed was calculated by subtraction of the eluted material from the amount of DNA used. With this procedure, between 2 to 5 mg of DNA were fixed per milliliter of Sepharose. The DNA-Sepharose column was washed with 5 M NaCl until no further A,,, could be eluted. The free active groups left on the Sepharose were blocked by washing with 3 vol of 0.05 M ethanolamine, and the suspension was kept until use in 0.02 M Tris-HCl buffer, pH 7.5, containing 2 mM sodium azide. After use the column was regenerated with 2 vol of 0.1 N NaOH and equilibrated with 5 vol of elution buffer. Optimum conditions for enzyme adsorption by the DNA-Sepharose column were obtained when the DNA was first heated for 5 min at 50” and then rapidly cooled at a concentration of 1 mg/ml. Denaturation of the DNA in aqueous solutions or the use of DNA solutions at concentrations higher than 1 mg/ml considerably decreased the amount of DNA that could be fixed to Sepharose. When the average DNA chainlength was reduced to 4 x lo4 daltons by ultrasonication (as measured by polyacrylamide-agarose gel electrophoresis), the enzyme was not adsorbed on the DNASepharose column, although the amount of DNA bound to the Sepharose was markedly high.
76
SALOMON
Preparation of histidyl-tRNA synthetase. All steps were performed at 4”. Mouse
or rat liver was washed thoroughly with a solution containing 10 n&f Tris-HCl, pH 7.5, 10 mM MgC12, 60 mM NH&l, 10 miV reduced glutathione, and 10% glycerol. The livers were homogenized for 15 set in an Ultra Turrax (Ika Werk, Breisgau, Germany) high-speed homogenizer with 3 vol of the same buffer solution. The homogenate was centrifuged for 10 min at 13,000g and then for 3 hr at 100,OOOg. The supernatant solution (S-100) was subjected to phase partition according to the procedure of Babinet (1967). The S-100 solution (600 ml) was mixed with 150 ml of a solution containing 10.6 g of dextran T500 (Pharmacia), 42.6 g of polyethylene glycol 6000 (Uniroyal), and 7.5 ml of 1.0 M TrisHCl buffer, pH 7.5, previously dissolved by boiling. The mixture was stirred for 40 min, centrifuged for 10 min at 13,000 g, and the supernatant was designated Phase I and was subsequently discarded. The pellet was suspended in a 70-ml solution containing 46 ml of 13.8% polyethylene glycol 6000, 1.4 ml of 1.0 M Tris-HCl, pH 7.5, 3.4 ml of 0.2 M reduced glutathione, and 19.2 ml of water. Sixteen grams of NaCl was then added to bring the salt concentration to 4 M, and the suspension was stirred for 30 min. After centrifugation for 10 min at 13,000 g, the supernatant (Phase II) was dialyzed for 3 hr against three changes of 1 liter of a solution containing 10 n-&f TrisHCl, pH 7.5; 1 miV reduced glutathione, and 10% glycerol, and the reduced glutathione concentration was adjusted to 50 mM. The dialyzed Phase II solution (120 ml) was diluted with 1 vol of a solution containing 10 m&f potassium phosphate buffer, pH 7.5, 1 m&f MgC&, 0.1 m&f EDTA, 10 mM reduced glutathione, and 5% glycerol, and applied on a DEAE-cellulose column (3 x 10 cm) pre-equilibrated with the same buffer. The column was washed with 100 ml of the same buffer, and the enzyme was eluted from the column with a 0.01 - 0.36 M (50-ml each) linear gradient of potassium phosphate buffer, pH 7.5, containing 1 mM MgC12, 0.1 n-& EDTA, 10 n&f reduced glutathione, and 5% glycerol, and 2-ml fractions were
ET AL.
collected. Histidyl-tRNA synthetase activity was recovered between 0.15-0.25 M, and the enzyme fraction was dialyzed for 1 hr against 2 liters of 10 mM potassium phosphate buffer, pH 6.8, containing 1 n&f reduced glutathione and 10% glycerol. The reduced glutathione and glycerol were adjusted to final concentrations of 10 mM and 50%, respectively, and the enzyme fraction (87.5 ml) was kept at -20”. Ten milliliters of the above enzyme preparation was diluted with 1 vol of 10 n&f potassium phosphate buffer, pH 6.8, containing 0.1 n-&f DlT and 10% glycerol, and the solution was applied to 2 x 4-cm hydroxylapatite column (Bio-Rad), pre-equilibrated with the same buffer. The column was washed with 12 ml of the application buffer, and then eluted stepwise with 5-ml each of 0.1 M, 0.15 M, 0.20 M phosphate buffer containing DTT and glycerol. The enzyme activity was eluted with 0.25 M phosphate buffer containing D’M’ and glycerol. The histidyl-tRNA synthetase was purified about 145-fold and showed a final sp act of 77 units/mg of protein (Table 1). To remove trace amounts of nucleases, the enzyme preparation (12 ml) was diluted fourfold with 36 ml of 10 m&f TrisHCl buffer, pH 7.7, containing 0.1 mM EDTA, 0.1 n-&f DTT, and 5% glycerol. It was immediately applied to a 2 x 7.5-cm column of DNA-Sepharose. The column was washed with 20 ml of the above buffer. Histidyl-tRNA synthetase activity was eluted between 1.0-1.5 M NaCl, with a 50ml linear salt gradient of O-2 M NaCl in 0.01 M Tris-HCl buffer, pH 9.0, 0.1 miV DTT, 0.1 n&f EDTA, and 5% glycerol. The very dilute enzyme preparation obtained was extremely labile and was stabilized by TABLE PURIFICATIONOF
1
HwrmyL-tRN.4
SYNTHETASE
Fractions
Proteins hnghnl)
Total mtivity (units)
Specific activity (units/ mg of pmtein)
s-100 Phase II DEAE-cellulose Hydroxylapatite DNA-Sepharose
7.4 1.8 0.35 0.055 0.0055
2330 1100 550 372 151
0.5 5.1 18 71 69
AMINOACYLATION
the addition of iodoacetate-treated bovine serum albumin (Zimmerman and Sandeen 1966) and glycerol to a final concentration of 0.5 mg/ml and 50%, respectively. The iodoacetate treatment of serum albumin was necessary to inactivate its nuclease contamination. Enzyme assay. The reaction rate was measured in an incubation mixture (0.05 ml) containing 40 m&f Tris-HCl, pH 7.5, 2 mM ATP, 10 mM MgC12, 3 mM reduced glutathione, 140 n&f KCl, 0.5 &i of 13Hlhistidine (58 Ci/mmole) or 0.1 &i of [14Clhistidine (342 mCi/mmole), 30 pg of tRNA, and 0.002-0.01 units of enzyme. Incubation was carried out at 30” for 10 min, and the reaction was stopped by immersing the tubes in an ice bath; 0.5 ml of bovine serum albumin solution (0.25 mg) and 7 ml of cold 5% trichloroacetic acid were then added. The precipitate was filtered through glass fiber filters (Whatman GF/C), washed three times with cold 5% trichloroacetic acid, and counted in a scintillation counter with toluene-based scintillation fluid. One unit of enzyme is defined as the amount which incorporates 1 nmole of histidine in 60 min, and the specific activity is expressed as enzyme units per milligram of protein. When the extent of the aminoacylation was assayed, the incubation mixture contained 5-15 pg of tRNA and 0.01-0.06 units of enzyme, and the reaction was carried out for 20 min at 30”. The histidine acceptance capacity of TMV RNA was assayed in the absence of KC1 in the reaction mixture. N-acetylation
of TMV
histidyl-RNA.
The ester bond of TMV histidyl-RNA was stabilized by treatment with N-hydroxysuccinimide acetylester to form TMV Nacetylhistidyl-RNA as previously described (Daniel et al., 1970; Salomon and Littauer, 1974). Protein determination. Protein was determined by the method of Lowry et al. (19511, with crystalline bovine serum albumin as the standard. Dilute protein solutions were concentrated prior to protein determination by the addition of 2 vol of ethanol and precipitation overnight at -20”. The precipitate was collected by cen-
OF
TMV
77
RNA
trifugation and dissolved in 0.01 ml of 0.1 N NaOH by incubating for 30 min at 37”. Preparation
of RNA,,
and RNA,,,.
One milliliter of TMV RNA or tRNA solution (2.2 mg/ml) was supplemented with 0.15 ml of 1 M sodium acetate buffer, pH 5.0, and then mixed with 0.35 ml of freshly prepared 0.1 M NaIO,. The solution was kept in the dark for 30 min at room temperature and precipitated with 0.3 ml of 5 M NaCl followed by 3.0 ml of ethanol. Excess periodate in the ethanolic supernatant was titrated with ethylene glycol (Tal et al., 1972). Oxidized TMV-RNA and tRNA were reduced by the addition of 1 M NaBH, to a final concentration of 0.07 A4 and 0.25 M Tris-HCl, pH 8.0. This mixture was incubated at room temperature until bubbles ceased to emerge (about 90 min), and the RNA was precipitated with ethanol. Phosphorolysis assay of TMV RNA. The reaction mixture contained 20 pg of TMV RNA, 30 m&f Tris-HCl buffer, pH 8.0, 1 mM Na,EDTA, 10 mM MgCl*, 20 n-&f potassium phosphate buffer (pH 8.0), containing carrier-free [32P]orthophosphate (about lo* cpm/pmole), 5 pg of purified polynucleotide phosphorylase (1.25 units), and 5 mM dCDP, in a final volume of 30 ~1. The mixture was incubated at o”, and at various time intervals 5-~1 aliquots were removed and applied onto DEAE-cellulose paper sheets (Whatman DE 811, and the 32P-labeled nucleoside diphosphates were chromatographed as previously described (Soreq et al., 1974). Preparation of phosphorolyzed TMV RNA. The reaction mixture was as de-
scribed above except for the omission of ls2Plorthophosphate and the addition of 160 pg of TMV RNA, and 20 pg of purified polynucleotide phosphorylase (5 units), in a final volume of 270 ~1. The mixture was incubated at 0”. At various time intervals, 40-~1 aliquots were removed, and 2 vol of 95% ethanol were added to precipitate the RNA. Infectivity assay of TMV RNA. Serial dilutions of intact TMV RNA were applied to half leaves of Nicotiana glutinosa L. Five microliters of the corresponding dilutions of different treated RNA samples
78
SALOMON
were applied to the opposite half leaves. In addition, samples of each preparation were incubated at 37” with a solution containing 0.15 M NaCl, 0.015 M trisodium citrate, and 10 pg/ml of pancreatic-RNAse extracted with phenol and then applied to another set of half leaves. This was done to rule out the possibility that positive infectivity could be due to the presence of residual whole TMV molecules in the RNA preparation. Polyacrylamide-agarose resis. Gel electrophoresis
ET AL.
Mgz+ concentration required for acylation of tRNA and TMV RNA with histidine. On the other hand, KC1 inhibited the reaction with TMV RNA. Figure 2 demonstrates that at 20 mM KCl, the aminoacylation of
gel electropho-
was performed according to Peacock and Dingman (1968), and the gels were stained with “stains all” (Dahlberg et al., 1969). RESULTS
Properties of purified enzyme. TMV RNA was shown to be aminoacylated with histidine by extracts from either yeast or KB cells @berg and Philipson, 1972; Carriquiry and Litvak, 1974). In comparable tests carried out in this laboratory, rat and mouse liver extracts were found to contain aminoacylation activity while E. coli MRE 600 extract was totally inactive (Salomon and Littauer, 1974). However, since the histidyl-tRNA synthetase preparation did contain endonucleotic activity (see below), it was impossible to dissociate the aminoacylation reaction of TMV RNA with histidine from the fragmentation reaction. A prerequisite for the separation of these two processes is the acquisition of nucleasefree histidyl-tRNA synthetase. Such an enzyme was purified from rat liver extracts employing phase partition, DEAEcellulose, and hydroxylapatite chromatography, as well as affinity chromatography, on a DNA-Sepharose column (Table 1). Although the affinity chromatography step did not enrich the enzyme preparation, it was found to be essential for final removal of trace amounts of nucleases. With this enzyme preparation, the requirements for the TMV RNA aminoacylation reaction were characterized and compared with those for tRNA. The optimal pH range for histidine incorporation with both types of RNA was found to be broad between 7.5-8.5. As shown in Fig. 1, there was no essential difference in the optimal
FIG. 1. Effect of MgCl, on acylation to TMV RNA and mixtures contained 35 wg of purified enzyme, the indicated tions, and were incubated for
the rate of histidine tRNA. The reaction RNA, 0.003 units of MgCl, concentra10 min at 30”.
FIG. 2. Effect of KC1 on the rate of histidine acylation to TMV RNA and tRNA. The reaction mixture contained 35 pg of RNA, 0.003 units of purified enzyme, the indicated KC1 concentrations, and was incubated for 10 min at 30”.
AMINOACYLATION
TMV RNA was inhibited to 50%, whereas reaction with tRNA was stimulated by KC1 with an optimal concentration at 140 mM. Figure 3 shows a time curve for TMV RNA aminoacylation using the purified enzyme preparation. The extent of histidine acylation of TMV RNA was about 0.3 mole/mole of RNA, which is similar to previously reported values (Carriquiry and Litvak, 19741, which were attained with crude yeast enzyme preparations. The extent of histidine acylation could not be increased by lowering the temperature or pH of the reaction mixture. The reason for incomplete acylation of TMV RNA with histidine has been recently shown to be due to a heterogeneity of the TMV RNA population, some of the RNA chains being defective at the 3’-terminus (Littauer et al., 1975). Presence of nuclease contamination in the purified enzyme preparation. In pre-
vious studies,
the size of the aminoacylI
I
I
.
TMVRNP
TMV
I
RNAor
,./--Al, I IO
I Time
I
I 20
(min)
FIG. 3. The effect of periodate oxidation on the aminoacylation capacity of TMV RNA. 0 - 0, Untreated TMV RNA; A-A, TMV RNA,,,; 0- - -0, a 1:l mixture ,of TMV RNA and TMV RNA,. The extent of reaction was measured with 15 fig of RNA and 0.06 units of purified enzyme. In the latter case, the attachment of histidine was expressed as Wlhistidine, cpm/Fg of unoxidized TMV RNA present in the above mixture.
OF
TMV
RNA
79
FIG. 4. Polyacrylamide-agarose gel electrophoresis of TMV A’-acetyl [3H]histidyl-RNA prepared with a partially purified histidyl-tRNA synthetase. TMV RNA was aminoacylated with 13H]histidine with partially purified rat liver enzyme (DEAEcellulose fraction). The resulting [3H]histidyl-RNA was reacted with N-hydroxysuccinimide acetyl ester to yield N-acetyl [3H]histidyl-RNA. About 10 pg of N-acetyl 13H1histidyl-RNA was applied to 1.7% polyacrylamide-0.5% agarose slab gel, and electrophoresis was carried out for 2.5 hr at 0” at 11.4 V/cm. Radioactivity was measured on l-mm slices as previously described (Salomon and Littauer, 1974). In adjacent slots untreated TMV RNA, E. coli rRNA and tRNA markers were run simultaneously.
ated RNA molecule has been estimated by sucrose gradient centrifugation or gel IXtration (Oberg and Philipson, 1972; Litvak et al., 1973). In our work we preferred to use acrylamide gel electrophoresis. For electrophoresis, the ester bond of TMV histidyl-RNA was stabilized by treatment with N-hydroxysuccinimide acetylester to form TMV N-acetyl-histidyl-RNA (Salomon and Littauer, 1974). Using ZV-acetyl [3Hlhistidyl-RNA molecules, we have been able to estimate their size by monitoring the radioactivity of the sliced gel. Figure 4 shows the electrophoretic migration of TMV RNA which was aminoacylated with 13Hlhistidine using a partially purified enzyme preparation (DEAE-cellulose fraction). The labeled product migrated as a broad band with an apparent molecular weight between 0.4 x lo5 to 1.8 x 10”. It is obvious that considerable degradation of the TMV RNA chains took place during the aminoacylation reaction. On the other hand, when the purified enzyme preparation (DNA-Sepharose fraction) was employed for the aminoacylation of TMV RNA, the labeled N-acetylhistidyl-RNA corn&rated with the marker TMV RNA as demonstrated by its staining
80
SALOMON
profile (Fig. 5). Thus, no fragmentation of TMV RNA occurred during the aminoacylation with the purified enzyme preparation. Oxidation and reduction of TMV RNA. Although size determinations by polyacrylamide gel electrophoresis are very accurate (Peacock and Dingman, 1968), they do not exclude the possibility that a few nucleoside residues are cleaved, at or near the 3’-terminus. In order to determine whether the 3’-terminus of TMV RNA is indeed the site for histidine aminoacylation, we employed a chemical approach, in which the RNA was oxidized with sodium periodate. The 3’4erminal sequence of TMV RNA has been determined to be GCCCA-OH (Steinschnieder and FrankelConrat, 1966a). Therefore, periodate will oxidize TMV RNA at the 3’-adenosine, the expected site of histidine attachment, to form the dialdehyde derivative (TMV RN&,). Figure 3 shows that oxidation with periodate abolished the aminoacylation capacity of TMV RNA as well as that of rat liver tRNA, which served as a control. Addition of TMV RN&, had no effect on the aminoacylation of untreated TMV RNA, thus showing that no excess periodate remained attached to the isolated TMV RNA,,. An aliquot of the oxidized TMV RNA was analyzed by polyacryl-
FIG. 5. Polyacrylamide-agarose gel electrophoresis of TMV N-acetyl [3H]histidyl-RNA prepared with a nuclease-free histidyl-tRNA synthetase. TMV RNA was aminoacylated with [$H]histidine by a nuclease-free rat liver histidyl-tRNA synthetase (DNA-Sepharose fraction) and treated as described in the legend to Fig. 4. The smooth line represents the optical density at 570 nm of the stained RNA (“stains all”), and the dotted line shows the radioactivity distribution.
ET AL.
Time(min)
FIG. 6. The effect of cleavage of TMV RNA and TMV RN&,, on histidine aminoacylation capacity. The reaction mixture contained 350 pg of untreated TMV RNA (O-O], or 320 Fg TMV RNA, (A-A), and 40 n&f Tris-HCl, pH 7.5, 2 n-&f ATP, 10 mM MgCl,, 3 u&f reduced glutathione, and 10 ~1 of Phase II extract. After incubation for 30 min at 37” the RNA was extracted with phenol-chloroform mixture and precipitated with 2 vol of ethanol in the presence of 1 M NaCl. The RNA precipitates were dissolved in water and aliquots were removed for the assay. The extent of reaction was measured with 15 pg of RNA and 0.06 units of purified enzyme.
amide-agarose gel electrophoresis to ascertain that the viral RNA was not degraded during the oxidation process, and this was found to be the case. Thus, the results of these experiments would support the notion that the aminoacylation site is located at the 3’-terminal adenosine. We have also attempted to determine whether fragmentation of TMV RNA with a crude rat liver RNase would increase the number of sites available for acylation with histidine or any other amino acids. We argued that if such sites exist, cleavage of oxidized TMV RNA, which itself is inactive, would expose these sites for aminoacylation. TMV RN&, and nonoxidized TMV RNA were incubated with a crude rat liver extract (Phase II fraction). Analysis by gel electrophoresis showed that both preparations were cleaved and migrated as a broad band between the 16 S RNA and the tRNA markers. Figure 6 shows a time
AMINOACYLATION
OF
curve for histidine acylation of cleaved TMV RNA,,, as compared with cleaved, nonoxidized TMV RNA. It is clear from this figure, as compared with Fig. 3, that in both RNA preparations no new sites for aminoacylation are exposed by the cleavage treatment. The TMV RN&, remained as inactive as before treatment, and the histidine-acceptor capacity of the nonoxidized TMV RNA was unaltered. In addition, cleaved TMV RNA did not show acceptor activity for amino acids other than histidine (data not shown). Reduction of tRN&, with sodium borohydride converts the terminal dialdehyde group to a dialcohol lacking a covalent bond between the CZ’ and Cs’ of the terminal adenosine. It is known (Tal et al., 1972) that some, although not all, of the isoaccepting tRN&, species regain their aminoacylation capacity upon reduction. It was therefore of interest to examine whether reduction of TMV RNA,, would restore its histidine-acceptance capacity. Figures 7A and B show that both TMV RNbweci and rat liver tRNAXTed were unable to accept histidine, whereas reduction of tRNA,,, did restore most of its phenylalanine-acceptor activity. Oxidized TMV RNA was previously
5
IO
15
5
IO
15
5
IO
15
Ttmehn)
FIG. 7. The effect of oxidation and reduction of TMV RNA on the histidine aminoacylation capacity. TMV RNA and rat liver tRNA were oxidized with NaIO,. A portion of the oxidized material was then reduced with NaBH, as described in Materials and Methods. (A) Aminoaclyation of TMV RNA with histidine. (B) Aminoacylation of rat liver tRNA with histidine. (C) Aminoacylation of rat liver tRNA with phenylalanine. 0 - 0, Untreated RNA, A - A, oxidized RNA; A-A, oxidized and reduced RNA. The extent of reaction was measured with 15 pg of RNA and 0.06 units of purified enzyme.
TMV
60
81
RNA I
I
I
I
FIG. 8. The effect of oxidation and reduction on the infectivity of TMV RNA. Five microliters of the indicated RNA solutions were applied to the half leaves.
found to be noninfective (Steinschnieder and Frankel-Conrat, 196613). These results were confirmed by us, as is shown in Fig. 8. In addition, reduction of TMV RNA,,, did not restore its infectivity to tobacco leaves. Limited
phosphorolysis
of TMV
RNA.
To rule out the possibility that periodate oxidation affected parts of the RNA molecule other than the 3’-terminus, we employed an enzymatic approach, in which a few nucleoside residues are removed from the 3’-terminus. The method is based on the ability of polynucleotide phosphorylase to phosphorolyse long polynucleotides starting at the 3’-end by a processive mechanism. Using a 1:l molar ratio of enzyme to substrate, a synchronous mode of phosphorolysis can be established. Inclusion of 13*Plorthophosphate in the phosphorolysis medium leads to an incorporation of the label into the p-phosphate moieties of the generated nucleoside diphosphates, the quantities of which enable the extent of phosphorolysis to be followed (Soreq et al., 1974). When TMV RNA was incubated with polynucleotide phosphorylase for 30 set at O”, between 5 to 10 nucleoside residues were removed from the 3’terminus. This treatment eliminated the infectivity of TMV RNA (Fig. 9) and also reduced its 13Hlhistidine-acceptor capacity to a value of 500 cpm/lO c(g of RNA, as compared with 27,000 cpm/lO ,ug for the untreated molecules.
82
SALOMON
FIG. 9. The abolition of infectivity by limited phosphorolysis of TMV RNA with polynucleotide phosphorylase. Phosphorolyzed TMV RNA was prepared by incubating TMV RNA with polynucleotide phosphorylase for 30 set at 0” as described under Materials and Methods. Five microliters of the indicated RNA solutions were applied to the half leaves. DISCUSSION
The overall requirements for histidine charging of both TMV RNA and tRNA were found to be similar, except that the presence of KC1 was shown to stimulate aminoacylation of tRNA, but strongly inhibited that of TMV RNA (Fig. 1). This may suggest that the binding between the TMV RNA and the enzyme is weak and, therefore, readily dissociated by increasing salt concentrations, perhaps due to a difference in the secondary and/or tertiary structure of the aminoacylation region, or a masking of this region by other parts of the TMV RNA molecule in the presence of salt. The high molecular weight TMV RNA molecules appear to contain regions which are highly resistant to nuclease degradation. During incubation with various rat liver enzyme preparations, these regions survive degradation, and the viral RNA is cleaved into long fragments. Depending on the enzyme fraction and incubation conditions, the size of the fragments may vary between 3 x lo4 to 1.8 x 10” daltons. Prolonged incubation with the crude rat liver extract (Phase II fraction) resulted in fragments with a discrete size range that migrated on gel electrophoresis in a range between the tRNA and 5 S RNA markers;
ET AL.
smaller oligonucleotides, however, were absent. Moreover, fragmented viral RNA has the same histidine-acceptance capacity as intact TMV RNA (Figs. 3 and 6). This indicates that the aminoacylation region of TMV RNA, like tRNA, is relatively resistant to nuclease action. It also suggests that no new sites for aminoacylation are exposed during the incubation period. The latter suggestion was reaffirmed by the absence of amino acid acceptance of fragmented TMV RN&, (Fig. 6). The situation may be different when TMV RNA is fragmented with a crude tobacco cell-free extract (Sela, 1972). The presence of nuclease contamination in aminoacyl-tRNA synthetase preparations made it impossible to distinguish between the cleavage and charging reaction, and no answer could be given as to the precise location and number of the aminoacylation site(s). For this reason, the above described preparation of a nucleasefree histidyl-tRNA synthetase became essential. Using the nuclease-free enzyme, we were able to show by gel electrophoresis that the aminoacylated TMV RNA corn&grates with intact TMV RNA, suggesting that the histidine-acceptor site is located at or near the 3’-terminus. To demonstrate more conclusively that the 3’-terminus of TMV RNA is indeed the only site for aminoacylation, periodate oxidation of the 3’-ribose was performed. It was found that periodate oxidation entirely abolished the aminoacylation capacity of TMV RNA. It should be noted that when a crude aminoacyl-tRNA synthetase preparation was employed, TMV RN&, molecules could partially regain their acceptor capacity. Apparently, under these conditions, replacement of the oxidized terminal adenosine does occur, thereby restoring some of the RNA chains to their original form. Thus, the examination of the amino-acid-acceptor capacity of periodate-oxidized RNA molecules can only be performed with relatively purified enzyme prhparations. The possibility that periodate can affect parts of the molecule other than the 3’terminus cannot be excluded a priori. This is particularly relevant since in many
AMINOACYLATION
viral RNAs a modified structure has been detected at the 5’-terminus, which is susceptible to periodate oxidation (cf., Muthukrishnan et al., 1975; Zimmern, 1975). To rule out such a possibility, 5 to 10 nucleoside residues were removed from the 3’terminus of TMV RNA by limited phosphorolysis with E. coli polynucleotide phosphorylase. This treatment also eliminated the histidine-acceptor capacity of TMV RNA. The specificity of the highly purified exonuclease used in this experiment excludes any possibility of damage to other parts of the viral-RNA molecule and enabled us to determine the precise location of the aminoacylated site, which is the 3’-terminus of the TMV RNA. In contrast to the phenylalanine-acceptor capacity of tRN&,,d, both TMV RN&-m and tRN&x+.ed were unable to accept histidine (Fig. 7). It would be interesting to examine whether such a parallelism in response to the oxidation-reduction reactions would also hold for other plant virus RNAs, in which the corresponding isoaccepting tRN&, regains its aminoacylation capacity upon reduction. A case in point would be brome mosaic virus RNA. It has already been shown that BMV RN&, has lost its tyrosine-acceptor ability (Shih et al., 1974), but whether it will be regained upon reduction, similar to the situation in E. coli tRNA,,, (Tal et al., 1972), remains to be determined. A complete analogy between the loss of infectivity of TMV RNA,, TMV RN&X+.ed, and phosphorolyzed TMV RNA, and the reduction in their aminoacylation capacity was observed. These observations show that the same site is essential for maintaining both aminoacylation capacity and infectivity of the viral RNA, and may indicate that the viral aminoacyl-RNA is involved in the recognition site of viralRNA replicase. ACKNOWLEDGMENT This National 332220.
work was supported, Cancer Institute,
in part, Contract
by the U.S. No. NOICP
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a4
SALOMON
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ET
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