- Email: [email protected]
In vitro methylation of tRNA by extracts of Chlamydomonas reinhardi
ARCHIVES
OF
BIOCHEMISTRY
BIOPHYSICS
137, 409-414 (1970)
Methylation
of tRNA
AND
In Vitro
Chlamydomonas C. WELLS Department
of Biology,
Received
University
November
AND
by Extracts
of
reinhardi B. G. MOORE
of Alabama,
12, 1969; accepted
University, January
Alabama
36486
16, 1970
We have recently demonstrated that soluble extracts of C. reinhardi methylate heterologous transfer ribonucleic acid (tRNA) substrates in vitro using S-adenosylL-methionine (SAM) as the methyl donor. As with bacterial and animal systems that have been studied, no methylation of homologous tRNA occurred in vitro. Methyldeficient bacterial tRNA was not a significantly better substrate than its methylated counterpart or fully methylated t,RNA from another bacterial strain. The enzymes were more active at a pH of 8.2 and a temperature of 37” than at cultural conditions (pH 7&22”). No exogenous stimulation of the methylases was shown by K+, Na+, or NHa+ ions. Methylated products from the in vitro methylated bacterial tRNA were a methylcytosine (probably a 2’.O-methyl), I-methylguanine, N2-dimethylguanine, thymine, 6-methyladenine, and I-methyladenine. Nearly SOY0 of the methylation occurred on guanine residues of t,he tRNA.
Transfer ribonucleic acids (tRNA) UYUally contain numerous methylated components in addition to the four major purines/ pyrimidine (1, 2). S-Adenosyl-L-methionine (SARI) has been shown to be the methyl donor for this transmethylation which occurs at the polynucleotide level (3). Xethylating enzymes from bacteria, yeasts, and animal tissues exhibit species specificity due to differences, both qualitative and quantitative, in the methylated components present (4, 5). Although definite functions have not been assigned to the role of these minor components, Shugart et al. (6) have suggested that aminoacylation reactions are dependent on the full complement of methyl groups being present in a particular species of tRNA. Conformational structures of tRFJ\JA may be regulated by the methylated nucleotides as predicted by the sequence studies of Baguley and Staehelin (5). Whether methSlated and other minor nucleotides can impart enough molecular specificity to tRNA to regulate species differentiation as suggested by Hancock (7) remains to be seen.
Since there appears to be this widespread species variation of methylated nucleotides in tRNA, we were interested in whether enzymes from C. reinhardi cells could transmethylate tRNA using SARI as the methyl donor. MATERIALS
AND
METHODS
Chemicals and reagents. Biochemicals were obtained from either Sigma Chemical Co., St. Louis, MO. or Calbiochem, Inc., Los Angeles, Calif. MethylJ~C-SAM, having a sp act of 46.4 mCi/mmole, was obtained from International Chemical Corp., Irvine, Calif. Methylated adenine and guanine standards were the gift of Dr. G. Elion, Wellcome Laboratories, Tuckahoe, N.Y. N4-Methylcytosine was obtained from Dr. J. Fox, Sloan-Kettering Institute, New York, N. Y. All other chemicals were of reagent grade purity obtainable from commercial sources. Cultivation of cells. Chlamydomonas reinhardi was grown in Brist)ol’s inorganic salts medium (8) at room temperature under continuous light and aerated wit,h a 1% Cop-air mixture. Preparation of eztracts. C. reinhardi cells were harvested in late log phase in the cold by continuous-flow centrifugation, washed with 0.01 M tris (hydroxymethyl) aminomethane (Tris) buffer (pH
410
WELLS
AND
7.5), and then either frozen for later use or broken immediately for extraction of soluble proteins. No difference in activity of extracts from frozen and unfrozen cells was noted. Cells were disrupted by grinding with twice their wet weight of alumina for 15 min in the cold. The soluble proteins were then extracted with 20 ml of 0.01 M Tris buffer (pH 7.5) containing 0.01 M MgClg. The alumina was removed by centrifugation at 25,000g for 10 min, and the supernatant fraction was then centrifuged for 2 hr at 100,OOOg. This high-speed supernatant fraction was used as the source of enzymes for all assays. All extraction procedures were carried out at O-5”, and assays were run on the same day that the enzymes were extracted. Protein concentrations were determined by a spectrophotometric method (9). Preparation of substrate RNA. tRNA substrates from C. reinhardi and E. coli strains B and KIOWA were prepared by phenolic extraction (10) of cell Methyl-deficient tRNA was isolated extracts. described (11). from E. coli Kr2W6 as previously Assays. Methylation of tRNA was assayed by measuring the amount of radioactivity incorporated into the tRNA substrate from Y-methylSAM. The routine assay contained: 109 pmoles Tris buffer; 10 pmoles GSH; 10 gmoles MgC12; 2.5 mpmoles 14C-methyl-SAM (0.1 /*Ci); 0.5 mg tRNA; 0.5 mg soluble protein; water to a final voume of 1 ml (final pH was 8.2). Assay mixtures were incubated at 37” for 30 min except in experiments designed to test the effects of temperature and time. After incubation, the assay mixtures were cooled in an ice bath and 0.5 ml 6 N HCl was added, followed by 1 ml of 20% trichloracetic acid (TCA). The acid-insoluble materials were sedimented by centrifugation, and the pellet was washed four times with cold 5% TCA. The washed pellets were dissolved in 2 ml of 2 N NHeOH, and aliquots of 0.1-0.5 ml were mixed with 10 ml of Beckman’s cocktail D scintillation fluid and counted in a Beckman CPM-100 liquid scintillation counter to less than 5% error. DPM were obtained by use of internal standards except with the data obtained from the experiments designed to identify the methylation products. When appropriate, substrate blanks (minus LRNA) were run as controls, and these values were used to adjust the experimental determinations. Chromatographic procedures. Methylated components of tRNA for chromatographic separation were prepared by incubating 10 mg methyl-deficient tRNA, 5 &i of ‘%-methyl-SAM, 5 mg C. reinhardi protein, 400 pmoles Tris buffer (pH 8.2), 40 pmoles GSH, and 40 pmoles MgC& in a total volume of 4 ml for 1 hr at 37’. Methylsted tRNA for acid hydrolysis was extracted from the assay mixture with 80% phe-
MOORE nol, and the tRNA was precipitated from the aqueous phase with 2 vol of 957 ethanol and washed four times with 95% ethanol. The precipitate was then heated with 5 ml of 1 N HCl for 30 min in a boiling water bath and the hydrolyzate was dried in wacuo. Before applying to a Dowex 50-X4:490 (H+) ion-exchange column, the hydrolyzed tRNA was redissolved in 5 ml water, neutralized, and a 0.1~ml aliquot was removed for counting. The purines and the pyrimidine nucleotides were eluted with a O-4 N HCl gradient, and aliquots of each 5-ml fraction were assayed for ‘%-content. Elution of the major tRNA components was followed by monitoring the column eluent at 254 rnp. The monophosphates, which eluted in one peak, were rechromatographed on a Dowex 1 column to effect separation of cytosine monophosphate (CMP) and uridine monophosphate (UMP). The CMP and UMP peaks, with their associated radioactivity, were then concentrated to dryness and hydrolyzed to cytosine and uracil by heating at 100” in 3 ml of 35% HClOe for 60 min. The acid was removed by addition of an equivalent amount of KOH. These samples and the adenine and guanine peaks from the Dowex 50 column were concentrated to dryness in qacuo and taken up in a small volume of dilute HCl. Aliquots from each of the radioactive peaks were then applied to cellulose thin-layer plates and chromatographed in an isopropanol: HCl: Hz0 (680:168:152-v:v:v) system (10). The plates were then either exposed to X-ray film, or the radioactive spots were located by dividing each sample lane of migration into l-cm strips, eluting the counts in each strip with 1 ml of distilled water and assaying aliquots of each for 1%. RESULTS
The initial
experiments, which detect tRNA-methylating
were
de-
activities of cell-free extracts of C. reinhardi, were carried out using the assay conditions for bacterial tRNA methylases (11). Incorporation of 14C-methyl group was detected with this assay system (Table I). There was a 12-fold difference between the substrate blank and the experimental determination. Variation of the soluble protein added to the assays established a proportionality between methylation and protein quantity as expected if the results represented true methylation. Also derived from these experiments were the observations that the methylation system was pH- and temperature-dependent. Although the organism grows best at a medium pH of 7.0, the same signed
to
tRNA
METHYLASES
methylation reactions run at this pH gave negligible 14C-methyl uptake into tRNA; however, at pH 8.2, there was considerable 14C incorporated. The optimal temperature for methylase activity was also higher at 37” than at 22”, the incubation temperature of the algal cells. Although these results might suggest that bacterial contaminants were responsible for the methylation, rigorous observance of aseptic techniques during the culture of the alga and testing for bacteria before harvesting of the algal cells eliminated this possibility. TABLE
I
DP>MONSTR~\TION OF tRNA METHYL~TING ACTIVITY BY EXTRACTS OF C. reinhardi AND ENVIRONMENTAL EFFECTS ON METHYL~TION~ Enzyme extract A
B
Protein (mp)
PTI
1.0 1.0 1.0 0.5 1.0 3.0 3.0
8.2 8.2 8.2 8.2 8.2 8.2 7.0
1760b 21920 10022 9720 15740 16560 280
a Assay conditions were those described in Methods. The assay time was 30 min (this is referred to later as maximum assay conditions for a 30.min period). b No ULNA added. This control was not run with the other enzyme extract (B).
P-l -
TABLE
r------7
1/----J
20 40 60 80 ASSAY TIME( min)
100 120
FIG. 1. Time dependency of tRNA methylation by C. reinhardi enzymes. Assays were performed as described in Methods except that the time was varied.
II
METHYLATION OF DIFFERENT tRNA SUBSTRATES BY C. reinhardi EXTRACTS Sourceof tRNh
Endogenousb C. reinhardi E. coli K12W6: E. coli K12W6: E. coli B Yeast Rat liver
i I 2596 2662 20020 19932 215GOi 4070 3960
Methyl-deficient Methylated
0 66 17424 17336 18964 1474 1364
a DPM of 14C incorporated into 0.5 mg of the tRNA’s in 30 min by 0.54 mg of C. reinhardi-soluble protein. b This represents the DPM incorporated by the extract in the absence of exogenous tRNA.
TemperatureDPM of 1°C incorporated 0 37 37 22 37 37 37 37
411
OF C. reinhardi
TABLE
III
EFFXTSOF~ONSOKTHE C.reinhardi
METHYUSES
DPM incorporatedundermaximum assayconditions” K+ NC++ NHI+ 0 0.025 0.050 0.075 0.100 0.150 0.200
8,000 7,900 7,860 5,480 5,680 2,940 2,020
8,000 7,760 6,440 5,680 4,780 3,040 1,820
a Two different batches of enzymes these experiments. Slightly higher noted with the extract used to test ammonium ions. Values are adjusted blanks.
12,940 13,220 13,060 10,580 10,800 were used in activity was the effects of for substrate
The incorporation of methyl groups into the submethylated tRNA was linear with time up until about 60-90 min (Fig. 1). Although the submethylated bacterial tRNA is used exclusively for assaying tRNA methylases from heterologous sources, we were interested in the methylating activity of C. reinhardi extracts on other tRKA substrates. The results in Table II show the rates of methylation on different tRNA but not necessarily the maximum extent of methylation. Essentially no methylation occurred in the homologous system, which is consistent with other known systems except
412
WELLS AND MOORE
s 40. m 2
20.
6
10
I
I 20 TUBE
50
I
30 NUMBER
40
FIG. 2. Dowex 50 (H) column chromatography of I%-methyl-tRNA tained by acid hydrolysis. The locations of the major components (UMP, are indicated by squares in the upper parts of the figure.
20
40 60 TUBE NUMBER
80
FIG. 3. Dowex tography
1 (formate) column chromaof the UMP-CMP region of Fig. 2.
the rat spleen DNA methylases, which can apparently effect homologous methylations (12). The rate of submethylated tRNA methylation was no different from that of its fully methylated counterpart. E. coli B tRNA also methylated just as well as the methyl-deficient tRNA from E. coli K12W6. However, yeast and rat liver tRNA methylated at a rate about one-twelfth that of t,he bacterial tRNA’s. Studies with mammalian and yeast tRNA methylases (13, 14) have shown that certain ionic conditions influence the in vitro methylation assays. We observed varying degrees of inhibition with sodium, potassium, and ammonium ions rather than stimulation (Table III). Ammonium acetate-containing assay mixtures of Rodeh et al. (13) and Kaye and Leboy (15) also did not give
u
2
4
6
DISTANCE
components obCMP G, and A)
8 (cm)
10
12
14
FIG. 4. Cellulose TLC of the W-methyl guanines, adenines, and uracil. Aliquots of the concentrated peaks were spotted and developed (see Methods). Broken lines represent the adenine derivatives. Abbreviations are: G, guanine; A, adenine; U, uracil; I-MG, I-methylguanine; DMG, N2-dimethylguanine; l-MA, I-methyladenine; 6-MA, 6-methyladenine; T, thymine.
highly enhanced methylase activities in reference to our basic assay system (the former inhibited and the latter gave about 33 % stimulation). Attempts to demonstrate the in vitro methylation products were next undertaken. When the acid hydrolyzate of tRNA was applied to a Dowex 50 column and eluted with a O-4 N HCl gradient, we observed the results shown in Fig. 2. Three distinct areas of radioactivity were obtained. One with the UMP-CMP region, one and POSsibly two in the guanine region (the shoulder on the main peak was later shown to be a
tRNA
METHYLASES
distinct peak), and one in the adenine region. When the entire UMP-CMP region was concentrated and applied to a Dowex 1 column to effect separation of the pyrimidine nucleotides, the results shown in Fig. 3 were obtained. Radioactivity was associated wit’h the CMP peak and after the UMP peak. After correcting for 14C lost in the rechromatography process of the nucleotides and calculating the total 14C in each radioactive region, the percentages of 14Cmethyl groups incorporated into cytosine, uracil, adenine, and guanine residues were 2.9, 15.8, 1.5, and 79.8. Tentative identification of the methylated derivatives was established by cellulose thinlayer chromatography (TLC). Figure 4 sho\vs the identification of I-methylguanine and N2-dimethylguanine (l-methyl had nearly twice the 14Cas the N-methyl derivative), the thymine residue and the 6-methyl and 1-methyladenine derivatives (the 6methyl derivative had about eight times more associated 14C than did the l-methyl component). The behavior of the cytosine derivative, after perchloric acid hydrolysis, on cellulose TLC did not correspond to any of the cytosine derivatives (cytosine, 5methyl or N4-methyl cytosines) (Fig. .!?a). Instead, the radioactivity was in the area of thymine. We then prepared another batch of labeled tRNA, hydrolyzed it with acid, and obtained the CMP peak by Dowex 1 (formate) column chromatography and ran this in the TLC system (Fig. Sb). The radioactivity was now located with the C,lIP region. We now thought that perhaps the perchloric acid hydrolysis had deaminated the cytosine derivative to a uracil derivative. However, when authenic CNP and s-methyl CMP were carried through the same procedure, no uv material was found in the uracil area of the TLC plates. The alternative identity was then that the compound was a 2’-o-methyl ribose derivative. When the perchloric acid hydrolysis product was cochromatographed with ribose, almost exact coincidence of ribose color (aniline oxalate) and radioactivity was observed. These results tended to show that the methylation of the cytosine residues
OF C. reinhardi
a v) 800$600 3400 LL w 200. 2 ii!
b
A
3-76
I 4 ). 1, 8)
n
,o + 0”6002 0 400. 2co 10 , 12 ,
DISTANCE (cm) FIG. 5. (a). Behavior of the methylcytosine derivative obtained by perchloric acid hydrolysis of the radioactive peak from the Dowex 1 column. (b). TLC of the W-methyl CMP obtained by acid hydrolysis of tRNA followed by Dowex 1 column chromatography. Abbreviations are: C, cytosine; 5-MC, 5-methylcytosine; T, thymine.
occurred on the ribose rather than pyrimidine moiety of this nucleotide.
the
DISCUSSION
The soluble extracts of the alga, C. reinpossessed tRNA-methylating activities, on the bacterial tRNA substrates in vitro with 14C-methyl-SAM as the methyl donor. The effects of pH on the methylations were not surprising since this is an often-observed phenomenon with enzyme extracts of cells. However, the increased activity of the enzymes at 37” over that of 22” was worthy of note (Table I). The lower activity probably reflects the in viva situation, whereas the increased activity at 37” may reflect increased kinetics due to changes in the catalysis parameters; methylases and/or tRIC’A. The variation of methylation with time (Fig. 1) was interesting from this viewpoint; the methylation of the bacterial tRNA by the algal enzymes was much slower than with the homologous system (11). This is most likely due to at hardi,
414
WELLS AND MOORE
least two principles: (1) algal methylases having to seek out their methylating position on the bacterial tRNA and (2) lower IeveIs of the enzymes in algae as compared to bacteria. The failure to demonstrate hypermethylase activity by the addition of ammonium ions may have interesting overtones. Although one of the other assay mixtures used did enhance methylation slightly, we are not sure that it was the ammonium acetate alone since it had a diierent buffer and antioxidant. Bacterial enzymes and rat liver enzymes have both been shown to be stimulated by the addition of NHd+ to the assay mixture used in these experiments (Moore, B. G., unpublished results). NH,+ has been postulated as an uncoupler of in vivo inhibitors (16) of tRNA methylases. If this hypothesis is correct, the algal cells may not have developed these control mechanisms or the levels are so low that the assay method does not detect their action. The algal enzymes methylated bacterial tRNA’s at a much faster rate than tRNA from yeast and rat liver (Table II). These results were acceptable once the results of the batch methylations were shown by column chromatography (Figs. 2 and 3). Nearly 80% of the level was found in guanine residues. Bacterial tRNA have thymine as the predominant “minor component” (10) whereas rat liver and yeast tRNA have more methylated cytosines, adenines, and guanines (1, 2). Thus, more sites were available on the bacterial tRNA for the algal methylases than the other heterologous substrates tested. The methylation products, which reflect the nature of the algal tRNA methylases but not perhaps the quantitative levels, since a heterologous, partially methylated substrate was used, were distinct from those of the enteric bacteria and higher organisms such as yeast and animals (the rat liver comparison).
We speculate that the high methylated guanine content of the algal tRNA (if the in vivo situation is similar) imparts specificity to these plant enzymes. We plan to survey some other algae which are more and less related to bacteria than C. reinhardi to see if variations exist with stages of evolution of cellular activities with these groups, ACKNOWLEDGMENTS This research was supported in part, by University of Alabama Research Committee GrantIn-Aid 592 and by a N.D.E.A. fellowship to one of us (C.W.). We thank Dr. W. H. Darden, Jr., for assistance in the algal culture technique and other helpful suggestions. REFERENCES 1. SMITH, J. D., AND DUNN, D. B., Biochem. J. 72, 294 (1959). 2. HALL, R. H., Biochemistry 3, 876 (1964). 3. FLEIS~NER, E., AND BOREK, E., Proc. Nat. Acad. Sci. U.S.A. 48, 1199 (1962). 4. SRINIVASAN, P. R., AND BOREK, E., Proc. Nat. Acad. Sci. U.S.A. 49, 529 (1963). 5. BAGULEY, B. C., AND STAEHELIN, M., Biochemistry 7, 45 (1968). 6. SHUGART, L., NOVELLI, G. D., AND STULBERC~, M. P., Biochim. Biophys. Acta167, 83 (1968). 7. HANCOCK, R. L., Evolution 22, 835 (1968a). 8. BOLD, H. C., Bull. Torrey Bot. Club 76, 101 (1949). 9. WADDELL, W. J., J. Lab. Clin. Med. 48, 311 (1956). 10. HURWITZ, J., GOLD, M., AND ANDERS, M., J, Biol. Chem. 239, 3462 (1964). 11. MOORE, B. G., AND SMITH, R. C., Can. J. Biochem. 47, 561 (1969). 12. KALOUSEK, F., AND MORRIS, N. R., J. Biol. Chem. 244, 1157 (1969). 13. RODEH, R., FELDMAN, M., LITTAUER, U. B., Biochemistry 6, 451 (1967). 14. HANCOCK, R. L., Cancer Res. 28, 1223 (1968b). 15. KAYE, A. M., AND LEBOY, P. S., Biochim. Biophys. Acta 167, 289 (1968). 16. KERR, S. J., Fed. Proc. 28, 866 (1969).
OF
BIOCHEMISTRY
BIOPHYSICS
137, 409-414 (1970)
Methylation
of tRNA
AND
In Vitro
Chlamydomonas C. WELLS Department
of Biology,
Received
University
November
AND
by Extracts
of
reinhardi B. G. MOORE
of Alabama,
12, 1969; accepted
University, January
Alabama
36486
16, 1970
We have recently demonstrated that soluble extracts of C. reinhardi methylate heterologous transfer ribonucleic acid (tRNA) substrates in vitro using S-adenosylL-methionine (SAM) as the methyl donor. As with bacterial and animal systems that have been studied, no methylation of homologous tRNA occurred in vitro. Methyldeficient bacterial tRNA was not a significantly better substrate than its methylated counterpart or fully methylated t,RNA from another bacterial strain. The enzymes were more active at a pH of 8.2 and a temperature of 37” than at cultural conditions (pH 7&22”). No exogenous stimulation of the methylases was shown by K+, Na+, or NHa+ ions. Methylated products from the in vitro methylated bacterial tRNA were a methylcytosine (probably a 2’.O-methyl), I-methylguanine, N2-dimethylguanine, thymine, 6-methyladenine, and I-methyladenine. Nearly SOY0 of the methylation occurred on guanine residues of t,he tRNA.
Transfer ribonucleic acids (tRNA) UYUally contain numerous methylated components in addition to the four major purines/ pyrimidine (1, 2). S-Adenosyl-L-methionine (SARI) has been shown to be the methyl donor for this transmethylation which occurs at the polynucleotide level (3). Xethylating enzymes from bacteria, yeasts, and animal tissues exhibit species specificity due to differences, both qualitative and quantitative, in the methylated components present (4, 5). Although definite functions have not been assigned to the role of these minor components, Shugart et al. (6) have suggested that aminoacylation reactions are dependent on the full complement of methyl groups being present in a particular species of tRNA. Conformational structures of tRFJ\JA may be regulated by the methylated nucleotides as predicted by the sequence studies of Baguley and Staehelin (5). Whether methSlated and other minor nucleotides can impart enough molecular specificity to tRNA to regulate species differentiation as suggested by Hancock (7) remains to be seen.
Since there appears to be this widespread species variation of methylated nucleotides in tRNA, we were interested in whether enzymes from C. reinhardi cells could transmethylate tRNA using SARI as the methyl donor. MATERIALS
AND
METHODS
Chemicals and reagents. Biochemicals were obtained from either Sigma Chemical Co., St. Louis, MO. or Calbiochem, Inc., Los Angeles, Calif. MethylJ~C-SAM, having a sp act of 46.4 mCi/mmole, was obtained from International Chemical Corp., Irvine, Calif. Methylated adenine and guanine standards were the gift of Dr. G. Elion, Wellcome Laboratories, Tuckahoe, N.Y. N4-Methylcytosine was obtained from Dr. J. Fox, Sloan-Kettering Institute, New York, N. Y. All other chemicals were of reagent grade purity obtainable from commercial sources. Cultivation of cells. Chlamydomonas reinhardi was grown in Brist)ol’s inorganic salts medium (8) at room temperature under continuous light and aerated wit,h a 1% Cop-air mixture. Preparation of eztracts. C. reinhardi cells were harvested in late log phase in the cold by continuous-flow centrifugation, washed with 0.01 M tris (hydroxymethyl) aminomethane (Tris) buffer (pH
410
WELLS
AND
7.5), and then either frozen for later use or broken immediately for extraction of soluble proteins. No difference in activity of extracts from frozen and unfrozen cells was noted. Cells were disrupted by grinding with twice their wet weight of alumina for 15 min in the cold. The soluble proteins were then extracted with 20 ml of 0.01 M Tris buffer (pH 7.5) containing 0.01 M MgClg. The alumina was removed by centrifugation at 25,000g for 10 min, and the supernatant fraction was then centrifuged for 2 hr at 100,OOOg. This high-speed supernatant fraction was used as the source of enzymes for all assays. All extraction procedures were carried out at O-5”, and assays were run on the same day that the enzymes were extracted. Protein concentrations were determined by a spectrophotometric method (9). Preparation of substrate RNA. tRNA substrates from C. reinhardi and E. coli strains B and KIOWA were prepared by phenolic extraction (10) of cell Methyl-deficient tRNA was isolated extracts. described (11). from E. coli Kr2W6 as previously Assays. Methylation of tRNA was assayed by measuring the amount of radioactivity incorporated into the tRNA substrate from Y-methylSAM. The routine assay contained: 109 pmoles Tris buffer; 10 pmoles GSH; 10 gmoles MgC12; 2.5 mpmoles 14C-methyl-SAM (0.1 /*Ci); 0.5 mg tRNA; 0.5 mg soluble protein; water to a final voume of 1 ml (final pH was 8.2). Assay mixtures were incubated at 37” for 30 min except in experiments designed to test the effects of temperature and time. After incubation, the assay mixtures were cooled in an ice bath and 0.5 ml 6 N HCl was added, followed by 1 ml of 20% trichloracetic acid (TCA). The acid-insoluble materials were sedimented by centrifugation, and the pellet was washed four times with cold 5% TCA. The washed pellets were dissolved in 2 ml of 2 N NHeOH, and aliquots of 0.1-0.5 ml were mixed with 10 ml of Beckman’s cocktail D scintillation fluid and counted in a Beckman CPM-100 liquid scintillation counter to less than 5% error. DPM were obtained by use of internal standards except with the data obtained from the experiments designed to identify the methylation products. When appropriate, substrate blanks (minus LRNA) were run as controls, and these values were used to adjust the experimental determinations. Chromatographic procedures. Methylated components of tRNA for chromatographic separation were prepared by incubating 10 mg methyl-deficient tRNA, 5 &i of ‘%-methyl-SAM, 5 mg C. reinhardi protein, 400 pmoles Tris buffer (pH 8.2), 40 pmoles GSH, and 40 pmoles MgC& in a total volume of 4 ml for 1 hr at 37’. Methylsted tRNA for acid hydrolysis was extracted from the assay mixture with 80% phe-
MOORE nol, and the tRNA was precipitated from the aqueous phase with 2 vol of 957 ethanol and washed four times with 95% ethanol. The precipitate was then heated with 5 ml of 1 N HCl for 30 min in a boiling water bath and the hydrolyzate was dried in wacuo. Before applying to a Dowex 50-X4:490 (H+) ion-exchange column, the hydrolyzed tRNA was redissolved in 5 ml water, neutralized, and a 0.1~ml aliquot was removed for counting. The purines and the pyrimidine nucleotides were eluted with a O-4 N HCl gradient, and aliquots of each 5-ml fraction were assayed for ‘%-content. Elution of the major tRNA components was followed by monitoring the column eluent at 254 rnp. The monophosphates, which eluted in one peak, were rechromatographed on a Dowex 1 column to effect separation of cytosine monophosphate (CMP) and uridine monophosphate (UMP). The CMP and UMP peaks, with their associated radioactivity, were then concentrated to dryness and hydrolyzed to cytosine and uracil by heating at 100” in 3 ml of 35% HClOe for 60 min. The acid was removed by addition of an equivalent amount of KOH. These samples and the adenine and guanine peaks from the Dowex 50 column were concentrated to dryness in qacuo and taken up in a small volume of dilute HCl. Aliquots from each of the radioactive peaks were then applied to cellulose thin-layer plates and chromatographed in an isopropanol: HCl: Hz0 (680:168:152-v:v:v) system (10). The plates were then either exposed to X-ray film, or the radioactive spots were located by dividing each sample lane of migration into l-cm strips, eluting the counts in each strip with 1 ml of distilled water and assaying aliquots of each for 1%. RESULTS
The initial
experiments, which detect tRNA-methylating
were
de-
activities of cell-free extracts of C. reinhardi, were carried out using the assay conditions for bacterial tRNA methylases (11). Incorporation of 14C-methyl group was detected with this assay system (Table I). There was a 12-fold difference between the substrate blank and the experimental determination. Variation of the soluble protein added to the assays established a proportionality between methylation and protein quantity as expected if the results represented true methylation. Also derived from these experiments were the observations that the methylation system was pH- and temperature-dependent. Although the organism grows best at a medium pH of 7.0, the same signed
to
tRNA
METHYLASES
methylation reactions run at this pH gave negligible 14C-methyl uptake into tRNA; however, at pH 8.2, there was considerable 14C incorporated. The optimal temperature for methylase activity was also higher at 37” than at 22”, the incubation temperature of the algal cells. Although these results might suggest that bacterial contaminants were responsible for the methylation, rigorous observance of aseptic techniques during the culture of the alga and testing for bacteria before harvesting of the algal cells eliminated this possibility. TABLE
I
DP>MONSTR~\TION OF tRNA METHYL~TING ACTIVITY BY EXTRACTS OF C. reinhardi AND ENVIRONMENTAL EFFECTS ON METHYL~TION~ Enzyme extract A
B
Protein (mp)
PTI
1.0 1.0 1.0 0.5 1.0 3.0 3.0
8.2 8.2 8.2 8.2 8.2 8.2 7.0
1760b 21920 10022 9720 15740 16560 280
a Assay conditions were those described in Methods. The assay time was 30 min (this is referred to later as maximum assay conditions for a 30.min period). b No ULNA added. This control was not run with the other enzyme extract (B).
P-l -
TABLE
r------7
1/----J
20 40 60 80 ASSAY TIME( min)
100 120
FIG. 1. Time dependency of tRNA methylation by C. reinhardi enzymes. Assays were performed as described in Methods except that the time was varied.
II
METHYLATION OF DIFFERENT tRNA SUBSTRATES BY C. reinhardi EXTRACTS Sourceof tRNh
Endogenousb C. reinhardi E. coli K12W6: E. coli K12W6: E. coli B Yeast Rat liver
i I 2596 2662 20020 19932 215GOi 4070 3960
Methyl-deficient Methylated
0 66 17424 17336 18964 1474 1364
a DPM of 14C incorporated into 0.5 mg of the tRNA’s in 30 min by 0.54 mg of C. reinhardi-soluble protein. b This represents the DPM incorporated by the extract in the absence of exogenous tRNA.
TemperatureDPM of 1°C incorporated 0 37 37 22 37 37 37 37
411
OF C. reinhardi
TABLE
III
EFFXTSOF~ONSOKTHE C.reinhardi
METHYUSES
DPM incorporatedundermaximum assayconditions” K+ NC++ NHI+ 0 0.025 0.050 0.075 0.100 0.150 0.200
8,000 7,900 7,860 5,480 5,680 2,940 2,020
8,000 7,760 6,440 5,680 4,780 3,040 1,820
a Two different batches of enzymes these experiments. Slightly higher noted with the extract used to test ammonium ions. Values are adjusted blanks.
12,940 13,220 13,060 10,580 10,800 were used in activity was the effects of for substrate
The incorporation of methyl groups into the submethylated tRNA was linear with time up until about 60-90 min (Fig. 1). Although the submethylated bacterial tRNA is used exclusively for assaying tRNA methylases from heterologous sources, we were interested in the methylating activity of C. reinhardi extracts on other tRKA substrates. The results in Table II show the rates of methylation on different tRNA but not necessarily the maximum extent of methylation. Essentially no methylation occurred in the homologous system, which is consistent with other known systems except
412
WELLS AND MOORE
s 40. m 2
20.
6
10
I
I 20 TUBE
50
I
30 NUMBER
40
FIG. 2. Dowex 50 (H) column chromatography of I%-methyl-tRNA tained by acid hydrolysis. The locations of the major components (UMP, are indicated by squares in the upper parts of the figure.
20
40 60 TUBE NUMBER
80
FIG. 3. Dowex tography
1 (formate) column chromaof the UMP-CMP region of Fig. 2.
the rat spleen DNA methylases, which can apparently effect homologous methylations (12). The rate of submethylated tRNA methylation was no different from that of its fully methylated counterpart. E. coli B tRNA also methylated just as well as the methyl-deficient tRNA from E. coli K12W6. However, yeast and rat liver tRNA methylated at a rate about one-twelfth that of t,he bacterial tRNA’s. Studies with mammalian and yeast tRNA methylases (13, 14) have shown that certain ionic conditions influence the in vitro methylation assays. We observed varying degrees of inhibition with sodium, potassium, and ammonium ions rather than stimulation (Table III). Ammonium acetate-containing assay mixtures of Rodeh et al. (13) and Kaye and Leboy (15) also did not give
u
2
4
6
DISTANCE
components obCMP G, and A)
8 (cm)
10
12
14
FIG. 4. Cellulose TLC of the W-methyl guanines, adenines, and uracil. Aliquots of the concentrated peaks were spotted and developed (see Methods). Broken lines represent the adenine derivatives. Abbreviations are: G, guanine; A, adenine; U, uracil; I-MG, I-methylguanine; DMG, N2-dimethylguanine; l-MA, I-methyladenine; 6-MA, 6-methyladenine; T, thymine.
highly enhanced methylase activities in reference to our basic assay system (the former inhibited and the latter gave about 33 % stimulation). Attempts to demonstrate the in vitro methylation products were next undertaken. When the acid hydrolyzate of tRNA was applied to a Dowex 50 column and eluted with a O-4 N HCl gradient, we observed the results shown in Fig. 2. Three distinct areas of radioactivity were obtained. One with the UMP-CMP region, one and POSsibly two in the guanine region (the shoulder on the main peak was later shown to be a
tRNA
METHYLASES
distinct peak), and one in the adenine region. When the entire UMP-CMP region was concentrated and applied to a Dowex 1 column to effect separation of the pyrimidine nucleotides, the results shown in Fig. 3 were obtained. Radioactivity was associated wit’h the CMP peak and after the UMP peak. After correcting for 14C lost in the rechromatography process of the nucleotides and calculating the total 14C in each radioactive region, the percentages of 14Cmethyl groups incorporated into cytosine, uracil, adenine, and guanine residues were 2.9, 15.8, 1.5, and 79.8. Tentative identification of the methylated derivatives was established by cellulose thinlayer chromatography (TLC). Figure 4 sho\vs the identification of I-methylguanine and N2-dimethylguanine (l-methyl had nearly twice the 14Cas the N-methyl derivative), the thymine residue and the 6-methyl and 1-methyladenine derivatives (the 6methyl derivative had about eight times more associated 14C than did the l-methyl component). The behavior of the cytosine derivative, after perchloric acid hydrolysis, on cellulose TLC did not correspond to any of the cytosine derivatives (cytosine, 5methyl or N4-methyl cytosines) (Fig. .!?a). Instead, the radioactivity was in the area of thymine. We then prepared another batch of labeled tRNA, hydrolyzed it with acid, and obtained the CMP peak by Dowex 1 (formate) column chromatography and ran this in the TLC system (Fig. Sb). The radioactivity was now located with the C,lIP region. We now thought that perhaps the perchloric acid hydrolysis had deaminated the cytosine derivative to a uracil derivative. However, when authenic CNP and s-methyl CMP were carried through the same procedure, no uv material was found in the uracil area of the TLC plates. The alternative identity was then that the compound was a 2’-o-methyl ribose derivative. When the perchloric acid hydrolysis product was cochromatographed with ribose, almost exact coincidence of ribose color (aniline oxalate) and radioactivity was observed. These results tended to show that the methylation of the cytosine residues
OF C. reinhardi
a v) 800$600 3400 LL w 200. 2 ii!
b
A
3-76
I 4 ). 1, 8)
n
,o + 0”6002 0 400. 2co 10 , 12 ,
DISTANCE (cm) FIG. 5. (a). Behavior of the methylcytosine derivative obtained by perchloric acid hydrolysis of the radioactive peak from the Dowex 1 column. (b). TLC of the W-methyl CMP obtained by acid hydrolysis of tRNA followed by Dowex 1 column chromatography. Abbreviations are: C, cytosine; 5-MC, 5-methylcytosine; T, thymine.
occurred on the ribose rather than pyrimidine moiety of this nucleotide.
the
DISCUSSION
The soluble extracts of the alga, C. reinpossessed tRNA-methylating activities, on the bacterial tRNA substrates in vitro with 14C-methyl-SAM as the methyl donor. The effects of pH on the methylations were not surprising since this is an often-observed phenomenon with enzyme extracts of cells. However, the increased activity of the enzymes at 37” over that of 22” was worthy of note (Table I). The lower activity probably reflects the in viva situation, whereas the increased activity at 37” may reflect increased kinetics due to changes in the catalysis parameters; methylases and/or tRIC’A. The variation of methylation with time (Fig. 1) was interesting from this viewpoint; the methylation of the bacterial tRNA by the algal enzymes was much slower than with the homologous system (11). This is most likely due to at hardi,
414
WELLS AND MOORE
least two principles: (1) algal methylases having to seek out their methylating position on the bacterial tRNA and (2) lower IeveIs of the enzymes in algae as compared to bacteria. The failure to demonstrate hypermethylase activity by the addition of ammonium ions may have interesting overtones. Although one of the other assay mixtures used did enhance methylation slightly, we are not sure that it was the ammonium acetate alone since it had a diierent buffer and antioxidant. Bacterial enzymes and rat liver enzymes have both been shown to be stimulated by the addition of NHd+ to the assay mixture used in these experiments (Moore, B. G., unpublished results). NH,+ has been postulated as an uncoupler of in vivo inhibitors (16) of tRNA methylases. If this hypothesis is correct, the algal cells may not have developed these control mechanisms or the levels are so low that the assay method does not detect their action. The algal enzymes methylated bacterial tRNA’s at a much faster rate than tRNA from yeast and rat liver (Table II). These results were acceptable once the results of the batch methylations were shown by column chromatography (Figs. 2 and 3). Nearly 80% of the level was found in guanine residues. Bacterial tRNA have thymine as the predominant “minor component” (10) whereas rat liver and yeast tRNA have more methylated cytosines, adenines, and guanines (1, 2). Thus, more sites were available on the bacterial tRNA for the algal methylases than the other heterologous substrates tested. The methylation products, which reflect the nature of the algal tRNA methylases but not perhaps the quantitative levels, since a heterologous, partially methylated substrate was used, were distinct from those of the enteric bacteria and higher organisms such as yeast and animals (the rat liver comparison).
We speculate that the high methylated guanine content of the algal tRNA (if the in vivo situation is similar) imparts specificity to these plant enzymes. We plan to survey some other algae which are more and less related to bacteria than C. reinhardi to see if variations exist with stages of evolution of cellular activities with these groups, ACKNOWLEDGMENTS This research was supported in part, by University of Alabama Research Committee GrantIn-Aid 592 and by a N.D.E.A. fellowship to one of us (C.W.). We thank Dr. W. H. Darden, Jr., for assistance in the algal culture technique and other helpful suggestions. REFERENCES 1. SMITH, J. D., AND DUNN, D. B., Biochem. J. 72, 294 (1959). 2. HALL, R. H., Biochemistry 3, 876 (1964). 3. FLEIS~NER, E., AND BOREK, E., Proc. Nat. Acad. Sci. U.S.A. 48, 1199 (1962). 4. SRINIVASAN, P. R., AND BOREK, E., Proc. Nat. Acad. Sci. U.S.A. 49, 529 (1963). 5. BAGULEY, B. C., AND STAEHELIN, M., Biochemistry 7, 45 (1968). 6. SHUGART, L., NOVELLI, G. D., AND STULBERC~, M. P., Biochim. Biophys. Acta167, 83 (1968). 7. HANCOCK, R. L., Evolution 22, 835 (1968a). 8. BOLD, H. C., Bull. Torrey Bot. Club 76, 101 (1949). 9. WADDELL, W. J., J. Lab. Clin. Med. 48, 311 (1956). 10. HURWITZ, J., GOLD, M., AND ANDERS, M., J, Biol. Chem. 239, 3462 (1964). 11. MOORE, B. G., AND SMITH, R. C., Can. J. Biochem. 47, 561 (1969). 12. KALOUSEK, F., AND MORRIS, N. R., J. Biol. Chem. 244, 1157 (1969). 13. RODEH, R., FELDMAN, M., LITTAUER, U. B., Biochemistry 6, 451 (1967). 14. HANCOCK, R. L., Cancer Res. 28, 1223 (1968b). 15. KAYE, A. M., AND LEBOY, P. S., Biochim. Biophys. Acta 167, 289 (1968). 16. KERR, S. J., Fed. Proc. 28, 866 (1969).