Isolation and chracterization of m5U-methyltransferase from Escherichia coli.

The tRNA-modifying enzyme, S-adenosylmethionine: tRNA (uridine-5)-methyltransferase, has been purified essentially to homogeneity from an Escherichia coli strain containing an elevated level of this enzyme. A rapid, efficient method has been developed for the purification, consisting of polyethyleneimine precipitation to remove nucleic acids, followed by phosphocellulose and Blue Sepharose affinity chromatography. The enzyme is a single polypeptide chain of molecular weight 42,000. It has a pH optimum of 8.4, a Km of 12.5 microM for S-adenyosyl-L-methionine, and a Km of 1.1 microM for wheat germ tRNAGly1. The ability of the enzyme to methylate a variety of tRNA substrates including prokaryotic, eukaryotic, mitochondrial, and chloroplastic tRNAs has been characterized.

' The abbreviations used are: m5U-methyltransferase, S-adenosylmethionine:tRNA (uridine-5)-methyltransferase; m5U or T, ribothymidine; mcmo5U-methyltransferase, S-adenosylmethionine:tRNA (uridine-5-oxyacetic acid)-methylester methyltransferase; Ado-Met, S-adenosyl-L-methionine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid Tricine, N-tris(hydroxymethy1)methylglycine; SDS, sodium dodecyl sulfate; Ado-Hcy, S-adenosylhomocysteine; A260 Crude tRNA from E. & (171, bovine liver (18) and rabbir liver ( 8 ) . and pure cRNAc:y from h e a t germ (19.20) were isoleled se previously published. Oicfyosrelium discoidem mlfochandria were isolated 8s described (21). The loicochondriel tRNA was purified by D U E cellulose chromafoRraphy 88 described by Palatnit g g . (   the nucleic acid (usually 0.3 to 0.45%) can readily be determined. For example, the extract is brought to 0.2% PEI as described. After the PEI-treated extract is stirred for 15 min a small aliquot is removed and centrifuged in a S o~a l l SS-34 rotor at 10,000 rprn (12,000 X g) for 15 min. A clear, golden supernatant indicates that the nucleic acid precipitation is complete. A cloudy supernatant indicates that more Polymin P is required, and should be added as described above. Az,/ AZm ratios at this point in the purification are not useful, since the Polymin P interferes strongly with these readings. Following nucleic acid removal by Polymin P precipitation and centrifugation, solid (NH&S04 was added to the clear golden supernatant to bring it to 85% of saturation. After the (NH4)2S04 dissolved, the precipitated extract was stored at -20°C for at least 2 h and then centrifuged at 12,000 rpm for 30 min in a S o~a l l GSA rotor. The resulting pellets were stored at -20°C until further use.
Phosphocellulose Chromatography-The (NH4)2S04 pellets of the extract following PEI precipitation were each dissolved in 20 to 40 ml of dialysis buffer containing 10 mM Tris-HC1, pH 7.6,O.l mM Na2EDTA, 10% glycerol, and 0.5 mM dithiothreitol. The sample was dialyzed, usually for a total of about 24 h, against several changes of this buffer in a ratio of 10 volumes of dialysis buffer to 1 volume of sample. The dialysis was continued until the addition of a few drops of 1 M BaC12 to a 1-ml aliquot of the buffer failed to produce a white precipitate of BaS04.
As the concentration of (NH4)zS04 in the extract decreases during dialysis, a white precipitate usually forms in the sample. This is readily removed, with little or no loss of enzymic activity, by centrifugation at 12,000 rpm for 15 min in a SON^ GSA rotor.
The dialysate, having an Azm of 20, an of 13, and a volume of 443 ml , was applied to a 450-ml phosphocellulose column (2.6 X 85 cm) previously equilibrated with 0.1 M potassium phosphate, pH 6.5, in PC buffer containing 10% glycerol, 0.5 m~ Na2EDTA, and 0.5 m~ dithiothreitol. Following sample application, the column was washed with 50 ml of 0.1 M potassium phosphate, pH 6.5, in PC buffer and then with 400 ml of 0.15 M potassium phosphate, pH 6.5, in PC  Fig. 1. Most of the protein was not retained on the phosphocellulose column. The enzyme, however, was retained on the column, and was eluted subsequently with approximately 0.2 M potassium phosphate. Fractions containing the m5U-methyltransferase activity were pooled, and the sample brought to 85% of (NH4),S04 saturation and centrifuged as described above. This purification step was very effective, achieving a more than 40-fold purification.
It should be noted that the potassium phosphate concentration required to elute the enzyme may vary with different lots of phosphocellulose. New lots should therefore be tested prior to use on a large scale. Furthermore, the enzyme activity has been observed occasionally to split into two peaks upon phosphocellulose chromatography. The reason for this is not known. Splitting was found to vary with different lots of phosphocellulose and the occurrence of splitting did not appear to affect the properties of the m"U-methyltransferase. Splitting is believed to be a column artifact and not to represent two forms of the m'U-methyltransferase enzyme since, on those occasions when splitting was observed, the enzyme mcmo5U-methyltransferse was also found to be split. Moreover, both enzymes split in the same proportions. In addition, there is no evidence for two forms of m'U-methyltransferase as judged by a single sharp peak observed upon gel fdtration chromatography on Ultrogel AcA 54, and one major protein band on SDS-polyacrylamide gels.
Blue Sepharose Affinity Chromatography-One of three (NH4)2S04 pellets from the m"U-methyltransferase activity peak of the phosphocellulose column was dialyzed into dialysis buffer as described above for phosphocellulose chromatography. When aLl of the (NH4),S04 was removed, the sample was then dialyzed into 10 mM potassium phosphate, pH 7.0, 2 mM NarEDTA, 20% glycerol, and 0.5 mM dithiothreitol, resulting in 4.5 ml of sample having an ArM, of 5.6 and an of 3.6. An aliquot of this sample (PC enzyme) was removed for further purification and the remainder was frozen at -70°C in aliquots for future use. PC enzyme (1.5 m l ) was applied to a 3-ml Blue Sepharose column (0.6 X 10.5 cm), previously equilibrated with 2 mM NasEDTA, 20% glycerol, and 0.5 mM dithiothreitol (BSC buffer) containing 10 mM potassium phosphate, pH 7.0. The column was washed successively with 3 rnl of 10 mM potassium phosphate, pH 7.0, in RSC buffer, 9 ml of 40 mM potassium phosphate, pH 7.0, in BSC buffer, and 12 ml of 10 mM potassum phosphate, pH 7.0, in BSC buffer. The enzyme was then affinity eluted with 12 ml of BSC buffer containing 10 mM potassium phosphate and wheat germ tRNA"" I . a t a concentration of 1 AZM, unit/ml. The column was then washed with 6 m l of 10 mM potassium phosphate, pH 7.0, in BSC buffer. The affinity elution step does not remove all of the enzyme from the column. The remaining m'U-methyltransferase (approximately 40% of the total) was eluted from the column with a 12-1111 wash of 200 mM potassium phosphate, pH 7.0, in BSC buffer. This enzyme is considerably less pure than the affinity-eluted enzyme. One-milliliter fractions were collected and assayed as described above. The affinity-eluted m'U-methyltransferase activity peak was pooled as shown in Fig. 2a. The enzyme was then freed of tRNA and concentrated by the use of a small ( 1 . 5 4 ) DEAE-cellulose column built in a 5-ml pipette. The affinity-eluted enzyme (16 m l ) was applied to the small DEAE-cellulose column which was previously equilibrated with 10 mM Tris-HC1, pH 7.6, 1 mM Na,EDTA, 10% glycerol, and 0.5 mM dithiothreitol (DEAE-buffer) con-  taining 10 mM KC1, and the column was washed with 4 ml of DEAE-buffer containing 10 n m KCl. The enzyme was then eluted with 0.22 M KC1 in DEAE-buffer. The fractions were assayed, and the activity peak pooled (Fig. 26). The enzyme was pipetted into prefrozen Eppendorf tubes and stored a t -70°C until needed. Since the enzyme at this stage is quite sensitive to freeze-thawing, it is usually stored in 5O-pl aliquots which are thawed once for use. The enzyme is stable at -70°C for at least 6 months. To recover the tRNA for reuse, the small DEAE-cellulose column was washed with 1 M KC1 in DEAE-buffer, and the tRNA precipitated with 2 volumes of ethanol.
The complete purification procedure is summarized in Table I.

Determination of the Molecular Weight and Purity
SDS-polyacrylamide gel electrophoresis of the affinityeluted Blue Sepharose enzyme resulted in only a single major band of M , = 42,000 (Figs. 3a, and 4a). The densitometer tracing of this gel (Fig. 4) shows that the enzyme is essentially homogeneous, with only traces of impurities present. This enzyme preparation was also chromatographed on Ultrogel AcA 54. A single major protein peak was observed which coincided with the m'U-methyltransferase activity peak, indicating that the major band on the gel was the m5U-methyltransferase (data not shown).
The m'U-methyltransferase was also chromatographed together with molecular weight standards on Ultrogel AcA 54 in the absence of SDS. Under these conditions, the enzyme was found to have a M , of 38,000 (Fig. 36).

Optimization of pH and Ionic Strength
Using the highly purified enzyme, the effect of pH on m5Umethyltransferase activity has been determined over a range of pH 7.6 to pH 8.8 with both Hepes and Tricine buffers. Both buffers resulted in the same activity, and with either buffer, an optimum of pH 8.4 is observed (data not shown). A 25% inhibition of enzyme activity occurs at pH 7.8 and pH 8.8. The effect of ammonium acetate and spermidine on m5Umethyltransferase has also been investigated. Ammonium acetate has a broad optimal concentration range around 50 mM, at which concentration the methylation rate is enhanced 30% relative to the rate in the absence of ammonium acetate. The optimal concentration of spermidine in the presence of 50 mM ammonium acetate has been determined to be 20 mM, at which concentration a slight stimulation (1.0-to 1.1-fold) is observed (data not shown).

Specificity of Methylation
The specificity with which m5U-methyltransferase either from E. coli SNOl (pTN015) or E. coli MRE 600 forms m5U has been confirmed by 2-dimensional thin layer chromatography. When the methylated tRNA, either wheat germ tRNA?IY, bovine liver tRNAVa', or Dictyostelium discoideum vegetative stage mitochondrial tRNA, was digested to mononucleotides and chromatographed as described above, the only methylated product was m5U (data not shown). Moreover, it has previously been shown that all of the m5U produced by the methylation of wheat germ tRNA?'Y with m5Umethyltransferase of E. coli MRE 600 is present in the normal position that m5U occupies in tRNA, i.e. the 23rd nucleotide from the 3'-end of the molecule in the sequence G T W (6,20).

Methylation of Various tRNA Substrates
A variety of tRNA substrates have been methylated by the highly purified m5U-methyltransferase. The extent of methylation of these tRNAs has been determined (Table II), along with that of wheat germ tRNA?ly and bovine liver tRNAV"', both of which are known to competely lack m5U (4). Unfractioned E . coli tRNA, presumably because all of the molecules already possess m5U, is not a substrate for the enzyme.
The initial rates of methylation have also been determined for these tRNAs (Fig. 5). The data points represent the per cent of complete methylation which has occurred in the times indicated for each substrate.

Kinetic Studies
The initial velocities were determined for the methylation of wheat germ tRNA?'Y over a tRNA concentration range of 60 to 2000 nM and an Ado-Met concentration of 2.5 to 30 PM. Duplicate assays were incubated for 2 min which is within the linear range of the reaction, and lines were fitted to the data points by unweighted least squares analysis. From the data, the K, values have been determined to be 12.5 p~ for Ado-Met and 1.1 PM for wheat germ tRNA?IY.

DISCUSSION
Blue Sepharose and Affi-Gel Blue (Bio-Rad) have been used by a number of investigators (37-44) for the purification of enzymes which interact with nucleic acids and related molecules. The enzymes have an affinity for the dye, Cibacron Blue F3G-A, which is bound to the agarose matrices in these resins. Since only 60% of the m5U-methyltransferase was removed from the Blue Sepharose column by the affinity elution process described above, attempts were made to improve the recovery. Affinity elution of the enzyme with a wheat germ tRNA?IY gradient (0 to 1 A2w/ml in 10 mM potassium phosphate, pH 7.0, in BSC buffer) improved neither the purity nor the yield of the enzyme (data not shown), while attempts to affinity elute more of the enzyme by raising the tRNA concentration in the buffer to 2. 5 units/ml resulted in enzyme of lower purity (Fig. 4, Scan b).
In view of the ease and simplicity of the purification scheme described here, an attempt was made to extend the procedure to other tRNA methyltransferases. Preliminary results from studies on the purification of mcmo5U-methyltransferase from E. coli SNOl (pTN015) indicate that this enzyme can also be purified by chromatography on Blue Sepharose resin. The Blue Sepharose procedure reported here, and variations thereof, may indeed serve as a general method for the purification of tRNA methyltransferases.
The molecular weight of m5U-methyltransferase was determined by SDS-polyacrylamide gel electrophoresis to be 42,000. A M, of 38,000 was determined for this enzyme upon gel fitration on Ultrogel AcA 54 in the absence of SDS. This indicates that the native form of the enzyme is probably a monomer. These values are in excellent agreement with a M , of 42,000, which has recently been observed for m5U-methyltransferase synthesized in a minicell system directed by a 2.9kilobase E. coli chromosomal DNA fragment containing the structural gene for m5U-methyltransferase (27).
E. coli m5U-methyltransferase catalyzes the synthesis of m5U in E. coli tRNAs at a specific uridine residue 23 nucleotides from the 3'-end of the molecule. The enzyme has a broad substrate range and is capable of methylating a relatively large number of tRNAs from a wide range of different organisms as illustrated in Table 11. Indeed there is no known tRNA which has a uridine at the 23rd position from the 3'end which is not a substrate for this enzyme. This enzyme has even been shown to form m5U in viral tRNA-like moieties at a site corresponding to that occupied by m5U in tRNA (45,46). Apparently, the m5U-methyltransferases of higher eukaryotes, such as wheat germ and mammals, have a narrower substrate range, since in each case, there is a subset of tRNAs which lack m5U and have an unmodified uridine instead (4,6,

8).
Kinetic studies with highly purified m5U-methyltransferase gave a K,,, value of 12.5 ~L M for Ado-Met which is 30-fold greater than that reported for another highly purified methyltransferase, the 1-methyladenosine methyltransferase of rat liver (11). Higher K , values have been previously observed for Ado-Met with E. coli as compared to mammalian tRNA methyltransferases (1). The isoelectric point (PI, 4.8) and the sensitivity of the m5U-methyltransferase to Ado-Hcy (50% inhibition of the methylation rate at an Ado-Hcy concentration of 2 p~) have previously been determined for m5U-methyltransferase isolated from E. coli MRE 600 (47). Table I1 shows the extent of m5U formation with highly purified m5U-methyltransferase and a variety of tRNAs. As expected, Bacillus subtilis and E. coli tRNAs are either very poor substrates or are not methylated at all by this enzyme. The other prokaryotic tRNA studied, from Anacystis nidulans, is clearly a suitable substrate and methylates to an extent about 10-fold greater than that of B. subtilis. Among the eukaryotic tRNAs studied, D. discoideum total tRNA and rabbit liver tRNA are both substrates, with rabbit liver tRNA being methylated to about twice the extent of D. discoideum tRNA. It is interesting that, of the two organelle tRNAs studied, the spinach chloroplast tRNA is methylated to only a very limited extent, comparable to that of prokaryotic tRNA, while D. discoideum mitochondrial tRNA is quite a good substrate, being methylated to an extent more than 10fold higher than spinach chloroplast tRNA. The methylation of mitochondrial tRNA was not totally unexpected, since two mitochondrial tRNAs recently sequenced, Neurospora crassa tRNAr" (48) and yeast tRNAPh' (49), have a uridine at the 23rd position from the 3'-end of the molecule.
Studies of the kinetics of methylation (Fig. 5) show that the prokaryotic tRNAs studied are methylated with more rapid kinetics than the eukaryotic species tested. An exception to this is the chloroplast tRNA, which methylates with similar kinetics to B. subtilis and A . nidulans tRNAs. This may imply structural similarities between certain chloroplast tRNAs and those of prokaryotes. On the other hand, D. discoideum mitochondrial tRNA is methylated with kinetics quite dissimilar to the rapid kinetics observed with spinach chloroplast and prokaryotic tRNAs. of E. coli SNOl (pTN015). Drs. M. Freundlich, K. Nakamura, and M.