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Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 528-530, February 1975

Biosynthesis of Ribosylthymine in the Transfer RNA of Streptococcus faecalis: A Folate-Dependent Methylation Not Involving S-Adenosylmethionine (/tRNA modification/formate/[methyl-'4Clmethionine/GlT'C-loop) ANN S. DELK AND JESSE C. RABINOWITZ Department of , University of California, Berkeley, Calif. 94720 Communicated by Bruce Ames, November 18, 1974

ABSTRACT Ribosylthymine is not present in tRNA of to produce 5'-monophosphate, which is sub- Streptococcus faecalis grown in the absence of folic , sequently used in DNA synthesis. Because of the folate although this methylated residue does occur in the tRNA of this when it is grown in the presence of folate. requirement for T formation in S. faecalis, it appeared possible We have found that, unlike other methylated residues in that methylation of RNA in this organism, particularly in RNA of S. faecalis and other , the methyl moiety the of T, involves a folate derivative and not of ribosylthymine of the tRNA of folate-sufficient S. S-adenosylmethionine. faecalis is not derived from S-adenosylmethionine. Pre- liminary evidence suggests that a folate derivative serves MATERIALS AND METHODS as the methyl donor in this methylation. Unless stated otherwise, S. faecalis (ATCC 8043) and Esche- When grown in the presence of folic acid, Streptococcus richia coli (MRE 600) were grown for several generations in faecalis, like other prokaryotes, initiates synthesis the presence of folic acid in 10 ml of a medium identical to with fMet-tRNAfMet. However, when this bacterium is one described (2, 12), except that it contained 3 g/liter of grown in folate-free medium, initiation proceeds with non- vitamin-free Bacto-casamino (Difco) containing ap- formylated Mlet-tRNAfMet (1-3) in a manner that, at least proximately 0.1 g of L-methionine, the supplemental methio- superficially, resembles the initiation process in nine was omitted, and one of the following labeled compounds (4). The ability of folate-deficient S. faecalis to initiate protein (Amersham Searle) was added: L- [methyl-14C]methionine, synthesis with nonformylated Met-tRNAfMet has been shown 10-50,Ci, 56 mCi/mmol; sodium [14C]formate, 20-250 uCi, to be due to a difference in the tRNAfMet of cells grown in the 54 mCi/mmol; sodium ['H ]formate, 100 uACi, 250 mCi/mmol. absence of folate, relative to that tRNAfMet species found in Cells were harvested by centrifugation during late logarithmic folate-sufficient cells (3). We have recently shown that this phase. Because the S. faecalis wall is difficult to disrupt, difference resides in a single modification of the tRNAfMet cells were treated with lysozyme (0.4 mg in 0.2 ml), quickly molecule (5). In tRNAfMet of cells grown in the presence of frozen and thawed at 370 several' times, and sonicated. folate, ribosylthymine (T), which is 5-methyluridine, occurs Sodium dodecyl sulfate (1%0 final concentration) and carrier in the sequence GT;C in loop IV. In the tRNAfMet of cells tR.NA (6 A260 units) were added, and the tRNA was isolated grown in the absence of folate, the sequence GUVC is found. by phenol extraction and DEAE-cellulose column chroma- The remainder of the sequence is identical for the tography (3). That material eluting between 0.2 and 1.0 M two tRNAs. Furthermore, we have shown that the absence of NaCl was precipitated by the addition of two volumes of T is not unique to tRNAfmet. Every tRNA synthesized in ethanol, dissolved in 0.2 M ammonium acetate, pH 4.5, and folate-deficient S. faecalis appears to lack T (5). These findings reprecipitated with ethanol. The tRNA was routinely digested led us to investigate the biosynthesis of T in S. faecalis. with 3 jg of pancreatic RNase (Worthington), 3 jg of Ul It is currently accepted that S-adenosylmethionine serves RNase (13) (a gift of Drs. A. Blank and C. A. Dekker), and as the methyl donor for every methylation of RNA occurring 3 units of T1 RNase (Sankyo) in 0.01 M Tris-HCl, pH 7.0, at the level (6-9). Assuming this to be the case in at 370 for 6-12 hr. In one experiment, the tRNA was hy- S. faecalis, the effect of the absence of folate on the ability of drolyzed in 0.3 M NaOH, 370, 18 hr. Where indicated, the S. faecalis to synthesize T is surprising. Since S. faecalis tRNA was treated with 50 ,g/ml of pancreatic DNase in requires methionine in the , even in the pres- 0.01 M Tris-HCl, pH 7.0, 0.005 M MgCl2, at 370 for 18 hr, ence of folate (10), both folate-sufficient and folate-deficient and collected by ethanol precipitation before RNase hydroly- media were supplemented with equivalent amounts of sis. The 3'- were fractionated by two-dimensional methionine. Thus, it seemed unlikely that the absence of T thin-layer chromatography (14) on cellulose-coated plates in folate-deficient cells is due to insufficient methionine for the (Eastman, 6064). The four major nucleotides were located methylation reaction. by their ultraviolet absorption and the radioactive nucleotides On the other hand, folate derivatives are recognized as by autoradiography (Kodak, Blue Brand x-ray film). methyl donors in certain reactions (11). Of particular signifi- The nucleotide composition of S. faecalis tRNA was also cance, 5,10-methylenetetrahydrofolate serves as the methyl examined by labeling the tRNA in vivo with 321p as described donor in the conversion of 5'-monophosphate (5) or by labeling periodate-treated with KB'H4 (15). The ability of S. faecalis to use glycine and homocysteine Abbreviations: f.\Iet-tRNAfMet, N-formylmethionyl-tRNAfMet; was T, ribosylthymine or 5-methyluridine; Tp, ribosylthymine 3'- for synthesis of serine and methionine, respectively, one de- . tested in a folate-sufficient medium similar to the 528 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Biosynthesis of Ribosylthymine 529

scribed (2, 12), except that a mixture of amino acids lacking a b either serine or methionine, but containing glycine or homo- cysteine, respectively, was substituted for Bacto-casamino AOH I AOH 0 0 acids-and no additional amino acids were added. The occur- AQ rence of radioactivity in the amino acids of S. faecalis grown i AflC in the presence of [3H]formate was examined by analysis of T oT protein extracted with hot (90') 5% trichloroacetic acid. .. SZ_ Nucleic acid was removed from the acid-insoluble material Gg O by exhaustive digestion with snake venom phosphodiesterase in 0.01 M TrisHCl, pH 8.9, 0.001 M EDTA (Na+), and reprecipitation of the protein with cold 5% trichloroacetic -It.~ ~ ~ ~ ~ I acid. The sample was hydrolyzed in 6 M HOl at 1100 for 22 ---2 hr, and analyzed on a Beckman Automatic Amino-Acid FIG. 1. Autoradiograph of the fractionation of the 3'-nucleo- Analyzer (model 117). tides of tRNA isolated from cells grown in the presence of [methyl- 14C]methionine. (a) E. coli; (b) S. faecalis. (1) First dimension: RESULTS isobutyric acid-NH40H; (2) isopropanol-HCl-H20 (14). Out- In agreement with previously published results, Fig. la lines indicate position of nucleotides located by UV absorption. shows that when E. coli is grown in the presence of [methyl- Nucleotides are designated by standard letter designations. 14Cjmethionine, Tp is found to be the most abundant labeled The radioactive material below A is 7-methylguanosine 3'-phos- residue in tRNA. On the other hand, although Tp was shown phate; that above and to the right of A is probably a methylated by 32p labeli'ng to be present in the tRNA of S. faecalis grown 3'-phosphate. in the presence of folate, no radioactive label from methionine digested to 5'- with pancreatic DNase appears in Tp of S. faecalis, as shown in Fig. lb. Labeled and snake venom phosphodiesterase and to 3'-deoxyribo- 7-methylguanosine 3'-phosphate, methyladenosine 3'-phos- nucleotides with micrococcal and spleen phospho- phate, and several unidentified products are clearly dis- diesterase, and the products were separated by thin-layer tinguishable. The presence of methylated G and A in S. chromatography. No ultraviolet-absorbing material coincided faecalis tRNA was confirmed by both the 32p and KB3H4 with Tp. analytical methods. They were shown to be present in the Analysis of 32P-labeled bulk tRNA indicated that when tRNA of folate-deficient, as well as folate-sufficient, S. S. faecalis is grown in the absence of folate, there is a trace faecalis. amount of Tp (or a nucleotide with similar chromatographic As shown in Fig. 2, when S. faecalis is grown in folate- properties in the solvent system described in Fig. 1). RNA sufficient medium containing [14C]formate, the radioactive fractions predominantly containing rRNA were isolated from label found in tRNA appears almost exclusively in Tp. The cells grown in the presence and in the absence of folate. radioactive material remaining at the origin was not suscepti- Both samples were shown to contain Tp (or a similar nucleo- ble to hydrolysis by snake venom phosphodiesterase and is, tide) in approximately equal amounts. No T could be detected therefore, not nucleic acid. No label from ['4C]formate is in purified tRNAfMet of folate-deficient cells by either the 32p detectable in the methylated residues that derive their methyl or KB3H4 method. moieties from methionine. Although growth studies substituting glycine and homo- DISCUSSION cysteine for serine and methionine, respectively, showed that These data show that methionine, and consequently S- S. faecalis requires the addition of the latter two amino acids adenosylmethionine, does not serve as the methyl donor in for optimal growth, analysis of the protein of cells grown in the biosynthesis of T of the tRNA of Streptococcus faecalis. the presence of [3H]formate indicated that folate-sufficient On the other hand, methionine is the source of the methyl S. faecalis can utilize formate for the biosynthesis of serine but not of methionine. However, any radioactive serine formed from ['4C]formate is diluted into a large pool of nonradioactive , since unlabeled serine, 0.4 g/liter, was added to the growth medium. To exclude the possibility that the radioactivity derived Oc T from formate actually occurs in , the , present as the result of hydrolysis of contaminating DNA, the tRNA from cells grown in the EG u presence of [w4C]formatewas analyzed in the following manner. (i) The tRNA was treated extensively with DNase, precipitated twice with ethanol, digested with RNases, and fractionated by thin-layer chromatography. Material migrat- ing with Tp contained radioactive label. (ii) The tRNA was hydrolyzed with alkali. (The of DNA is stable under alkaline conditions.) Again radioactive material - 2 corresponding to Tp was observed. (iii) The tRNA was sub- FIG. 2. Autoradiograph of fractionation of the 3'-nucleotides jected to chromatography without prior hydrolysis. All of tRNA isolated from S. faecalis grown in the presence of [14C]- radioactive material remained at the origin. (iv) DNA was formate. Designations are as in the legend of Fig. 1. Downloaded by guest on September 29, 2021 530 Biochemistry: Delk and Rabinowitz Proc. Nat. Acad. Sci. USA 72 (1975)

moiety of other methylated residues found in the tRNA of based exclusively on the incorporation of methionine in vivo both folate-sufficient and folate-deficient S. faecalis. It appears into the modified residue, while perhaps appropriate for E. that the trace amount of T found in crude preparations of coli, must be considered with reservation for other organisms. tRNa is of rRNA origin and that the biosynthesis of T of We thank Mr. Joseph M. Romeo for analysis of rRNA with rRNA is not dependent upon the presence of folate. It is KB3H4. This work was supported by NIH Grant AM-2109 from speculated that methionine serves as the source of the methyl the National Institute of Arthritis, and Digestive moiety of T of rRNA. Diseases. A.S.D. is the recipient of a NIH PHS Postdoctoral Formate is utilized by S. faecalis for the biosynthesis of T Fellowship (1 F22 AM00906-01). of tRNA. The incorporation of ['4C]formate into T occurs 1. Samuel, C. E., D'Ari, L. & Rabinowitz, J. C. (1970) J. Biol. only in the presence of folate. Under folate-free growth condi- Chem. 245, 5115-5121. tions synthesis of T does not occur in tRNA. S. faecalis cannot 2. Samuel, C. E., Murray, C. L. & Rabinowitz, J. C. (1972) J. Biol. Chem. 247, 6856-6865. incorporate ["4C]formate into methionine and, consequently, 3. Samuel, C. E. & Rabinowitz, J. C. (1974) J. Biol. Chem. the modified residues that derive their methyl moieties from 249, 1198-1206. methionine do not become labeled with [14C]formate. Al- 4. Lucas-Lenard, J. & Lipmann, F. (1971) Annu. Rev. Bio- though S. faecalis can utilize formate for serine biosynthesis, chem. 40, 409-448. 5. Delk, A. S. & Rabinowitz, J. C. (1974) Nature 252, 106-109. any radioactive serine formed from [14C]formate is diluted 6. Borek, E. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, into a large pool of the unlabeled amino acid. 139-148. These data suggest that a folate derivative carrying a one- 7. Cantoni, G. L. (196.5) in Transmethylation and Methionine carbon moiety may be the direct source of the methyl group Biosynthesis, eds. Shapiro, S. K. & Schlenk, F. (University T S. exclude the possibility that of Chicago Press, Chicago), pp. 21-32. of of faecalis tTNA and 8. Starr, J. L. & Sells, B. H. (1969) Physiol. Rev. 49, 623-669. S-adenosylmethionine serves as the donor. In S. faecalis, 9. Kerr, S. J. & Borek, E. (1973) in The , ed. Boyer, formate is activated by the formation of 10-formyltetrahydro- P. D. (Academic Press, New York), Vol. 9, pp. 167-195. folate (1). This derivative may be enzymatically reduced to 10. Snell, E. E. & Guirard, B. M. (1943) Proc. Nat. Acad. Sci. 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofo- USA 29, 66-73. 11. Rabinowitz, J. C. (1960) in The Enzymes, eds. Boyer, P. D., late, both of which have been shown to as methyl Lardy, H. & Myrback, K. (Academic Press, New York), donors (11). The participation of a corrinoid coenzyme as the Vol. 2, pp. 185-252. methyl transferring agent, as occurs in the synthesis of 12. Difco Manual (1953) (Difco Laboratories, Inc., Detroit), methionine by N5-methyltetrahydrofolate-homocysteine 9th ed., pp. 225-226. 13. Kenney, W. C. & Dekker, C. A. (1971) Biochemistry 10, methyltransferase (16), should also be considered. 4962-4970. It has previously been assumed that S-adenosylniethionine 14. Nishimura, S. (1972) in Progress in Nucleic Acid Research serves as the methyl donor in every methylation of RNA and Molecular , eds. Davidson, J. N. & Cohn, W. E. (6-9). Based on analyses involving the incorporation of (Academic Press, New York), Vol. 12, pp. 49-85. it has been 15. Randerath, K. & Randerath, E. (1971) in Procedures in methionine in vivo into nucleic acid residues, Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. reported that Bacillus subtilis tRNA contains abnormally low (Harper and Row, New York), Vol. 2, pp. 796-812. quantities of T (17, 18). We (19) have recently shown that T 16. Taylor, R. T. & Weissbach, H. (1973) in The Enzymes, ed. is present in the tRNA of B. subtilis to the extent of one T Boyer, P. D. (Academic Press, New York), Vol. 9, pp. 121- per tRNA molecule. However, the methyl moiety of T in 165. 17. Arnold, H. & Kersten, H. (1973) FEBS Lett. 36, 34-38. B. subtilis tRNA (19), like that of S. faecalis tRNA, is not 18. Klagsbrun, M. (1973) J. Biol. Chem. 248, 2612-2620. derived from methionine. In view of these findings, analysis 19. Romeo, J. M., Delk, A. S. & Rabinowitz, J. C. (1975) of the methylated nucleotide content of nucleic acids that is Biochem. Biophys. Res. Comm., in press. Downloaded by guest on September 29, 2021