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Biosynthesis of Ribosylthymine in the Transfer RNA of Streptococcus

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Biosynthesis of Ribosylthymine in the Transfer RNA of Streptococcus 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 (thymine/tRNA modification/formate/[methyl-'4Clmethionine/GlT'C-loop) ANN S. DELK AND JESSE C. RABINOWITZ Department of Biochemistry, University of California, Berkeley, Calif. 94720 Communicated by Bruce Ames, November 18, 1974 ABSTRACT Ribosylthymine is not present in tRNA of to produce thymidine 5'-monophosphate, which is sub- Streptococcus faecalis grown in the absence of folic acid, sequently used in DNA synthesis. Because of the folate although this methylated residue does occur in the tRNA of this organism 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 organisms, the methyl moiety the biosynthesis 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 protein 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 acids (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 eukaryotes 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 cell 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 nucleotide 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 polymer 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 growth medium, 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'-nucleotides 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 deoxyuridine 5'-monophosphate (5) or by labeling periodate-treated nucleosides 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- phosphate. 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 adenosine 3'-phosphate. in the presence of folate, no radioactive label from methionine digested to 5'-deoxyribonucleotides 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 nuclease 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.
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