Volume 3 no.10 October1976
Nucleic Acids Research
Identification of the minor guanylated tRNA of rabbit reticulocytes
Walter R. Farkas and David Chernoff* University of Tennesse Memorial Research Center, Knoxville, IN 37920, USA
Received 7 July 1976
ABSTRACT Two of the tRNA's found in rabbit reticulocytes are substrates for a post-transcriptional modification leading to the incorporation of guanine into the polynucleqtidv chain. The major guanylated tRNA was previously In the present report we show that the minor identified as tRNA H1-S and that just as in the case of tRNAWHis), guanylated tRNA is the ?uanine is located in an internal position. There are onlv two tRNA s8n) in reticulocytes. We further show that one of these, the'one that is not 1abz led with guanine, contains the hypermodified base known as Q. tRNA( en) does not contain Q. INTRODUCTION Incubation of rabbit, sheep and human reticulocytes with radioactive guanine results in the uptake of the guanine into tRNA (1,2). Since reticulocytes do not synthesize RNA (3), the labeling of the tRNA could only be explained by a post-transcriptional modification of tRNA (1,2). Indeed, an enzyme was found (4) and purified (5) that catalyzed the incorporation of guanine into tRNA. The requirements of the enzyme are simple. The only cofactor requirement is for a monovalent cation, a tRNA that is an acceptor and the base guanine (4). The reaction appears to proceed by the excision of a base from an internal position within the polynucleotide chain of tRNA and replacement of that base by guanine (4). The reaction is highly specific for the tRNA and also for a particular site within the tRNA (1). In reticulocytes, only two tRNA's undergo guanylation; (the major guanylated tRNA has been identified as tRNA(HI")) (1,2,4,5); the other quanylated tRNA has until now not been identified. In this renort we show that the minor guanylated tRNA of rabbit reticulocytes is tRNA(A2n) Reticulocytes contain three tRNA (Hi8); a major one, tRNA (Hi) which elutes early, a minor one, tRNA(Hia) which elutes afterwards during RPC-5 chromatogwhich precedes tRNA 2 rapy (1,4,5,11) and a very small peak,tRNA1 is found in rabbit liver. Smith et al. have but not tRNA(His), tRNA(His), 3 2 reported the identical situation for tRNA~~~~~~~(Asn) , namely two tRNA (Asn) in
iRNAWJ84),
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2521
Nucleic Acids Research reticulocytes. Of these, a major one elutes early and a minor one comes off but not tRNA(A8n), is found in rabbit liver (6). RPC-5 later. Again Because of these biochemical similarities and the fact that the position at which Smith and McNamara reported tRNA(A8n) to elute from RPC-5 was close to where the minor guanylated tRNA was eluted, we decided to test the possibility that tRNA(Aan) is the minor guanylated tRNA. His and Asn are part of the tetrad of amino acids whose cognate tRNA's contain the hypermodified base known as Q (7,8). The other two amino acids are Asp and Tyr. For these four amino acids, there exist two isoacceptors that arise from identical transcripts. These isoacceptors differ in that one form contains Q (a derivative of G) (8), and the other isoacceptor contains G in place of Q. In Drosophila the relative amounts of the Q form and the G form of these tRNA may be important for normal morphogenesis (8). In another report we showed that tRNA(His) contains Q (5). In this report we show that tRNA 1 and not the guanylated tRINA( contains Q. MATERIALS AND METHODS had the specific activities [8-14C]Guanine, [3H] and [ of 50 mCi/mmol, 37 Ci/mmol and 84 mCi/mmol respectively were purchased from Schwarz/Mann Corporation. The packing for RPC-5 columns was purchased from Miles Laboratories. The capacity of the resin to retain tRNA was unsatisfactory and it was recoated with adogen 464 (9). Preparation of radioactive guanylated tRNA and asparaginyl tRNA: In order to prepare [ 14C] guanylated tRNA, rabbit reticulocytes were incubated with [14 C]guanine as previously described (1,2). Asparaginyl tRNA was prepared by incubating rabbit reticulocytes (1.5 ml packed cells) in 0.015 M Tris, pH 7.4; 0.01 M KC1; 0.15 M NaCl, 0.09 M NaHCO3; 5.6 mM glucose 0.68 mM Ferrous ammonium sulfate; 3,000 u/ penicillin; 19 amino acids minus asparagine 0.5 mM each and 13 iiCi [IH]L-asparagine in a final volume of 7.8 ml. The cells were incubated for 20 min at 370. The cells incubated with [ and [3H]asparagine were pooled and transferred to an ice bath. One-half volume of 0.10 M sodium acetate pH 5.0, 0.10 M NaCl, 0.01 M EDTA was added and this was followed by the addition of two volumes of water-saturated phenol. The suspension was shaken vigorously for 5 min and then centrifuged for 10 min at 10,000 x g. The aqueous layer was collected and the phenol phase extracted with an equal volume of the pH 5.0 acetate buffer. The aqueous phases were pooled and 2.2 volumes of 95% ethanol was added and the RNA permitted to precipitate for at least 3 hr at -15°. The RNA was collected by centrifugation, dried in vacuo and dissolved in 1.0 M NaCl
tRNA(Asn),
).,
14C]asparagine
4C]guanine
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Nucleic Acids Research at 00. Insoluble material was removed by centrifugation at 10,000 x g for 15 min and diluted with 2 volumes of 0.01 M sodium acetate, pH 4.5; 0.01 M MgCl2; 0.002 M mercaptoethanol 0.40 M NaCl (RPC-5 buffer). Reversed phase-5 chromatography of tRNA: The solution containing the [14C] guanylated and [3H]asparaginyl tRNA's was layered onto an RPC-5 column 0.8 x 25 cm that had been equilibrated with 0.40 M NaCl dissolved in the RPC-5 buffer. The column was developed as previously described (5,9) with a linear gradient from 0.50 M to 0.70 M NaCl dissolved in the RPC-5 buffer. There were 50 ml in each chamber of the gradient maker and the pressure was maintained at 300 psi. Samples containing 1.1 ml were collected. The entire samples were counted as previously described (1,2) or in experiments where the tRNA was to be used for further experiments, 20 ul aliquots were pipetted onto 3 MM paper discs which were processed according to Bollum before
counting (10). Chromatography of tRNA on DEAE Sephadex: The preparation of [14C] guanylated and [3HlAsn tRNA was scaled up three-fold. The [3H)Asn tRNA peak which coincided with [14C] guanylated tRNA after RPC-5 chromatography was pooled and precipitated with 2 volumes of ethanol. The tRNA was collected and dissolved in 0.02 M sodium acetate pH 4.5; 0.008 M magnesium acetate; 0.30 M NaCl. The tRNA solution was layered onto a .5 x 25 cm column of DEAE-Sephadex A-25 equilibrated with this buffer at 4°. The DEAE-Sephadex column was developed with a linear gradient with 100 ml of the above buffer in the mixing chamber and 100 ml of 0.02 M sodium acetate pH 4.5; 0.003 M magnesium acetate; 1.0 M NaCl in the other chamber. The flow rate was 25 ml per hr and 3.25 ml fractions were collected. The fractions were prepared for scintillation counting as previously described (1,2). Reaction of Asn tRNA with periodate: Reticulocytes were incubated in one case with [14C]Asn and in the other with [3H]Asn. The incubation conditions described above were scaled up three-fold. The aminoacyl tRNA's were precipitated-with ethanol as described above. After precipitation with ethanol, the [3H] and [14C] tRNA's were each dissolved in 2 ml of 0.10 M sodium acetate pH 5.0. Aliquots were taken for counting; the [3H]Asn tRNA contained 57,600 cpm and the [14C]Asn tRNA 87,960 cpm. Sodium metaperiodate 0.10 M was added to the [14C]Asn tRNA to a concentration of 0.02 M. The [3HlAsn tRNA which acted as control received an equal volume of H20. The two solutions were maintained at 0° for 60 min with gentle stirring. The 10 reaction was stopped by the addition of sucrose to 1.0 M, and the [3H] and [14C labeled tRNA's were pooled. Low molecular weight impurities were 2523
Nucleic Acids Research removed by adsorbing the tRNA's to a DEAE-cellulose column (0.7 x 8 cm) equilibrated with 0.25 M NaCl; 0.01 M MgCl2; 0.001 M disodium EDTA adjusted to pH 4.9. The column was washed with 150 ml of this solution. The tRNA was eluted with 0.70 M NaCl dissolved in the same buffer. One ml fractions were collected and-the tRNA located by monitoring the column effluent at 260 nm. The fractions containing tRNA were pooled and diluted with an equal volume of RPC-5 buffer and the tRNA was now analyzed by RPC-5 chromatography. RESULTS RPC-5 chromatography of reticulocyte tRNA charged with [3H]asparagine and guanylated with [ 14Cguanine: Reticulocytes from the same rabbit were labeled with [14C]guanine in the presence of the 20 unlabeled amino acids and also with [.3 H]asparagine in the presence of 19 cold amino acids minus asparagine. The two incubation mixtures were placed in an ice bath, the pH adjusted to 5.0. The samples were then pooled and the tRNA extracted as described above. The pooled tRNA's were chromatographed on RPC-5, and as seen in Fig. 1, asparaginyl tRNA2 cochromatographed with guanylated tRNA1. Asn tRNA, 3000 _
2700
2400
-3 Ii
2100
z1800 _
(-1500 z
01200
900r 600 1
300
10
20
30
40
50
60
70
FRACTION NUMBER
Fig. 1: RPC-5 chromatography of
tRNA: After incubation cyte [IH]asparagine, the tRNA was column.
2524
(
[14C] guanylated reticulo[3H]asparaginyl, of reticulocytes with [14C]guanine and
extracted and chromato raphed on an RPC-5 ) is the [3H]asparagine, (---) is the guanine.
Nucleic Acids Research and guanylated tRNA2, which is in fact Ri8 tRNA3 (5), are also seen in Fig. 1. Chromatography of [3H]asparaqinyl tRNA2 and [ 14C] guanylated tRNAl on DEAE-Sephadex: The peak corresponding to asparaginyl tRNA2 - [14C] guanylated tRNA1 after RPC-5 chromatography was located and precipitated with 2 volumes of ethanol and chromatographed on DEAE-Sephadex. One-tenth volume of 90% trichloroacetic acid was added to each fraction and the precipitated tRNA's prepared for counting. As seen in Fig. 2, the [3H] and [14C] peaks 1000 _ 900 _ l l
800 _
II 700_ zz600
~-500
z
0
400 300 _
200
100
_ 10
20
30 40 50 FRACTION NUMBER
60
70
Fig. 2: Chromatography of pooled [3HJAon tRNA2: [14C] guanylated tRNAl on DEAE Sephadex. The [l C] and [3H]-containing fractions eluted from an RPC-5 column were located by taking 20 ul aliquots from each fractions. The fractions corresponding to [3Hl4sn tRNA2 and [14C]guanine tRNA1 were pooled, precipitated with two volumes of ethanol and chromatographed on DEAE Sephadex. (-) is the [3H]asparagine, (---) is the
[14C]guanine.
coincide, showing that reticulocyte guanylated tRNA1 is probably identical to tRNA(A8n). The reason that there are fewer [3H] counts than [14C] after DEAE-Sephadex chromatography is probably due to deacylation of Asn tRNA during the manipulation after RPC-5 chromatography. The guanine residue incorporated into tRNA(2Sn) is in an internal position: The guanylated tRNA labeled with [14C]guanine was located after elution from an RPC-5 column and the first peak was then digested with 0.3 M KOH at 2525
Nucleic Acids Research
370 for 18 hr. The digest was neutralized with Dowex 50 (H+) and then spotted onto Whatman 3 MM paper. The paper was developed with a solvent that separates molecules on the basis of charge (11). As seen in Fig. 3, all of the radioactive guanine incorporated in 250 Z 200 %%150 z 100 50-
0
OQCG 0
2
CD
2',3'GMP 4
6
8
Guo
10 12 14 16 18 20 22 24 26
C M FROM ORIGIN Fig. 3: The guanine residu is incorporated into an internal position in the tRNA: The first ['4C] guanylated tRNA to elute from an RPC-5 column was hydrolyzed with 0.3 M KOH for 18 hr at 370 and then neutralized with Dowex 50 H+. After the resin was removed by filtration, the filtrate was concentrated to 0.1 ml at 370 in a stream of dry N2. The sample was streaked onto Whatman 3 MM paper. Appropriate markers, GR, GMP and pGp were also spotted. The chromatogram was developed with ethanol: 1 M ammonium acetate, 70:30. After drying, the markers were located under mineral light and the track containing the hydrolyzed tRNA was cut into 1 cm strips which were placed into scintillation vials and counted.
tRNA(Asn) was converted to material that cochromatographed with 2',3' GMP after alkaline digestion, indicating that the guanine was incorporated into an internal position within the polynucleotide chain (2). Were the guanine incorporated into the 3' position, the radioactivity would be in the nucleoside region of the chromatogram. If the guanine were incorporated at the 5' end, the label would have been found in pGp. Periodate oxidation of Aen tRNA: Since Asn is one of the four amino acids whose cognate tRNA's contain either guanine or Q in the first position of the anticodon, it was of importance to determine which of the two tRNA(A8n) contained Q. In order to distinguish between the Q form and G form, we took advantage of the fact that Q contains vicinal hydroxyl groups and, therefore, reacts with IOi. 4C]An tRNA and [3H]Asn tRNA were prepared and the tRNA reacted with IOi. The was treated identically but Io4 was not added. The two Aen tRNA's were then pooled and analyzed on an RPC-5 column. Fig. 4 shows that IO caused a disappearance of Ain tRNA1, but
[14C]Asn
2526
[3H]Asn
Nucleic Acids Research 900 _
_ 450
800 _
"
400
700 _
-
350
600 _
-
300!
5 500 _
2505
F 400
_ 200F
I
200
j
II
100
100
~~~~~0
50
5
10 IS 20 25 30 35 40 45 50 55 60 65 70 75 FRACTION NUMBER
Chromatography of periodate-treated Asn tRNA on RPC-5: [14C] and [3H]Asn tRNA were prepared by incubating reticulocytes with [14C] and [3H]Asn respectively. The ['4C]Asn tRNA was treated with IOi, the [3H]Asn tRNA served as a control. The two labeled tRNA's were pooled and chromatographed on RPC-5. (---) represents [14C] IOi-treated tRNA. ( -) represents the [3H] control.
Fig: 4:
not of Asn tRNA2, indicating that tRNA(Ain) is probably the Q-containing form in reticulocytes. If the Q forms and G forms of tRNA are homogeneic, the Q form would be expected to elute from RPC-5 columns before the G form, due to the extra positive charge imparted by the secondary amino group contained in Q (7).
DISCUSSION The fact that the minor guanylated reticulocyte tRNA has turned out to be specific for asparagine is especially interesting, since along with histidine (the major guanylated reticulocyte tRNA), they are part of the tetrad of amino acids whose cognate tRNA's contain Q. For each of these amino acids, there are two major isoaccepting tRNA's that are homogeneic. One of the two isoacceptors (the one that elutes early from RPC-5 columns) contains Q; the other isoacceptor contains G in place of Q. Nishimura et al. have postulated that the guanylating enzyme may be responsible for converting the Q form of tRNA to the G form (7). DuBrul and Farkas have, however, shown that the Q form of tRNA(Hia) is not the substrate for the guanylating enzyme. It is the G form that is the substrate (5). At least in the reticulocyte the role of the guanylating enzyme is not to convert the Q form of tRNA's to the G form. The fact that the role of the guanylating enzyme is not to remove Q from within the polynucleotide chain and replace it with G is further indicated by the fact that yeast tRNA, which does not contain Q (12), is an excellent substrate for the guanylating enzyme (4) and that rabbit liver tRNA which contains exclusively the Q form and not the G form of tRNA(Asn) and 2527
Nucleic Acids Research
tRNA(Hi8)
is not a substrate for the guanylating enzyme (4). The tRNA's for Asn and His of mammalian cells appear to be closely related. In the reticulocyte there are two isoacceptors for each of these amino acids. [Actually, a very small peak which elutes just prior to the tRNA(Hi8) has been reported (5).] The more abundant form contains Q and elutes early from RPC-5 columns. The less abundant form which elutes after the major form is the one that undergoes the guanylation reaction (5). In liver there is only one tRNA (the Q form) for both of these amino acids (6). Furthermore, for each of these amino acids there are two codons which differ by a pyrimidine in the third letter CAU and CAC for His and MU and MC for Asn (13). At present there are three possibilities to be considered: 1) that the two reticulocyte tRNA's that are substrates in the guanylation reaction and also have an isoacceptor that contains Q may be merely coincidental; and 2) that the guanylation reaction may be one of a series of events in the posttranscriptional modification of a guanine residue leading to the synthesis of Q at the macromolecular level; and 3) that the two major tRNA for Asn and His are not the Q and G form but the Q form and the Q* form. Nishimura has shown that Q* is a derivative of Q found in mammalian cells but not in prokaryote tRNA (12). It is possible that the late eluting tRNAs for His and Asn contains Q* and not G and that the role of the guanylating enzyme is to replace Q*, and not Q, with G. ACKNOWLEDGEMENTS This work was supported by grant NP 186 from the American Cancer Society and GM 20546 from the U.S. Public Health Service. We also thank Mr. Tom Stanawitz and Mr. Don Lewis for expert technical assistance. *
Present address: Yale university, New Haven, CT, USA
REFERENCES 1 Farkas, W.R., Hankins, W.D. and Singh, R. (1973) Biochim. Biophys. Acta 294, 94-105 2 Hankins, W.D. and Farkas, W.R. (1970) Biochim. Biophys. Acta 213, 77-89 3 DeBellis, R.H., Gluck, N. and Marks, P.A. (1964) J. Clin. Invest. 43, 1329-1337 4 Farkas, W.R. and Singh, R.D. (1973) J. Biol. Chem. 248, 7780-7785 5 DuBrul, E.D. and Farkas, W.R. (in press) Biochim. Biophys. Acta 6 Smith, E.D.W., Meltzer, V.N. and McNamara, A.L. (1974) Biochim. Biophys. Acta 349, 366-375 7 Kasai, H., Ohashi, Z., Harada, F., Nishimura, S., Oppenheimer, N.J., Crain, P.F., Liehr, J.G., von Minden, D.L. and McCloskey, J.A. (1975) Biochemistry 14, 4198-4208 8 White, B., Tener, G.M., Holden, J. and Suzuki, D.T. (1973) J. Mol. Biol. 2528
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74, 635-651 Kelmers, A.D. and Heatherly, D.E. (1971) Anal. Biochem. 44, 486-495 Bollum, F. (1968) Methods in Enzymology XII, Part B, 169-172 Spahr, P.F. (1964) J. Biol. Chem. 239, 3716-3726 Kasai, H., Kuchino, Y., Nihei, K. and Nishimura, S. (1975) Nuc. Acid Res. 2, 1931-1939 Marshall, R.E., Caskey, C.T. and Nirenberg, M. (1967) Science 155, 820-826
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Nucleic Acids Research
Identification of the minor guanylated tRNA of rabbit reticulocytes
Walter R. Farkas and David Chernoff* University of Tennesse Memorial Research Center, Knoxville, IN 37920, USA
Received 7 July 1976
ABSTRACT Two of the tRNA's found in rabbit reticulocytes are substrates for a post-transcriptional modification leading to the incorporation of guanine into the polynucleqtidv chain. The major guanylated tRNA was previously In the present report we show that the minor identified as tRNA H1-S and that just as in the case of tRNAWHis), guanylated tRNA is the ?uanine is located in an internal position. There are onlv two tRNA s8n) in reticulocytes. We further show that one of these, the'one that is not 1abz led with guanine, contains the hypermodified base known as Q. tRNA( en) does not contain Q. INTRODUCTION Incubation of rabbit, sheep and human reticulocytes with radioactive guanine results in the uptake of the guanine into tRNA (1,2). Since reticulocytes do not synthesize RNA (3), the labeling of the tRNA could only be explained by a post-transcriptional modification of tRNA (1,2). Indeed, an enzyme was found (4) and purified (5) that catalyzed the incorporation of guanine into tRNA. The requirements of the enzyme are simple. The only cofactor requirement is for a monovalent cation, a tRNA that is an acceptor and the base guanine (4). The reaction appears to proceed by the excision of a base from an internal position within the polynucleotide chain of tRNA and replacement of that base by guanine (4). The reaction is highly specific for the tRNA and also for a particular site within the tRNA (1). In reticulocytes, only two tRNA's undergo guanylation; (the major guanylated tRNA has been identified as tRNA(HI")) (1,2,4,5); the other quanylated tRNA has until now not been identified. In this renort we show that the minor guanylated tRNA of rabbit reticulocytes is tRNA(A2n) Reticulocytes contain three tRNA (Hi8); a major one, tRNA (Hi) which elutes early, a minor one, tRNA(Hia) which elutes afterwards during RPC-5 chromatogwhich precedes tRNA 2 rapy (1,4,5,11) and a very small peak,tRNA1 is found in rabbit liver. Smith et al. have but not tRNA(His), tRNA(His), 3 2 reported the identical situation for tRNA~~~~~~~(Asn) , namely two tRNA (Asn) in
iRNAWJ84),
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2521
Nucleic Acids Research reticulocytes. Of these, a major one elutes early and a minor one comes off but not tRNA(A8n), is found in rabbit liver (6). RPC-5 later. Again Because of these biochemical similarities and the fact that the position at which Smith and McNamara reported tRNA(A8n) to elute from RPC-5 was close to where the minor guanylated tRNA was eluted, we decided to test the possibility that tRNA(Aan) is the minor guanylated tRNA. His and Asn are part of the tetrad of amino acids whose cognate tRNA's contain the hypermodified base known as Q (7,8). The other two amino acids are Asp and Tyr. For these four amino acids, there exist two isoacceptors that arise from identical transcripts. These isoacceptors differ in that one form contains Q (a derivative of G) (8), and the other isoacceptor contains G in place of Q. In Drosophila the relative amounts of the Q form and the G form of these tRNA may be important for normal morphogenesis (8). In another report we showed that tRNA(His) contains Q (5). In this report we show that tRNA 1 and not the guanylated tRINA( contains Q. MATERIALS AND METHODS had the specific activities [8-14C]Guanine, [3H] and [ of 50 mCi/mmol, 37 Ci/mmol and 84 mCi/mmol respectively were purchased from Schwarz/Mann Corporation. The packing for RPC-5 columns was purchased from Miles Laboratories. The capacity of the resin to retain tRNA was unsatisfactory and it was recoated with adogen 464 (9). Preparation of radioactive guanylated tRNA and asparaginyl tRNA: In order to prepare [ 14C] guanylated tRNA, rabbit reticulocytes were incubated with [14 C]guanine as previously described (1,2). Asparaginyl tRNA was prepared by incubating rabbit reticulocytes (1.5 ml packed cells) in 0.015 M Tris, pH 7.4; 0.01 M KC1; 0.15 M NaCl, 0.09 M NaHCO3; 5.6 mM glucose 0.68 mM Ferrous ammonium sulfate; 3,000 u/ penicillin; 19 amino acids minus asparagine 0.5 mM each and 13 iiCi [IH]L-asparagine in a final volume of 7.8 ml. The cells were incubated for 20 min at 370. The cells incubated with [ and [3H]asparagine were pooled and transferred to an ice bath. One-half volume of 0.10 M sodium acetate pH 5.0, 0.10 M NaCl, 0.01 M EDTA was added and this was followed by the addition of two volumes of water-saturated phenol. The suspension was shaken vigorously for 5 min and then centrifuged for 10 min at 10,000 x g. The aqueous layer was collected and the phenol phase extracted with an equal volume of the pH 5.0 acetate buffer. The aqueous phases were pooled and 2.2 volumes of 95% ethanol was added and the RNA permitted to precipitate for at least 3 hr at -15°. The RNA was collected by centrifugation, dried in vacuo and dissolved in 1.0 M NaCl
tRNA(Asn),
).,
14C]asparagine
4C]guanine
2522
Nucleic Acids Research at 00. Insoluble material was removed by centrifugation at 10,000 x g for 15 min and diluted with 2 volumes of 0.01 M sodium acetate, pH 4.5; 0.01 M MgCl2; 0.002 M mercaptoethanol 0.40 M NaCl (RPC-5 buffer). Reversed phase-5 chromatography of tRNA: The solution containing the [14C] guanylated and [3H]asparaginyl tRNA's was layered onto an RPC-5 column 0.8 x 25 cm that had been equilibrated with 0.40 M NaCl dissolved in the RPC-5 buffer. The column was developed as previously described (5,9) with a linear gradient from 0.50 M to 0.70 M NaCl dissolved in the RPC-5 buffer. There were 50 ml in each chamber of the gradient maker and the pressure was maintained at 300 psi. Samples containing 1.1 ml were collected. The entire samples were counted as previously described (1,2) or in experiments where the tRNA was to be used for further experiments, 20 ul aliquots were pipetted onto 3 MM paper discs which were processed according to Bollum before
counting (10). Chromatography of tRNA on DEAE Sephadex: The preparation of [14C] guanylated and [3HlAsn tRNA was scaled up three-fold. The [3H)Asn tRNA peak which coincided with [14C] guanylated tRNA after RPC-5 chromatography was pooled and precipitated with 2 volumes of ethanol. The tRNA was collected and dissolved in 0.02 M sodium acetate pH 4.5; 0.008 M magnesium acetate; 0.30 M NaCl. The tRNA solution was layered onto a .5 x 25 cm column of DEAE-Sephadex A-25 equilibrated with this buffer at 4°. The DEAE-Sephadex column was developed with a linear gradient with 100 ml of the above buffer in the mixing chamber and 100 ml of 0.02 M sodium acetate pH 4.5; 0.003 M magnesium acetate; 1.0 M NaCl in the other chamber. The flow rate was 25 ml per hr and 3.25 ml fractions were collected. The fractions were prepared for scintillation counting as previously described (1,2). Reaction of Asn tRNA with periodate: Reticulocytes were incubated in one case with [14C]Asn and in the other with [3H]Asn. The incubation conditions described above were scaled up three-fold. The aminoacyl tRNA's were precipitated-with ethanol as described above. After precipitation with ethanol, the [3H] and [14C] tRNA's were each dissolved in 2 ml of 0.10 M sodium acetate pH 5.0. Aliquots were taken for counting; the [3H]Asn tRNA contained 57,600 cpm and the [14C]Asn tRNA 87,960 cpm. Sodium metaperiodate 0.10 M was added to the [14C]Asn tRNA to a concentration of 0.02 M. The [3HlAsn tRNA which acted as control received an equal volume of H20. The two solutions were maintained at 0° for 60 min with gentle stirring. The 10 reaction was stopped by the addition of sucrose to 1.0 M, and the [3H] and [14C labeled tRNA's were pooled. Low molecular weight impurities were 2523
Nucleic Acids Research removed by adsorbing the tRNA's to a DEAE-cellulose column (0.7 x 8 cm) equilibrated with 0.25 M NaCl; 0.01 M MgCl2; 0.001 M disodium EDTA adjusted to pH 4.9. The column was washed with 150 ml of this solution. The tRNA was eluted with 0.70 M NaCl dissolved in the same buffer. One ml fractions were collected and-the tRNA located by monitoring the column effluent at 260 nm. The fractions containing tRNA were pooled and diluted with an equal volume of RPC-5 buffer and the tRNA was now analyzed by RPC-5 chromatography. RESULTS RPC-5 chromatography of reticulocyte tRNA charged with [3H]asparagine and guanylated with [ 14Cguanine: Reticulocytes from the same rabbit were labeled with [14C]guanine in the presence of the 20 unlabeled amino acids and also with [.3 H]asparagine in the presence of 19 cold amino acids minus asparagine. The two incubation mixtures were placed in an ice bath, the pH adjusted to 5.0. The samples were then pooled and the tRNA extracted as described above. The pooled tRNA's were chromatographed on RPC-5, and as seen in Fig. 1, asparaginyl tRNA2 cochromatographed with guanylated tRNA1. Asn tRNA, 3000 _
2700
2400
-3 Ii
2100
z1800 _
(-1500 z
01200
900r 600 1
300
10
20
30
40
50
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70
FRACTION NUMBER
Fig. 1: RPC-5 chromatography of
tRNA: After incubation cyte [IH]asparagine, the tRNA was column.
2524
(
[14C] guanylated reticulo[3H]asparaginyl, of reticulocytes with [14C]guanine and
extracted and chromato raphed on an RPC-5 ) is the [3H]asparagine, (---) is the guanine.
Nucleic Acids Research and guanylated tRNA2, which is in fact Ri8 tRNA3 (5), are also seen in Fig. 1. Chromatography of [3H]asparaqinyl tRNA2 and [ 14C] guanylated tRNAl on DEAE-Sephadex: The peak corresponding to asparaginyl tRNA2 - [14C] guanylated tRNA1 after RPC-5 chromatography was located and precipitated with 2 volumes of ethanol and chromatographed on DEAE-Sephadex. One-tenth volume of 90% trichloroacetic acid was added to each fraction and the precipitated tRNA's prepared for counting. As seen in Fig. 2, the [3H] and [14C] peaks 1000 _ 900 _ l l
800 _
II 700_ zz600
~-500
z
0
400 300 _
200
100
_ 10
20
30 40 50 FRACTION NUMBER
60
70
Fig. 2: Chromatography of pooled [3HJAon tRNA2: [14C] guanylated tRNAl on DEAE Sephadex. The [l C] and [3H]-containing fractions eluted from an RPC-5 column were located by taking 20 ul aliquots from each fractions. The fractions corresponding to [3Hl4sn tRNA2 and [14C]guanine tRNA1 were pooled, precipitated with two volumes of ethanol and chromatographed on DEAE Sephadex. (-) is the [3H]asparagine, (---) is the
[14C]guanine.
coincide, showing that reticulocyte guanylated tRNA1 is probably identical to tRNA(A8n). The reason that there are fewer [3H] counts than [14C] after DEAE-Sephadex chromatography is probably due to deacylation of Asn tRNA during the manipulation after RPC-5 chromatography. The guanine residue incorporated into tRNA(2Sn) is in an internal position: The guanylated tRNA labeled with [14C]guanine was located after elution from an RPC-5 column and the first peak was then digested with 0.3 M KOH at 2525
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370 for 18 hr. The digest was neutralized with Dowex 50 (H+) and then spotted onto Whatman 3 MM paper. The paper was developed with a solvent that separates molecules on the basis of charge (11). As seen in Fig. 3, all of the radioactive guanine incorporated in 250 Z 200 %%150 z 100 50-
0
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C M FROM ORIGIN Fig. 3: The guanine residu is incorporated into an internal position in the tRNA: The first ['4C] guanylated tRNA to elute from an RPC-5 column was hydrolyzed with 0.3 M KOH for 18 hr at 370 and then neutralized with Dowex 50 H+. After the resin was removed by filtration, the filtrate was concentrated to 0.1 ml at 370 in a stream of dry N2. The sample was streaked onto Whatman 3 MM paper. Appropriate markers, GR, GMP and pGp were also spotted. The chromatogram was developed with ethanol: 1 M ammonium acetate, 70:30. After drying, the markers were located under mineral light and the track containing the hydrolyzed tRNA was cut into 1 cm strips which were placed into scintillation vials and counted.
tRNA(Asn) was converted to material that cochromatographed with 2',3' GMP after alkaline digestion, indicating that the guanine was incorporated into an internal position within the polynucleotide chain (2). Were the guanine incorporated into the 3' position, the radioactivity would be in the nucleoside region of the chromatogram. If the guanine were incorporated at the 5' end, the label would have been found in pGp. Periodate oxidation of Aen tRNA: Since Asn is one of the four amino acids whose cognate tRNA's contain either guanine or Q in the first position of the anticodon, it was of importance to determine which of the two tRNA(A8n) contained Q. In order to distinguish between the Q form and G form, we took advantage of the fact that Q contains vicinal hydroxyl groups and, therefore, reacts with IOi. 4C]An tRNA and [3H]Asn tRNA were prepared and the tRNA reacted with IOi. The was treated identically but Io4 was not added. The two Aen tRNA's were then pooled and analyzed on an RPC-5 column. Fig. 4 shows that IO caused a disappearance of Ain tRNA1, but
[14C]Asn
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[3H]Asn
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_ 450
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"
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-
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Chromatography of periodate-treated Asn tRNA on RPC-5: [14C] and [3H]Asn tRNA were prepared by incubating reticulocytes with [14C] and [3H]Asn respectively. The ['4C]Asn tRNA was treated with IOi, the [3H]Asn tRNA served as a control. The two labeled tRNA's were pooled and chromatographed on RPC-5. (---) represents [14C] IOi-treated tRNA. ( -) represents the [3H] control.
Fig: 4:
not of Asn tRNA2, indicating that tRNA(Ain) is probably the Q-containing form in reticulocytes. If the Q forms and G forms of tRNA are homogeneic, the Q form would be expected to elute from RPC-5 columns before the G form, due to the extra positive charge imparted by the secondary amino group contained in Q (7).
DISCUSSION The fact that the minor guanylated reticulocyte tRNA has turned out to be specific for asparagine is especially interesting, since along with histidine (the major guanylated reticulocyte tRNA), they are part of the tetrad of amino acids whose cognate tRNA's contain Q. For each of these amino acids, there are two major isoaccepting tRNA's that are homogeneic. One of the two isoacceptors (the one that elutes early from RPC-5 columns) contains Q; the other isoacceptor contains G in place of Q. Nishimura et al. have postulated that the guanylating enzyme may be responsible for converting the Q form of tRNA to the G form (7). DuBrul and Farkas have, however, shown that the Q form of tRNA(Hia) is not the substrate for the guanylating enzyme. It is the G form that is the substrate (5). At least in the reticulocyte the role of the guanylating enzyme is not to convert the Q form of tRNA's to the G form. The fact that the role of the guanylating enzyme is not to remove Q from within the polynucleotide chain and replace it with G is further indicated by the fact that yeast tRNA, which does not contain Q (12), is an excellent substrate for the guanylating enzyme (4) and that rabbit liver tRNA which contains exclusively the Q form and not the G form of tRNA(Asn) and 2527
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tRNA(Hi8)
is not a substrate for the guanylating enzyme (4). The tRNA's for Asn and His of mammalian cells appear to be closely related. In the reticulocyte there are two isoacceptors for each of these amino acids. [Actually, a very small peak which elutes just prior to the tRNA(Hi8) has been reported (5).] The more abundant form contains Q and elutes early from RPC-5 columns. The less abundant form which elutes after the major form is the one that undergoes the guanylation reaction (5). In liver there is only one tRNA (the Q form) for both of these amino acids (6). Furthermore, for each of these amino acids there are two codons which differ by a pyrimidine in the third letter CAU and CAC for His and MU and MC for Asn (13). At present there are three possibilities to be considered: 1) that the two reticulocyte tRNA's that are substrates in the guanylation reaction and also have an isoacceptor that contains Q may be merely coincidental; and 2) that the guanylation reaction may be one of a series of events in the posttranscriptional modification of a guanine residue leading to the synthesis of Q at the macromolecular level; and 3) that the two major tRNA for Asn and His are not the Q and G form but the Q form and the Q* form. Nishimura has shown that Q* is a derivative of Q found in mammalian cells but not in prokaryote tRNA (12). It is possible that the late eluting tRNAs for His and Asn contains Q* and not G and that the role of the guanylating enzyme is to replace Q*, and not Q, with G. ACKNOWLEDGEMENTS This work was supported by grant NP 186 from the American Cancer Society and GM 20546 from the U.S. Public Health Service. We also thank Mr. Tom Stanawitz and Mr. Don Lewis for expert technical assistance. *
Present address: Yale university, New Haven, CT, USA
REFERENCES 1 Farkas, W.R., Hankins, W.D. and Singh, R. (1973) Biochim. Biophys. Acta 294, 94-105 2 Hankins, W.D. and Farkas, W.R. (1970) Biochim. Biophys. Acta 213, 77-89 3 DeBellis, R.H., Gluck, N. and Marks, P.A. (1964) J. Clin. Invest. 43, 1329-1337 4 Farkas, W.R. and Singh, R.D. (1973) J. Biol. Chem. 248, 7780-7785 5 DuBrul, E.D. and Farkas, W.R. (in press) Biochim. Biophys. Acta 6 Smith, E.D.W., Meltzer, V.N. and McNamara, A.L. (1974) Biochim. Biophys. Acta 349, 366-375 7 Kasai, H., Ohashi, Z., Harada, F., Nishimura, S., Oppenheimer, N.J., Crain, P.F., Liehr, J.G., von Minden, D.L. and McCloskey, J.A. (1975) Biochemistry 14, 4198-4208 8 White, B., Tener, G.M., Holden, J. and Suzuki, D.T. (1973) J. Mol. Biol. 2528
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74, 635-651 Kelmers, A.D. and Heatherly, D.E. (1971) Anal. Biochem. 44, 486-495 Bollum, F. (1968) Methods in Enzymology XII, Part B, 169-172 Spahr, P.F. (1964) J. Biol. Chem. 239, 3716-3726 Kasai, H., Kuchino, Y., Nihei, K. and Nishimura, S. (1975) Nuc. Acid Res. 2, 1931-1939 Marshall, R.E., Caskey, C.T. and Nirenberg, M. (1967) Science 155, 820-826
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