Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA

ARCHIVES Vol248,

OF BIOCHEMISTRY AND BIOPHYSICS No. 2, August 1, pp. 540-550,1986

Biosynthesis of 5-Methylaminomethyl-2-selenouridine, a Naturally Occurring Nucleoside in Escherichia co/i tRNA ARTHUR Section

J. WITTWER’

012 Intermediary Metabolism and Blood Institute,

AND

THRESSA

and Bioenergetics, National Institutes Received

Laboratory of Health,

February

C. STADTMAN2 of Biochemistry, Bethesda, Maryland

National 20892

Heart,

Lung,

20,1986

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/lOO mol tRNA) when selenium is present at about 1 PM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 PM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coZi cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATPdependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium. 0 1986 Academic Press. Inc.

Selenium is found in biomolecules as the result of both specific and nonspecific incorporation mechanisms. The chemical similarity of sulfur and selenium allows many of the enzymes of sulfur metabolism to catalyze with near equal efficiency the analogous reactions with selenium-containing substrates (1). This provides pathways for the nonspecific incorporation of selenium into macromolecules. An example is the facile synthesis of selenomethioninecontaining proteins by Escherichia coli and Clostm’dium kluyveri when selenomethionine or inorganic selenium is present (2,3). Certain enzymes, however, whose activity depends on the presence of selenocysteine

at a specific location in the polypeptide have been identified. In these cases, incorporation of selenium must occur by a unique mechanism rather than by random substitution for sulfur. Environmental S to Se ratios usually are lOOO-10,000 to 1, yet in the two best-characterized seleniumdependent enzymes, the cysteine to selenocysteine ratio is only 2 to 1 (4) or 4 to 1 (5). The number of active species of these enzymes which could be formed under normal conditions by chance replacement of cysteine with selenocysteine would be prohibitively small. How the selection of selenium from the environment and its specific incorporation into proteins take place are unknown, but of considerable interest. Selenium also is specifically incorporated into another class of sulfur-containing macromolecules, the amino acid transfer ribonucleic acids (tRNAs). Selenium-modified tRNA was first noted in E. coli, but

1 Present address: Monsanto Company, 800 N. Lindbergh Boulevard, St. Louis, MO. 6316’7. ‘To whom correspondence should be addressed: National Heart, Lung, and Blood Institute, NIH, Building 3, Room 108, Bethesda, Md. 20892. 0003-9861/86 Copyright All rights

$3.00

0 1966 by Academic Press, Inc. of reproduction in any form reserved.

540

SELENONUCLEOSIDE

was assumed to represent nonspecific substitution of selenium for sulfur (6-8). Additional studies, however, showed that incorporation of ‘5Se label into tRNAs of Clostridium sticklandii (9), E. coli (10, ll), and Methanococcus vannielii (12) was insensitive to the level of environmental sulfur. The seleno-tRNAs in these organisms that have been characterized to date have either lysine or glutamate acceptor specificity (11-13) and contain the selenonucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U)3 (14). This compound is the selenium analog of 5-methylaminomethyl-2-thiouridine (mnm5s2U), a thionucleoside found in E. coli tRNALys (15) and tRNAG’” (16). In an effort to define the mechanism whereby specific incorporation of selenium into this type of biomolecule occurs, the biosynthesis of the mnm5se2U residue in E. coli tRNA was studied. EXPERIMENTAL

PROCEDURES

Materials. Ha”SeOa and HZa5S04 were obtained from New England Nuclear. “SeOf was oxidized to ?SeOqwith Hz02 as described previously (11). [3H]Uridine was obtained from Schwarz-Mann and S-[methyl-3H]adenosylmethionine from Amersham. Purified E. coli tRNALYB and tRNAG’” were obtained from Sigma Chemical Company. Authentic mnm5seq and other nucleosides were prepared as previously described (14). Source and growth of E. coli strains. Wild-type E. coli K12 strain WGl was obtained and grown in a minimal glucose-nitrate medium as previously described (11). E. coli strains GM1 (parent) and GM19 (tmC mutant) (1’7) were obtained from Martin G. Marinus, Department of Pharmacology, University of Massachusetts (Worcester, Mass.). E. coli strains DEVl (parent) and DEV16 (trmE mutant) (18) were obtained from Dirk Elseviers, Department of Microbiology, New York Medical College (Valhalla, N.Y.). The mutant and parent strains were routinely cultured in Luria broth (LB broth from Quality Biological, Inc., Rockville, Md.). Labeling of E. coli with ?Se was done by anaerobic growth in the presence of 1 pM “SeOi(1 &i/ml) at 30°C unless otherwise indicated. Isolation of tRNA. E. coli cells were harvested from lo-ml cultures by centrifugation, washed once with

3 Abbreviations methyl-2selenouridine; methyl-2-thiouridine.

used:

mnm5sev, mnmss2U,

5-methylamino5-methylamino-

BIOSYNTHESIS

IN

E. coli

541

buffer A (20 mM sodium acetate, 10 mM MgC12, 1 mM dithiothreitol, pH 5.0), resuspended in 1 ml of buffer A, and extracted with 1 ml of 88% phenol by vortexing for about 1 h. After addition of 0.2 ml of 5 M NaCl, the mixture was centrifuged to separate phases. Two volumes of ethanol were added to the aqueous phase. After several hours at -2O”C, the precipitate was collected by centrifugation, dissolved in 1 ml of buffer A, and applied to a small (0.3 ml) column of DEAEcellulose (DE52, Whatman) equilibrated with buffer A. The column was washed with 1 ml of buffer A containing 0.3 M NaCl, and the tRNA eluted with 1 ml of 1 M NaCl in buffer A. As previously described, this procedure resulted in minimal (lo-15%) contamination with high-molecular-weight RNA (11). For ‘5Selabeled tRNA, selenium incorporation was estimated from the absorbance at 260 nm and radioactivity of the DEAE-cellulose column eluate or by direct HPLC nucleoside analysis (see below). Similar values were obtained by either method. For HPLC nucleoside analysis, the tRNA in the 1 M NaCl eluate was precipitated with ethanol and dissolved in 25 ~1 of water. HPLCnucleoside analysis Up to 3 Azso units of tRNA were hydrolyzed with nuclease Pl and bacterial alkaline phosphatase as described by Gehrke et al. (19), except that the incubations were performed under argon and in the presence of 1 mM dithiothreitol. Immediately prior to chromatography, 10 to 15 ~1 0.5 M Tris-HCl, pH 8.0, was added to the digestion mixture to improve the resolution of mnm5se2U and mnm5s2U. Without this addition, mnm5se2U eluted at the same position as mnm’s2u. For HPLC, the entire digestion mixture was injected onto a 0.4 X 30-cm PBondapak C-18 column (Waters Associates) equilibrated with 10 mM ammonium acetate, pH 5.3 (970/o), and methanol (3%). Chromatography was at room temperature and 1 ml/min flow rate. Elution of the column was performed with linear gradients of 3 to 5% methanol from 0 to 12.5 min, 5 to 20% methanol from 12.5 to 27.5 min, and 20 to 100% methanol from 27.5 to 30 min. The column was then washed with 100% methanol for 10 min before returning to the initial conditions. A Spectra-Physics SP8700 solvent delivery system and a Hewlett-Packard 1040A spectrophotometric detector were used. The eluate was monitored at 257 and 313 nm (8nm bandwidths) and both signals were referenced against the absorbance at 550 nm (loo-nm bandwidth). Radioactivity in the HPLC fractions was measured with a Beckman Gamma 5500 counter or by liquid scintillation counting in ACS counting fluid (Amersham) using an LKB RacBeta scintillation counter. Amounts of mnmsseaU and mnm”sS were determined from the integrated 313-nm absorbance of the appropriate peaks by using the molar absorptivities for mnm5se2U and mnm5saU at 313 nm of 17,000 cm-i M-' (14) and 5000 cm-i M-l, respectively. This latter value was determined from the uv spectrum of mnm5s2U under HPLC conditions. The absorbance of mnm6saU

542

WITTWER

AND

at 313 nm was one-third of its maximum absorbance at 275 nm, for which the molar absorptivity is reported to be 15,200 M-I cm-’ (20). Levels of mnm5se2U and mnm’sw were expressed as picomoles per Am unit of tRNA by calculating the A, units from the amount of pseudouridine recovered in each analysis. It was found experimentally that 1 Azso unit of bulk E. co.3 tRNA yielded 0.019 AZ7 unit of pseudouridine. For comparison, the pseudouridine nucleoside content of E. coli tRNA has been reported to be 1.77 to 2.16 mol% (21-23). These values correspond to a pseudouridine content of 0.017 to 0.021 AZ7 unit per Am unit of bulk tRNA if one assumes an average of ‘78 nucleoside residues per tRNA, 1.66 nmol tRNA per Am unit, and a molar absorptivity of 7500 cm-’ M-’ (24). Determination of selemmethionine and methionine in E. eoli protein Bulk E. coli “Se-labeled protein was isolated from the phenol layer that remained after isolation of r5Se]tRNA (see above). Dialysis of the radioactive phenol solution against 20 mM sodium acetate, pH 5.0,l mM dithiothreitol resulted in the formation of a precipitate that contained over 90% of the 76Se label. About 1 mg of this precipitate was hydrolyzed with HCl and the amino acids were determined by ion-exchange chromatography as previously described (25). Selenomethionine was estimated from the ?YJe radioactivity which eluted coincident with standard selenomethionine, and methionine was determined by fluorescence, using postcolumn derivatization with o-phthalaldehyde. In vitro methyl&ion of ‘5Se-bbeled GM19 tRNA. The reaction mixture (100 ~1) similar to that described by Marinus et al. (17) contained 1.9 Aw units of E. coli GM19 r5Se]tRNA, 100 nmol (2 j&i) S-[methyl3H]adenosylmethionine, 300 nmol dithiothreitol, 5 pmol Tris-HCl, pH 7.5, 1 pmol MgC12, 1 pmol KCl, and 10 pl of E. coli enzyme preparation (Am = 15). The enzyme preparation used was a crude aminoacyltRNA synthetase preparation, isolated from E. coli K12 as described previously (11). This preparation was found also to possess the desired methylase activity. After incubation at 3’7”C, reaction mixtures were diluted with 1 ml of 0.1 M sodium acetate, pH 5.0, containing 0.2 M NaCl, and the tRNA was purified using small columns of DEAE-cellulose. The ‘5Se- and 3Hlabeled nucleosides in digests of the tRNA samples were analyzed by HPLC as described above. In samples incubated for 1.5 and 3 h, 13 and 17%,respectively, of the mutant r’Se]nueleoside was converted to a ‘Hlabeled compound which coeluted with authentic mnm6se2U. The only 3H-labeled nucleosides that were detected in the digests eluted from the HPLC column at positions corresponding to mnm’se2u and mnm5sS.

RESULTS

Influence of selenium level cm amounts of mnm%$Uand mnm%?Uin tRNA. Labeling

STADTMAN

of E. co.3 tRNA by growth in the presence of low levels (0.1-l PM) of %eOgor 75Se0z- results in the synthesis of “Se-labeled mnm5se2U (14). Previous studies, conducted primarily with cultures in which cell division had been stopped by chloramphenicol (ll), had indicated that the incorporation of selenium into tRNA is relatively insensitive to variations in selenium or sulfur concentrations of the culture medium. To extend these observations, the effects of extremes of sulfur and selenium in the culture medium on the levels of mnm5se2U and mnm5s2U in E. co2i tRNA synthesized during normal growth were examined. In certain studies in which the unlabeled selenonucleoside was determined, it was necessary to measure low levels of mnm5se2U in the presence of appreciably higher amounts of mnm5s2U. It was found that as little as 5 pmol of the selenonucleoside could be measured by uv absorbance when the nucleosides in hydrolysates of bulk tRNA were separated by reversed-phase HPLC. As shown in Fig. 1, the absorbancies due to mnm5se2U and mnm5s2U are both readily apparent in the effluent from the column monitored at 313 of the selenonucleoside) in adnm W,,, dition to 257 nm. The levels of mnm5se2U and mnm5s2U in this preparation (Fig. 1) were calculated to be 67 and 95 pmol per Azso unit, respectively, based on the 313nm absorbance. Analysis of the tRNAs from bacteria cultured under a wide variety of conditions showed that there is an inverse relationship in the levels of mnm5se2U and its sulfur isolog (Table I) with the total of the two nucleosides remaining relatively constant. The maximum level of mnm5se2U was observed with 1 PM selenium in the medium and no further increase in amount of this selenonucleoside occurred when higher levels of selenium were added. In fact, at a high selenium concentration (100 PM) which inhibits growth somewhat, the selenonucleoside levels were considerably lower. The marked specificity of this selenium incorporation by E. coli is apparent when levels of mnm5se2U in tRNA are compared with levels of selenomethionine in bulk protein. Whereas the selenomethionine to methionine ratio in protein is

SELENONUCLEOSIDE

BIOSYNTHESIS

IN

E. co&i

543

20

Time,

min

FIG. 1. Nucleoside analysis of “Se-labeled tRNA from wild-type E. wli K12. [7SSe]tRNA was isolated from cells grown with 1 PM “SeOf(about 1 pCi/nmol), enzymatically hydrolyzed, and subjected to HPLC analysis as described under Experimental Procedures. The four major peaks of 25’7-nm absorbance are, in order of elution, cytidine, uridine, guanosine, and adenosine. The minor peak of 257-nm absorbance at about 4 min is pseudouridine. Peaks of 313-nm absorbance due to mnmsse2U (*) and mnm’s?J (#) are indicated. The prominent peak of 313-nm absorbance eluting near guanosine is due to I-thiouridine. A minor peak of 313-nm absorbance eluting at about 17 min is due to oxidized dithiothreitol present in the digestion mixture.

proportional to the Se to S ratio in the culture medium, maximal mnm5se2Usynthesis was observed at Se to S ratios of 1 to 8000. It is of interest that in the absence of added selenium no mnm5se2U was detected in the tRNA population. Apparently, the selenium content of the glucose-nitrate minimal medium used in these studies is extremely low. This could explain the failure to detect mnm5se2Uin numerous compositional studies of E. coli tRNAs reported in the literature. In spite of the well-documented requirement of selenium supplements for synthesis of formate dehydrogenases by E. coli (26, 27), selenium-deficient synthetic or semisynthetic media often have been used for culture of the cells from which the tRNAs were prepared. Some nondefined standard microbiological culture media contain sufficient available selenium for synthesis of mnm5se2U, but others such as Luria broth, which is commonly used for culture of E. coli, are deficient (Table II). Only when Luria broth was supplemented with 1 /IM SeOg- was the level of selenonucleoside synthesis similar to the maximal level obtained in selenium-

supplemented synthetic media (Table I) or certain of the nondefined media (Table II). There appears to be no requirement for anaerobic conditions for synthesis of mnm5se2U in tRNAs since similar levels were found in cells from cultures either incubated anaerobically or exposed to air (aerobic). Synthesis of selenonucleosides by E. coli treated with chloramphenicol. Although E. coli continues to synthesize tRNAs after cell division is stopped by the addition of chloramphenicol, it has been shown that treatment with this antibiotic results in the appearance of undermodified nucleosides in the tRNA population (23). At least three selenonucleosides, in addition to mnm5sezU,were detected in the r5Se]tRNA isolated from E. coli cells incubated for 2 h with chloramphenicol and 1 PM 75Se0$(Fig. 2). The levels of these additional r5Se]nucleosides, two of which eluted earlier in the HPLC profile, decreased with further incubation, while the relative amount of mnm5se2U increased. The total ‘5Se label in the tRNA remained constant during this time. These results imply that

544

WITTWER

AND

STADTMAN

TABLE

I

LEVELS OF mnm%e%J AND mnm5s2U IN tRNA, AND SELENOMETHIONINE AND METHIONINE IN PROTEIN, ISOLATED FROM E. coli GROWNWITH DIFFERENT AMOUNTS OF 75Se0:m or '5Se0iConcentration of Se added to the growth medium (pM) 0 0.1 (SeO:-) 0.5 1.0 1.0 10.0 100.0 0.1 1.0 10.0

(SeOf) (SeOz-) (SeO!-)* (SeO:-) (SeOq-) (SeOi-) (SeOg-) (SeOi-)

100.0 (SeO:-)

tRNA

(pmol/Azso

mnm’se2U

unit) mnm5saU

Se-Met

Protein: to Met

0

144

-6

4 27 63 41 46 12 35 55 45 33

60 114 106 134 95 67 65 72

1 to 1100

Note. All cultures were grown anaerobically in minimal medium containing 8 mM SOfNucleosides were determined by HPLC and amino acids by ion-exchange chromatography Experimental Procedures. D 40 pM SOi- instead of 8 mM. b Not determined.

chloramphenicol treatment results in the transient accumulation of 75Se-labeled nucleoside intermediates during the biosynthesis of mnm5se2U. However, under normal growth conditions these additional [75Se]nucleosides are not observed in wildtype E. coli. Synthesis of selenonucleosides by E. coli mutants. The high specificity with which selenium is incorporated into E. coli tRNA raises interesting questions as to the mechanism of biosynthesis of the selenonucleoside, mnm5se2U. Its structural similarity to the analogous thionucleoside, mnm5s2U, suggests that the two nucleosides may be synthesized by similar and perhaps partially shared pathways. The high specificity of mnm5se2U synthesis, however, requires a unique step whereby selenium is incorporated at the 2-position. The post-transcriptional modification reactions which lead to synthesis of mnm5s2U residues in E. coli lysine and glutamate tRNAs have been only partially elucidated. Studies with trmC mutants of E. coli (17, 28) or methyl-deficient tRNA (29) indicated that an S-adenosylmethionine-dependent reaction supplies the

ratio

1 to 130 1 to 9 -

except where as described

noted. under

terminal methyl group of the Ei-methylaminomethyl side chain. A different E. coli mutant (termed trmE) which lacks mnm5s2U (18) was reported to contain an unmodified 2-thiouridyl residue in its tRNA. To assess the effect of these mutations on selenonucleoside biosynthesis, E. coli trmC and tmzE mutants were grown in the presence of ‘5Se. HPLC analysis of nucleosides from r’Se]tRNAs isolated from the trmC and trmE parent strains indicated normal synthesis of mnm5se2U and mnm5s2U, similar to that seen in E. coli K12 (Fig. 1). However, r5Se]tRNA isolated from trmC mutant cultures showed the total absence of mnm5seS and mnm5s2U and the appearance of an increased peak of 313nm absorbance that eluted at the position of 2-thiocytidine (Fig. 3a). Rechromatography of this peak indicated the presence of a new seleno- and thionucleoside, each distinct from mnm5se2U and mnm5s2U but having similar uv spectra (data not shown). Selenium was incorporated to an equal extent into the tRNAs of both parent (56 pmol/Azso unit) and trmCmutant (58 pmol/ A260 unit) strains. The trmE mutant similarly showed a complete lack of mnm5se2U

SELENONUCLEOSIDE TABLE

BIOSYNTHESIS

II

BBL thiotone” BBL thiotone (aerobic) LB broth* LB broth + 1 pM SeOf LB broth + 1 pM SeOi(aerobic) Difco nutrient broth Difco beef extract Difco beef extract (aerobic)

545

co.5

61 59 12 55

6’7 45 153 74

55 45 4

85 93 133

2

144

Incwpwation of selenium into tRNA during normal growth. To gain insight into

pmol/AW unit of tRNA medium

E.

3H]methionine and a crude enzyme preparation from wild-type E. coli resulted in partial conversion of the mutant selenonucleoside to a 3H-labeled compound that coeluted with authentic mnm5se2U (data not shown). From these studies it is evident that, like sulfur, incorporation of selenium at the 2position of the uridyl precursor does not require a completed 5-methylaminomethyl side chain to be present. The greatly decreased selenonucleoside synthesis in the trmE mutant, however, suggests that the absence of a substituent at the 5-position may partially inhibit incorporation of selenium at the 2-position.

LEVELS OF mnm5sezU AND mnm5s*U IN tRNA FROM E. coli GROWN IN VARIOUS STANDARD MICROBIOLOGICAL MEDIA

Growth

IN

mnm5se2U

mnmVU

Note. Isolation of tRNA and analysis of nucleosides by HPLC were as described under Experimental Procedures. Commercial dry media were prepared as 1% solutions. All cultures were grown under anaerobic conditions except as indicated. ’ BBL thiotone from Baltimore Biological Laboratories is described as a high-sulfur peptone by the supplier. “LB broth (Luria broth) was from Quality Biological, Inc., Rockville, Md.

and mnm5s2U (Fig. 3b). In this case, however, significantly less selenium (10 pmol/ Azso unit) was present in the mutant tRNA than in that of the parent (65 pmol/Azm unit). The trmE mutant selenonucleoside eluted later in the HPLC profile, just after guanosine (Fig. 3b). The 313-nm absorbance of this nucleoside is not distinguishable because of the closely eluting 4thiouridine (Fig. 3b). A new peak of 313-nm absorbance eluting just before guanosine (Fig. 3b) may be the mutant thionucleoside, presumably 2-thiouridine. Preliminary characterization of the mutant selenonucleoside isolated from trmC tRNA (Fig. 3a) suggests that it is 5-aminomethyl-2-selenouridine. Cleavage of the sugar residue by anaerobic treatment with HCl yields a degradation product that coelutes with authentic 5-aminomethyl-2-selenouracil (A. Wittwer and L. Tsai, unpublished observations). As expected from similar studies with the thionucleoside from this mutant (17), incubation of the intact trmC tRNA with S-adenosyl[methyl-

the nature of the tRNA precursor which under normal conditions serves as substrate for the selenium incorporation reaction, selenium-deficient E. coli cultures were supplemented with 1 PM selenite under various conditions and the synthesis of mnm5se2U was monitored by HPLC analysis of tRNA digests. When both [3H]uridine and r5]selenite were added to stationary phase E. coli cultures, the level of mnm5se2U increased linearly from 0 to 3547 pmol/Az60 unit (about 2-2.5 mol Se/100

0

5

10

15 Time,

20

25

30

min

FIG. 2. HPLC elution profile of “Se-labeled nucleosides from tRNA synthesized by E. coli in the presence of chloramphenicol. Cells were grown to mid-log phase in the absence of added selenium. At zero time, 100 pg/ml chloramphenicol and 1 @M 75SeOf (1 &X/nmol) were added to each lo-ml culture. After incubation for 2 or 6 h at 30°C, cells were harvested, the r5Se]tRNA was isolated, and HPLC nucleoside analysis was performed as described under Experimental Procedures. The major peak of “Se label at 9.5 min coeluted with authentic mnm6se2U.

546

WITTWER

AND

0.03

0.02

i : i 0

L 0

10 Time,

20 min

30

FIG. 3. Nucleoside analysis of 75Se-labeled tRNA from E. wli mutants deficient in synthesis of mnm%?U. (a) h’. edi GM19 (a trmcmutant) and (b) E. wli DEV16 (a trmE mutant). r’Se]tRNA was isolated from cells grown with 1 piu ‘%eO%- (about 1 &i/nmol), enzymatically hydrolyzed, and subjected to HPLC as described under Experimental Procedures. The elution positions of pseudouridine, cytidine, uridine, guanosine, 4-thiouridine, and adenosine are as described for Fig. 1. The elution positions of mnm6se2U (*) and mnm5szU (#) in parallel HPLC runs of the parent strains of GM19 and DEV16 (GM1 and DEVl, respectively) are indicated by arrows in panels (a) and (b). The peak of 313-nm absorbance from 2-thiocytidine and coeluting mutant thio- and selenonucleosides from GM19 are indicated by an additional arrow in panel (a).

mol tRNA) over a 4-h period. During this time, newly synthesized tRNA (based on the level of 3H labeling) accounted for 1.3% of the total. Thus, at most, only one-half of the 75Se-tRNA formed under these conditions could have come from newly synthesized tRNA. However, the fraction of newly synthesized seleno-tRNA precursors was probably much lower, since tRNALy” and tRNAG’” species-in which are included the major E. coli seleno-tRNAs (ll)-represent about 10% of the bulk tRNA population (30, 31). Under these

STADTMAN

conditions, then, most of the mnm5se2Ucontaining tRNA must have been synthesized by the further modification of preformed, stable tRNAs. When the selenium was added to deficient mid-log-phase cultures of E. coli, mnm5se2U levels increased from zero to about 30 pmol/Azso unit during the first 120 min, whereas mnm5szU levels decreased from 150 to 120 pmol/A260 unit. A similar result was obtained when 5-fluorouracil and deoxythymidine were added to mid-log cultures along with the selenium to inhibit the formation of modified uridines, among them mnm5s2U, in the newly synthesized tRNA (32). In this case, mnm5se2U increased from zero to 28 pmol/A20, unit during the first 80 min and mnm5s2U decreased from 147 to 118 pmol/A260 unit. These results suggest that E. coli cells grown in selenium-deficient media contain a substantial pool of preformed tRNA molecules which can be modified with selenium to yield mnm5se2U residues. The reciprocal nature of mnm5se2U and mnm5s2U levels measured in these experiments, and in those reported in Tables I and II, suggests that mnm5s2Uitself is the residue that is modified to form mnm5sev. Loss of sulfur from “S-labeled mnm5.?U concomitant with the fomzation of mnm5se??ZTo determine whether mnm5s2U residues can actually serve as precursors of mnm5se2Uresidues in tRNA, the stability of the ?S-labeled nucleoside in the presence and absence of added selenium was compared. For this purpose sulfur-containing nucleosides of tRNA were labeled by growth of E. coli in media containing 200 PM 35SOz-without added selenium. At midlog phase the cells were washed and resuspended in fresh medium containing 8 mM unlabeled sulfate with and without 1 PM SeOg-. At time zero and 1 and 2 h later the amount of 35Slabel was determined in the three most abundant E. coli thionucleosides, 2-thiocytidine, 4-thiouridine, and mnm5s2U. As expected, unlabeled thionucleosides in newly synthesized tRNA diluted the 35Slabel in each thionucleoside and the amount of ?S per A260unit of tRNA decreased with time (Figs. 4a-c). The %S label in 2-thiocytidine (Fig. 4a) and in 4-

SELENONUCLEOSIDE

0

1

2

0

1

2

0

BIOSYNTHESIS

1

2

---.----.-O-------------------------~

'd d

.

4 -----------------------D-------------------------Q 0

1 Time,

2 hr

FIG. 4. Effect of selenium addition on the retention of ‘% in prelabeled thionucleosides and on the amount of mnm5sezU and mnm%% in tRNA. See text for details. The tRNA was isolated and hydrolyzed, and the %-labeled nucleosides were quantitated by HPLC as described under Experimental Procedures. The amount of “S label (normalized to a value of 100 at zero time) per Am unit of tRNA in (a) 2-thiocytidine, (b) 4-thiouridine, and (c) mnm6szU is given in each panel. In panel (d), circles indicate levels of mnm6s2U, and squares the corresponding amounts of mnm%e%J. In all four panels, dashed lines are drawn through the average values for samples incubated with no added selenium and solid lines are drawn through the average values for samples incubated with 1 pM SeO$-.

thiouridine (Fig. 4b) was similar throughout the time course and was unaffected by the selenium supplement. In contrast, added selenite markedly increased the loss of the % label from mnm5s2U (Fig. 4~) whereas, in the absence of selenite, dilution of 35S in mnm5s2U was similar to that in the other thionucleosides. This apparent accelerated loss of 35S from mnm5s2U was concomitant with increasing mnm5se2U levels and decreasing mnm5s2U levels (Fig. 4d). Clearly, if mnm5se2U residues are formed from preexisting %-labeled mnm5s2U residues, the 35S content of this thionucleoside should be less than that expected from dilution by de nova synthesis alone. If, on the other hand, mnm5se2U is derived exclusively from newly synthesized precursors, the observed reciprocal rela-

IN

E. wli

547

tionship between the thio- and selenonucleosides (Fig. 4d) dictates that a lower amount of unlabeled mnm5s2U would be synthesized in the presence of selenium than in its absence. The net result then would be a decrease in the dilution of the %l label in preformed mnm5s2U concomitant with mnm5se2U synthesis. Since this was not observed, the experimental results suggest that incorporation of selenium involves removal of sulfur from mnm5s2U residues and its replacement with selenium.

In vitro incorporation of selenium into tRNALys and tRNAG1”. In initial experiments to obtain more direct evidence that the mnm5s2U residue in tRNA can serve as the substrate for mnm5se2U synthesis, permeabilized E. coli cells were utilized. These preparations, in which endogenous tRNA served as the substrate, incorporated a low level of 75Se (about 0.5 pmol/A260 unit of tRNA) when incubated with 1 PM 75Se0z-. This incorporation of 75Se was stimulated 2- to 3-fold by the addition of ATP. It was decreased by a lo-fold molar excess of unlabeled selenate, selenite, or selenocysteine but not by an excess of cysteine or sulfite (Table III). Selenomethionine, which is not readily used as a selenium source for selenium-dependent formate dehydrogenase synthesis by various wild-type E. coli strains (34), also failed to decrease the extent of labeling of the tRNA. Analysis of the nucleosides in the 75Se-labeled tRNA by HPLC showed that the labeled selenonucleoside was exclusively mnm5se2U (data not shown). Results similar to those shown in Table III also were obtained using wild-type E. coli cells ruptured in a French pressure cell, but it was not possible to distinguish 75Se incorporation into endogenous tRNA present in the cell homogenate from that in added tRNALyS or tRNAG’“. To circumvent this problem experiments were carried out using broken cell preparations of the trrnE mutant of E. coli (strain DEV16) which is unable to synthesize mnm5s2U. Homogenates of this organism, when incubated with 75Se0;-, synthesized a labeled selenonucleoside that exhibited chromatographic properties identical to those of the

548

WITTWER TABLE

Complete -ATP +lo pM +lo pM +lo pM +lO NM +lo pM +lo PM

se@ se@SelenOCySteine

selenomethionine CySteine

so;-

STADTMAN

III

DISCUSSION

INCORPORATION OF “Se INTO tRNA PERMEABILIZED E. coli CELLS Incubation mixture composition

AND

IN

“Se in tRNA (cpm/Am unit) 12,716 4,376 4,966 2,304 1,906 12,384 12,828 10,271

Note. Mid-log cells were permeabilized by plasmolysis with 2 M sucrose, as described by Ben-Hamida and Gros (33) and suspended in l/25 the original culture volume. The complete incubation mixture (2 ml) contained 10 mM Tris-HCl, pH 8, 1 mM ATP, 1 pM ‘%eOf(5 pCi/nmol), 10 mM MgCla, and 1 ml permeabilized cells. Incubations were for 1 h at 3’7°C after which the tRNA was isolated and %e labeling measured as described under Experimental Procedures.

selenonucleoside synthesized by this mutant under normal growth conditions (Fig. 5a; compare with Fig. 3b). As implied above, this nucleoside may be 2-selenouridine. When purified tRNALy8 or tRNAG’” was added to serve as source of mnm5s2U, a second labeled selenonucleoside was formed (Fig. 5b) and this compound, when rechromatographed, coeluted with authentic mnm5sezU. Synthesis of this labeled selenonucleoside was dependent on added ATP. Addition of cysteine had no effect on incorporation of %e from labeled selenite, whereas dilution by excess unlabeled selenite was observed (Fig. 5~). Because the 5-methylaminomethyl group is not synthesized by the tmzE mutant, the r5Se]mnm5se2U produced by the homogenate must have been formed from mnm5s2U present in the added tRNALy” or tRNAG’“. This provides direct evidence for an enzymatic activity which can use mnm5s2Ucontaining tRNA as substrate for mnm5se2U synthesis. Cofactor requirements and the precise selenium donor for this reaction remain to be determined.

Studies with growing cultures and broken cell preparations of E. coli reported here indicate that mnm5s2U residues in lysine- and glutamate-accepting tRNA species may be converted directly to mnm5sezU residues in the presence of available selenium. The requirement of ATP for this process implies that the sulfur atom at position 2 of the modified uridine residue is activated to become a leaving group which then can be replaced by selenium. This would be analogous to the well-known reaction wherein a specific uridine residue in several E. coli tRNAs is converted to a 4thiouridine residue. In this two-step pro-

10

15

10

Time.

15

10

15

min

FIG. 5. HPLC elution profiles of 7sSe-labeled nucleosides demonstrating the in vitro synthesis of mnm5se2U. Mid-log-phase cells of E. coli, strain DEV16, were suspended in 10 mM MgCla, 10 mM TrisHCl, pH 8.0 (l/100 of the culture volume), and ruptured in a French pressure cell at 10,000 psi. For the experiments in panels (a) and (b), each incubation mixture (200 ~1) contained 150 ~1 of the broken cell preparation and 45 pM “Se@(2 pCi/nmol). For panel (a) the broken cell preparation and “SeOf were incubated alone (-) or supplemented with 5 mM ATP (- - -). For panel (b) incubation mixtures were further supplemented with 1 Am unit of tRNA”” in the presence (- - -) or absence (-) of 5 mM ATP, or 1 Am unit of tRNALY” + 5 mM ATP ( * . a). Incubation of 1 Am unit of tRNALYB in the absence of added ATP gave a result similar to that with tRNA”” (-). For the experiment in panel (c) each incubation mixture (70 ~1) contained 50 ~1 of broken cell preparation, 6 pM “SeOf(10 &i/nmol), 0.4 Am unit of tRNALYB, and 5 mM ATP (- - -) supplemented with 100 FM unlabeled Se@- (-) or 100 pM cysteine ( * * . ). After a l-h incubation at 37”C, the tRNA was isolated, digested to nucleosides, and analyzed by HPLC as described under Experimental Procedures. The elution position of mnmsseaU (*) is indicated by an arrow in each panel.

SELENONUCLEOSIDE

cess an initial reaction of the tRNA with one enzyme and ATP gives a product that is converted by a second enzyme to a 4thiouridine residue in the tRNA using cysteine as sulfur donor (35). In the case of the 2-selenonucleoside biosynthesis, it has been observed that under a variety of growth conditions the sulfur nucleoside and its selenium isolog appear to be maintained in a roughly reciprocal relationship. Moreover, when selenium is added to selenium-deficient cultures, there is a timedependent decrease in mnm5s2U and a corresponding increase in mnm5se2U. The mechanism by which the extent of this conversion is regulated is not known. At any given time the population of a particular amino acid-accepting tRNA species should consist of both aminoacylated and nonacylated forms, ribosome-bound molecules, and possibly molecules involved in strictly regulatory processes, e.g., bound to DNA sites. Preference for one of these forms as a substrate could be a basis of control of extent of selenium modification. Under a variety of conditions, the maximum amount of mnm5se2U found in this study was about 66 pmol/Azso unit, or 4 mol selenonucleoside/lOO mol tRNA, assuming 1 Am unit equals 1.66 nmol tRNA. Thus, a maximum of about 4% of the tRNA population can contain mnm5se2U. The selenotRNAs in E. coli have primarily lysine or glutamate acceptor specificity (ll), and tRNALyS and tRNAG’” species have been estimated to represent about 10% of the bulk E. coli population (29,30). This means that under selenium-sufficient conditions almost half of the total tRNALy” and tRNAG’” species may contain mnm5se2U instead of mnm5s2U Use of *mutant strains of E. coli unable to synthesize the complete 5-methylaminomethyl group of mnm5s2U showed that facile 75Se labeling of presumed precursors to give products tentatively identified as 5aminomethyl-2-selenouridine and 2-selenouridine can occur. Additional products, chromatographically distinct from mnm5se2U, also were detected in tRNAs of wild-type E. coli labeled with 75Se in the presence of chloramphenicol. These results indicate that replacement of sulfur at the

BIOSYNTHESIS

IN

E. coli

549

2-position of a uridine residue in tRNA with selenium does not require the presence of a complete methylaminomethyl group at position 5. Since the thionucleoside, mnm5s2U, occupies the first position of the anticodon (the “wobble” position) in E. coli tRNALyS (15) and tRNAG’” (16) and appears to be the normal precursor of mnm5se2U, the selenonucleoside must also occur in the same position of these tRNAs. This is consistent with the fact that mnm5se2U occupies the first position of the anticodon in tRNAG’” of C. sticklandii (36). Ribosome binding studies carried out with purified selenotRNALy” or seleno-tRNAG’” from E. coli (A. Whittwer and W.-M. Ching, unpublished observations) or purified seleno-tRNAG’” from C. sticklandii (36, 37) indicated little difference in recognition of synonymous codons ending in A and G. This is in contrast to results obtained with mnm5s2Ucontaining tRNAs, where lysine and glutamate codons ending in A bind significantly better than those ending in G (15, 16). Since E. coli appears to grow normally in selenium-deficient media even though unable to synthesize selenium-modified tRNAs, these observations suggest that the selenonucleoside may exert some subtle or highly specialized regulatory role. ACKNOWLEDGMENTS We thank Dr. Mark X. Sliwkowski and Mr. Joe N. Davis for assistance with amino acid analysis, and Dr. Lin Tsai for synthesis of mnm’sew and mnm%?J.

REFERENCES 1. STADTMAN, T. C. (1979) in Advances in Enzymology (Meister, A., ed.), Vol. 48, pp. l-28, Wiley, New York. 2. COWIE, D. B., AND COHEN, G. N. (1957) &o&m, Biqhys. Acta 26,252-261. 3. SLIWKOWSKI, M. X., AND STADTMAN, T. C. (1985) J. Bid Chem 260.3140-3144. 4. CONE, J. E., MARTIN DEL Rfo, R., AND STADTMAN, T. C. (1977) J. Biol Chmn. 262,5337-5344. 5. GUNZLER, W. A., STEFFENS, G. J., GROSSMAN, A., KIM, S.-M., kc, F., WENDEL, A., AND FLOHI?, L. (1984) Hqppe-Seyler ‘s 2. Physid Chem. 365, 195-212.

550

WITTWER

AND

6. SAELINGER, D. A., HOFFMAN, J. L., AND MCCONNELL, K. P. (1972) J. Afol Bid 69,9-17. ‘7. HOFFMAN, J. L., AND MCCONNELL, K. P. (1974) Biochim Biophys. Acta 366,109-113. 8. PRASADA RAO, Y. S., AND CHERAYIL, J. D. (1974) L$e Sci 14,2051-2059. 9. CHEN, C.-S., AND STADTMAN, T. C. (1980) Proc. Nati Ad Sci USA 77,1403-1407. 10. YOUNG, P. A., AND KAISER, I. I. (1979) Pi&t Physiol (Bethesda) 63,511-517. 11. WITTWER, A. J. (1983) J. Bill Chem 258, 86378641. 12. CHING, W.-M., WITTWER, A. J., TSAI, L., AND STADTMAN, T. C. (1984) Proc. Natl Acad Sti USA 81,57-60. 13. CHING, W.-M., AND STADTMAN, T. C. (1982) Proc Nat1 Acad Sci USA 79,374-377. 14. WI~WER, A. J., TSAI, L., CHING, W.-M., AND STADTMAN, T. C. (1984) Biochemistry 23,46504655. 15. CHAKRABURTTY, K., STEINSCHNEIDER, A., CASE, R. V., AND MEHLER, A. H. (1975) Nucleic Acids Res. 2,2069-2075. 16. OHASHI, Z., HARADA, F., AND NISHIMURA, S. (1972) FEBS L&t 20,239-241. 17. MARINUS, M. G., MORRIS, N. R., S&L, D., AND KWONG, T. C. (1975) J. Bacterial 12, 257-265. 18. ELSEVIERS, D., PETRULLO, L. A., AND GALLAGHER, P. J. (1984) Nucleic AczXs Res. 12,3521-3534. 19. GEHRKE, C. W., Kuo, K. C., MCCUNE, R. A., AND GERHARDT, K. 0. (1982) J. Chromatogr. 230,297308. 20. IKEDA, K., TANAKA, S., AND MIZUNO, Y. (1975) Chem. Pharm. Bull 23,2958-2964.

STADTMAN 21. BUCK, M., CONNICK, M., AND AMES, B. N. (1983) And B&hem 129,1-13. 22. BEST, A. N. (1978) J. Bacterid 133,240~250. 23. WATERS, L. C., SHUGART, L., YANG, W.-K., AND BEST, A. N. (1973) Arch. Biochem. Biophys. 156, 780-793. 24. SHAPIRO, R., AND CHAMBERS, R. W. (1961) J. Amex Chem. Sot 83,3920-3921. 25. HARTMANIS, M. G. N., AND STADTMAN, T. C. (1982) Proc, Nat1 Acad. Sei. USA 79,4912-4916. 26. PINSET, J. (1954) Biodem J. 57,10-16. 27. Cox, J. C., EDWARDS, E. S., AND DEMOSS, J. A. (1981) J. BacterioL 145,1317-1324. 28. BJ~RK, G. R., AND KJELLIN-STRPLBY, K. (1978) J. Bacttiol. 133,508-517. 29. TAYA, Y., AND NISHIMURA, S. (1973) Biochem. Bi+ phys. Res. Commun. 51.1062-1068. 30. IKEMURA, T. (1981) J. Mel Biol 146,1-21. 31. SPRINZL, M., AND CRAMER, F. (1975) Proc. Natl. Acad. Sci. USA 72,3049-3053. 32. KAISER, I. I. (1972) J. Mol. Biol. 71,339-350. 33. BEN-HAMIDA, F., AND GROS, F. (1971) Biochimie 53.71-80. 34. SHUM, A. C., AND MURPHY, J. R. (1972) J. BacterioL 110,447-449. 35. ABRELL, J. W., KAUFMAN, E. E., AND LIPSE~, M. N. (1971) J. Biol. Chem. 246,294-301. 36. CHING, W.-M., ALZNER-DEWEERD, B., AND STADTMAN, T. C. (1985) Proc. Natl. Acad. Sk USA 82, 347-350. 37. CHING, W.-M., TSAI, L., AND WITTWER, A. J. (1985) in Current Topics in Cellular Regulation (Shaltiel, S., and Chock, P. B., eds.), Vol. 27, pp. 497-507, Academic Press, New York.