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Modification of 4-thiouridine and phenylalanine transfer RNA with parachlormercuribenzoate
ARCHIVES
OF
BIOCHEMISTRY
Modification
AND
160, 86-90
BIOPHYSICS
of 4-Thiouridine
and
(1972)
Phenylalanine
Transfer
RNA
with
Parachlormercuribenzoate’ B. C. PAL,2 L. R. SHUGART, Biology
Division,
Oak
Received
Ridge
November
Ii. R. ISHAM,
National
Laboratory,
10, 1971;
accepted
R/I. P. STULBERG
AND
Oak
Ridge,
January
Tennessee
37830
27, 1972
While investigating the modification of the 4-thiouridine moiety in E. coli tRNAPhe with parachloromercuribeneoate with a view to further elucidate the biochemical function of the minor base and prepare tRNA labeled with a heavy metal at a specific site for X-ray crystallography, we observed that the paramercuribenzoate IS of Mg2+. Surprisingly moiety in the modified tRNAPhe . very labile in the presence this lability is not observed at the mononucleoside level when the reaction is carried out with the mercurated derivatives of 4-thiouridine or its methyl analog. Evidences for 1: 1 stoichiometry of the reaction of parachloromercuribenzoate with 4-thiouridine and for the covalent nature of the -Hg--Sbond in the mercurated nucleoside have been obtained by carrying out the reaction with N1-methyl4thiouracil and isolating and characterizing the product, 1-methyl4-thiouracilylparamercuribenzoic acid.
The discovery of the rare nucleoside 4-thiouridine in mixed tRNA from Escherichia coli by Lipsett (1) provided the impetus for numerous investigations into its chemistry and biological function. Sequence studies of (E. coli) tRNA (2-7) indicated that. 4-thiouridine occupies the eighth position from the 5’-terminus in these different tRNA’s. In the cloverleaf model (7) of these tRNA’s, 4-thiouridine appears as a nonhydrogen-bonded base, possibly indicating special reactivity and lability (4, 6). Previously we approximated the region in which the recognition site in (E. coli) tRN,4Phe was located, by inhibition studies with limit residues produced by the action of venom phosphodiesterase on the tRNAPh* (9). We further delineated the role of 5,6dihydrouridine and 4-thiouridine in the function of this site by their reaction with 1 Research sponsored by the U. Energy Commission under contract Union Carbide Corporation. 2 To whom to address correspondence.
[3Hlborohydride (10). In order to determine whether one or both of these nucleosides were necessary for the recognition phenomena, we have attempted a series of specific chemical modifications of 4-thiouridine in tRNA and its effect on the interaction with the enzyme. Although its sulfur probably exists in the thione rather than the thiol form in tRNA (II), it does respond readily to sulfhydryl reagents (12-15). In this paper we report studies on the reaction of parachloromercuribenzoic acid3 with model thiouridine-containing compounds and with (E. coli) tRNAPhe to determine the effect of this reaction upon enzymic aminoacylation and possibly to prepare a heavy-metal derivative for purposes of X-ray crystallography. * Abbreviations used are: p-ClHgBzOH, parachloromercuribenzoic acid ; p-HgBzOH-tRNA, tRNA treated with p-ClHgBzOH and dialyzed against water to remove unbound reagent. The parachloromercuribenzoic acid in various solutions may be combined principally with hydroxyl or other anions instead of chloride ion (17).
S. Atomic with the
86 Copyright
@ 1972 by Academic
Press,
Inc.
MODIFICATION EXPERIMENTAL
OF
tRNAPhe
WITH
SECTION
Unfractionated tRNA and purified tRNArhe from E. coli K-12M0 were obtained from A. D. Kelmers of this laboratory. Their preparation was as described by Kelmers et al. (16). The phenylalanine acceptance and terminal adenosine assay were 1000 and 1300 pmoles, respectively, per Azso unit4 of the tRNA. Bis-(1-8-u-ribosyl-4-thiouracil) disulfide was purchased from Cycle Chemical Corporation, Los Angeles, Ca. It was reduced to l-b-n-ribosyl-4thiouracil by sodium thiosulfate and desalted by passing through a P-2 gel column. [‘%I-Labeled p-ClHgBzOH, specific activity 10 Ci/mole was purchased from Schwarz BioResearch, Orangeburg, NY; its homogeneity and radiopurity were checked by chromatography on Whatman No. 1 paper in 2-propanol:HzO:NHa (60:30:10 by volume) (R,, 0.28) and by scanning for radioactivity in a Tracer Lab 4~ Scanner. Unlabeled commercial p-ClHgBzOH was purified by the method of Boyer (18) until chromatographitally homogeneous. l-Methyl-4-thiouracil was synthesized by the method of Fox et al. (19). Alkaline phosphatase and snake venom phosphodiesterase were obtained from Worthington Biochemical Corporation, New Jersey. The preparation and analysis of l-methyl-Qthiouracilyl-p-mercuribenzoic acid. 1-Methyl-4thiouracil, 284.4 mg (2 mmoles) in 2 ml of 1 N NaOH was treated with p-ClHgBzOH, 357 mg (1 mmole) in 3 ml of 1 N NaOH at room temperature for 10 min. The pH of the reaction mixture was adjusted to 5.0 with 0.5 N acetic acid, and a white precipitate was obtained. The precipitate was collected by filtration, resuspended in 300 ml of hot water, filtered, washed with hot water, and dried. The yield of pure l-methyl-4-thiouracilylp-mercuribenzoic acid was 325 mg (65% of theory) ; mp, 236” (dec) ;SS~O(pH 7), 17,960. Anal. Calcd for CnHloOaN2HgS: C, 31.13; H, 2.18; N, 6.05. Found: C, 31.68; H, 2.18; N, 6.25. The reaction of mixed tRNA with p-ClHgBzOH. Partition cells (compartments in series) with a 4.5-mm pathlength in each compartment (Precision Cells, Inc., New York) were used in these experiments. The reference cell: compartment 1 contained 0.1 ml HtO, 0.5 ml of 0.5 M phosphate buffer (pH 6.5), and 0.5 ml of a mixed tRNA solution (17.3 mg per ml); compartment 2 contained 0.5 ml of Hz0 and 0.5 ml of 0.5 M phosphate buffer. The sample cell: compartment 1 contained 0.5 ml 4 One absorbance unit is that amount of material in 1 ml of a solution that has an absorbance of 1.0 when it is measured with a LO-cm optical path at a particular wavelength.
F ‘ARACHLOROMERCURIBENZOATE
87
of 0.5 M phosphate buffer and 0.5 ml of tRNA solution; compartment 2 contained 0.6 ml of Hz0 and 0.5 ml of phosphate buffer. The difference specrum was recorded before and after addition of 0.1 ml of the solution of the mercurial (4.636 mg/2.6 ml of 0.1 N NaOH) in compartment 2 of the reference cell and compartment 1 of the sample cell. The treated tRNA was then dialyzed three times against 1000 ml of 0.001 M Mgz+ or three times against 1000 ml of water to eliminate excess mercuria1 . Treatment of puri$ed tRNAPhe with [W]pClHgBzOH. Purified tRNAPhe solution (148 A260 and 2.8 Aa units) was reacted with 0.25 ml of 1 M Tris buffer (pH 8) and 0.1 ml of [14C]p-ClHgBzOH (5 pmole) in 1 ml of 0.1 N NaOH. The reaction mixture was incubated for 1 hr at 45% and then dialyzed against water at room temperature. The specific activity of the dialyzate indicated incorporation of 1277 pmoles of reagent per As6,, unit of tRNAPhe. The modification of 4-thiouridine appears to be essentially quantitative when calculated on the basis of 1300 pmoles of tRNArhe per AMO unit. The reaction of p-HgBzOH-tRNAPbe with magnesium acetate. The p-HgBzOH-tRNArhe (9.08 A&ml) in 0.05 M Tris buffer, pH 8.2, was treated with 0.05 ml of 0.1 M magnesium acetate. The uv absorption spectra were recorded before and after the addition of magnesium acetate solution. Enzymatic hydrolysis of p-HgBzOH-tRNA. The p-HgBzOH-tRNA (50 A260 units in 0.55 ml) was treated with a solution containing 0.1 ml of 0.2 M ammonium acetate (pH 8.8), 0.125 ml of 0.3 M magnesium acetate, and 0.25 ml each of phosphodiesterase (95 mg/ml) and phosphatase (1 mg/ml). The reaction mixture was incubated at 46°C for 4 hr. Chromatographic isolation of the 4-[ (p-carboxyphenyl)mercurithio] &dine from the enzymic hydrolyzate of the p-HgBzOH-tRNAPhe. Chromatography of the nucleosides obtained from enzymitally hydrolyzed tRNA was performed at 5°C on a DEAE-cellulose column (Whatman DE-52,0.5 x 59 cm) previously equilibrated with 0.2 M triethylammonium bicarbonate solution (pH 7.5). Usually 25-40 A26a nm units of hydrolyzed tRNA in 1 ml were applied directly to the column and washed on with an equal volume of buffer. The sample was eluted with the same buffer at a flow rate of 0.5 ml/min, and the column eluant was monitored at 310 nm by a Beckman DB spectrophotometer equipped with a l-cm flow cell. Fractions (5 ml) were analyzed by the method of Stulberg et al. (20). The far uv spectrum of specified fractions were recorded at pH 7.5 after the fractions were concentrated by being evaporated to dryness and redissolved in water.
88
ET AL.
PAL
Aminoacylation of tRNA. The assay conditions and procedures for determining the total amount of phenylalanyl-tRNA formation were performed as outlined by Kelmers et al. (16). Unfractionated tRNA samples were assayed with a crude amino acid:tRNA ligase and the purified tRNAPhe samples with a purified phenylalanine: tRNA ligase (17). The uv absorption spectra were recorded in a Cary Recording Spectrophotometer Model 14 PM. RESULTS
AND
,O.1; FIG. methyl
1
DISCUSSION
Since neither the character nor the stoichiometry of the product of the reaction of 4-thiouridine with p-ClHgBzOH has been determined, we decided initially to carry out the reaction of the latter with the methyl analog of 4-thiouridine. The uv absorption spectrum of the reaction product at pH 7 showed a x,,, at 310 nm, as compared with aX max at 330 nm of l-methyll-thiouracil (Fig. 1). This compound has also been studied by X-ray crystallography, which shows that the mercury is covalently bonded to the sulfur (21). We also find that the compound readily undergoes thiolysis in the presence of fl-mercaptoethanol or dithiothreitol. The reaction of 4-thiouridine with the mercurial has been found to be very similar to that of its methyl analog.
290 WAVELENGTH
04
310 Inn,)
330
1. The uv-absorption spectrum - 4 - thiouracilyl - paramercuribenaoate phosphatebuffer at pH 7.
350
370
of
lin
-0.6
’
FIG. 2. The difference spectrum of tRNA before (A--A) and after treatment with p-ClHgBzOH. Details Experimental Section.
E. coli mixed (O---O) described
in
The uv absorption spectrum of mixed (E. coli) tRNA shows a small peak at 330 nm. At least two-thirds of this peak has been attributed to the 4-thiouridine moiety of the tRNA (G. Goldstein, 1970, personal communication). p-ClHgBaOH does not show any absorption at 330 nm, and an increase in A310and decreasein A,,o may be taken as indicative of the reaction of 4-thiouridine with the mercurial (Fig. 2), since the same spectral changes are obtained when 4-thiouridine or its methyl analog are treated with it. This agrees with observations made by Lipsett and Doctor in their studies on tRNATyr (12). The mixed (E. coli) tRNA after treatment with the mercurial was dialyzed against 0.001 M Mg2+ to remove excess reagent. Upon spectroscopic examination, the dialyzed tRNA unexpectedly revealed the regeneration of the peak at 330 nm, indicating that the p-HgBzOH had dissociated from the tRNA. We next attempted to confirm this observation with mononucleoside reactions. Neither p-ClHgBzOH-treated 4thiouridine nor its methyl analog (0.05 mM) showed any spectral change in the presence
MODIFICATION
OF
tRNAPtle
WITH
of as much as 0.5 mM magnesium chloride. Thus, it is apparent that 4-thiouridine dissociates readily from p-HgBzOH when it is a component of tRNA, as compared to its reactivity as a mononucleoside in solution. It is quite possible, however, that Mg2+ is altering the dissociation constant only slightly, such that a very small fraction of the mercurial is dissociated. Dialysis would, of course, disturb the equilibrium and eventually result in complete removal of the mercurial. To rule out this possibility, we studied the effect of addition of Mg2+ on the ultraviolet absorption spectrum of p-HgBzOH-tRNAPhe. The A310/A940 changed from 2.32 to 1.41 indicating the release of paramercuribenzoate from the modified tRNAPhe. This somewhat parallels the experience of Seno et al. (22), who found that Mg2+ does not affect the uv spectrum of 4-thiouridine but does affect profoundly the melting profile of dif200
T
160
7 P ;
120
F :: FE \ E 0 so. 7 .!.
40-
0 .A! 0
5 10 FRACTION NO.
15
FIG. 3. Isolation of the 4-([‘%](p-carboxyphenyl)mercurithio) uridine complex from after enzymic hydrolysis. Approximately nm units of onzymically hydrolyzed tionated tRNA which was pretreated with ClHgBzOH was chromatographed on a cellulose column. 2.5 X Los cpm applied covered.
tRNA 39 -4260 unfrac[XJ]pDEAEand re-
89
PARACHLOROMERCURIBENZOATE
I r r 300 WAVELENGTH
3
/ I 350 (nm)
I
I
, 41
1
FIG. 4. Far uv spectrum of the 4-([14C](pcarboxyphenyl)mercurithio) uridine complex recovered from enzymically hydrolyzed tRNA. Fractions No. 10-13 of Fig. 3 were pooled and concentrated, and the spectrum was recorded at pH 7.5.
ferent tRNA’s containing 4-thiouridine as followed by variation of A335with temperature. However, when we carried out the dialysis of the treated tRNA against water alone, the uv spectrum remained unaltered. The p-HgBzOH-tRNA thus obtained waa hydrolyzed to nucleosidesusing a mixture of venom phosphodiesterase and phosphatase, and the modified 4-thiouridine was then isolated by chromatography on DEAE-cellulose and identified spectrophotometrically. We then repeated the experiment with the (E. colz. tRNAPhe using [14C]p-CIHgBzOH. Isolation and characterization of the modified 4-thiouridine are shown in Figs. 3 and 4. All the radioactivity was associatedwith the peak whose position (Fig. 3) corresponds to that of modified 4-thiouridine. Furthermore, the spectrum of the material of this peak demonstrated the characteristic appearance of a X,,, at 310 nm and a decrease of the absorbance at 330 nm (Fig. 4). We attempted to assay the treated tRNAPhe for amino acid acceptance. Unfortunately the minimum amount of Mg2+ (0.0025 M) necessary for supporting activity was enough to
90
PAL
dissociate the p-HgBzOH from the tRNAPhe as indicated by the loss of 14C radioactivity upon dialysis of the modified tRNA (that) had been exposed to Mg2+) against water and also by the immediate change in the uv absorption spectrum (Experimental Section) before dialysis. The extent of aminoacylation of the tRNA that was dissociated from the p-HgBzOH was found to be the same as untreated tRNAPhe. Among a number of divalent cations tested, only Mg2+ and Mn2+ support phenylalanyl-tRNA formation to a significant extent in E. coli (23). Our attempts to replace Mg2+ with Mn2+ were thwarted because Mn2+ causesprecipitation in the obligate pH range utilized for the assay. Although the role of Mg”f in the biological activity of the nucleic acids and in their secondary and tertiary structures has been recognized for a long time (24), nothing is known about its binding sites in the tRNA. The displacement of the p-HgBzOH from the p-HgBzOH-tRNAPhe in the presence of Mg2f suggests that 4-thiouridine may be a binding site for this ion in tRNA, or that Mg2+ perturbs the tRNA structure sticiently to favor hydrolysis of the PCMB group. The amount of radioactivity of [‘“Cllabeled p-HgBzOH-tRNAPhe indicates the incorporation of 1 mole of the reagent per mole of tRNAPhe. The use of p-CIHgBzOH for specifically labeling a 4-thiouridine moiety of tRNA for X-ray crystallography would be useful only under conditions whereby this derivative could be stabilized in the presence of Mg2+. REFERENCES 1. LIPSETT, M. N. (1965) J. Biol. Chem. 240, 3975. 2. CORY, S., AND MARCKER, K. A. (1970). Eur. J. Biochem. 12. 177.
ET
AL. 3. DUBE:, 8. K., MARCK~R, K. A., CL.~RE~, B. F. C., AND CORY, S. (1968). Nature London 218, 232. 4. BARRELL, B. G., .~ND SINGER, F. (1969). Fed. Eur. Biol. Sac. Letf. 3,275. 5. GOODMAN, H. M., ABELSON, J., L.\NDY, A., BRENNER, S., AND SMITH, J. D. (1968) Nature London 217, 1019. 6. GASSEN, H. G., AND UZIEL, M. (1969). Abstr. Fed. Eur. Biol. Sot. Madrid 128, Biochemistrv 8. 1643. 7. F&I&, K., FUMIO, H., AND NISHIMURA, S. (1971) Biochemistry 10, 3277. 8. HOLLEY, R. W. (1966). Sci. Amer. 214,30. 9. STULBERG, M. P., AND ISHAM, K. R. (1967) Proc. Nat. Acad. Sci. U.S.A. 67,131O. 10. SHUGART, L., AND STULBERG, M. P. (1969) J. Biol. Chem. 244, 2806. 11. BROWN, D. J. (1962) “The Pyrimidines”, p. 485. Interscience, New York. 12. LIPSETT, M. N., AND DOCTOR, B. P. 0967) J. Biol. Chem. 242,4072. 13. CARBON, J., AND DAVID, H. (1968). Biochemistry 7,385l. 14. SCHEIT, K. H. (1968) Biochim. Biophys. Acta 166, 285. 15. SHUGART, L. Arch. Biochem. Biophys. (in press). 16. KELMERS, A. D., NOVELLI, G. D., AND STULBERG, M. P. (1965) J. Biol. Chem. 240,3979. 17. STULBERG, M. P. (1967) J. Biol. Chem. 242, 1060. 18. BOYER, P. D. (1954) J. Amer. Chem. Sot. 76, 4331. 19. FOX, J. J., PRAG, D. V., VEMPEN, I., DOERR, I., CHEONG, L., KNOLL, J. E., EDINOFF, M. L., BENDICH, A.! AND BROWN, G. B. (1959) J. Amer. Chem. Sot. 81,178. 20. STULBERG, M. P., ISHAM, K. R., AND STEVENS, A. (1969) Biochim. Biophys. Acla 186,297. 21. HAWKINSON, S. W., PAL, B. C., AND EINSTEIN, J. R. (1970) Abstr. of the Winter Meeting of the American Crystallography Association, March 2-5 (1970), New Orleans, La. 22. SENO, T., KOBAYASHI, M., AND NISHIMUR.~, S. (1969) Biochim. Biophys. Acta 174,71. 23. ItWIN, I. B., KELMERS, A. D., AND GOLDSTEIN, G. (1967) Anal. Biochem. 20,533. 24. FELSENFELD, G., AND MILES, H. T. (1967) Ann. Rev. Biochem. 36,497.
OF
BIOCHEMISTRY
Modification
AND
160, 86-90
BIOPHYSICS
of 4-Thiouridine
and
(1972)
Phenylalanine
Transfer
RNA
with
Parachlormercuribenzoate’ B. C. PAL,2 L. R. SHUGART, Biology
Division,
Oak
Received
Ridge
November
Ii. R. ISHAM,
National
Laboratory,
10, 1971;
accepted
R/I. P. STULBERG
AND
Oak
Ridge,
January
Tennessee
37830
27, 1972
While investigating the modification of the 4-thiouridine moiety in E. coli tRNAPhe with parachloromercuribeneoate with a view to further elucidate the biochemical function of the minor base and prepare tRNA labeled with a heavy metal at a specific site for X-ray crystallography, we observed that the paramercuribenzoate IS of Mg2+. Surprisingly moiety in the modified tRNAPhe . very labile in the presence this lability is not observed at the mononucleoside level when the reaction is carried out with the mercurated derivatives of 4-thiouridine or its methyl analog. Evidences for 1: 1 stoichiometry of the reaction of parachloromercuribenzoate with 4-thiouridine and for the covalent nature of the -Hg--Sbond in the mercurated nucleoside have been obtained by carrying out the reaction with N1-methyl4thiouracil and isolating and characterizing the product, 1-methyl4-thiouracilylparamercuribenzoic acid.
The discovery of the rare nucleoside 4-thiouridine in mixed tRNA from Escherichia coli by Lipsett (1) provided the impetus for numerous investigations into its chemistry and biological function. Sequence studies of (E. coli) tRNA (2-7) indicated that. 4-thiouridine occupies the eighth position from the 5’-terminus in these different tRNA’s. In the cloverleaf model (7) of these tRNA’s, 4-thiouridine appears as a nonhydrogen-bonded base, possibly indicating special reactivity and lability (4, 6). Previously we approximated the region in which the recognition site in (E. coli) tRN,4Phe was located, by inhibition studies with limit residues produced by the action of venom phosphodiesterase on the tRNAPh* (9). We further delineated the role of 5,6dihydrouridine and 4-thiouridine in the function of this site by their reaction with 1 Research sponsored by the U. Energy Commission under contract Union Carbide Corporation. 2 To whom to address correspondence.
[3Hlborohydride (10). In order to determine whether one or both of these nucleosides were necessary for the recognition phenomena, we have attempted a series of specific chemical modifications of 4-thiouridine in tRNA and its effect on the interaction with the enzyme. Although its sulfur probably exists in the thione rather than the thiol form in tRNA (II), it does respond readily to sulfhydryl reagents (12-15). In this paper we report studies on the reaction of parachloromercuribenzoic acid3 with model thiouridine-containing compounds and with (E. coli) tRNAPhe to determine the effect of this reaction upon enzymic aminoacylation and possibly to prepare a heavy-metal derivative for purposes of X-ray crystallography. * Abbreviations used are: p-ClHgBzOH, parachloromercuribenzoic acid ; p-HgBzOH-tRNA, tRNA treated with p-ClHgBzOH and dialyzed against water to remove unbound reagent. The parachloromercuribenzoic acid in various solutions may be combined principally with hydroxyl or other anions instead of chloride ion (17).
S. Atomic with the
86 Copyright
@ 1972 by Academic
Press,
Inc.
MODIFICATION EXPERIMENTAL
OF
tRNAPhe
WITH
SECTION
Unfractionated tRNA and purified tRNArhe from E. coli K-12M0 were obtained from A. D. Kelmers of this laboratory. Their preparation was as described by Kelmers et al. (16). The phenylalanine acceptance and terminal adenosine assay were 1000 and 1300 pmoles, respectively, per Azso unit4 of the tRNA. Bis-(1-8-u-ribosyl-4-thiouracil) disulfide was purchased from Cycle Chemical Corporation, Los Angeles, Ca. It was reduced to l-b-n-ribosyl-4thiouracil by sodium thiosulfate and desalted by passing through a P-2 gel column. [‘%I-Labeled p-ClHgBzOH, specific activity 10 Ci/mole was purchased from Schwarz BioResearch, Orangeburg, NY; its homogeneity and radiopurity were checked by chromatography on Whatman No. 1 paper in 2-propanol:HzO:NHa (60:30:10 by volume) (R,, 0.28) and by scanning for radioactivity in a Tracer Lab 4~ Scanner. Unlabeled commercial p-ClHgBzOH was purified by the method of Boyer (18) until chromatographitally homogeneous. l-Methyl-4-thiouracil was synthesized by the method of Fox et al. (19). Alkaline phosphatase and snake venom phosphodiesterase were obtained from Worthington Biochemical Corporation, New Jersey. The preparation and analysis of l-methyl-Qthiouracilyl-p-mercuribenzoic acid. 1-Methyl-4thiouracil, 284.4 mg (2 mmoles) in 2 ml of 1 N NaOH was treated with p-ClHgBzOH, 357 mg (1 mmole) in 3 ml of 1 N NaOH at room temperature for 10 min. The pH of the reaction mixture was adjusted to 5.0 with 0.5 N acetic acid, and a white precipitate was obtained. The precipitate was collected by filtration, resuspended in 300 ml of hot water, filtered, washed with hot water, and dried. The yield of pure l-methyl-4-thiouracilylp-mercuribenzoic acid was 325 mg (65% of theory) ; mp, 236” (dec) ;SS~O(pH 7), 17,960. Anal. Calcd for CnHloOaN2HgS: C, 31.13; H, 2.18; N, 6.05. Found: C, 31.68; H, 2.18; N, 6.25. The reaction of mixed tRNA with p-ClHgBzOH. Partition cells (compartments in series) with a 4.5-mm pathlength in each compartment (Precision Cells, Inc., New York) were used in these experiments. The reference cell: compartment 1 contained 0.1 ml HtO, 0.5 ml of 0.5 M phosphate buffer (pH 6.5), and 0.5 ml of a mixed tRNA solution (17.3 mg per ml); compartment 2 contained 0.5 ml of Hz0 and 0.5 ml of 0.5 M phosphate buffer. The sample cell: compartment 1 contained 0.5 ml 4 One absorbance unit is that amount of material in 1 ml of a solution that has an absorbance of 1.0 when it is measured with a LO-cm optical path at a particular wavelength.
F ‘ARACHLOROMERCURIBENZOATE
87
of 0.5 M phosphate buffer and 0.5 ml of tRNA solution; compartment 2 contained 0.6 ml of Hz0 and 0.5 ml of phosphate buffer. The difference specrum was recorded before and after addition of 0.1 ml of the solution of the mercurial (4.636 mg/2.6 ml of 0.1 N NaOH) in compartment 2 of the reference cell and compartment 1 of the sample cell. The treated tRNA was then dialyzed three times against 1000 ml of 0.001 M Mgz+ or three times against 1000 ml of water to eliminate excess mercuria1 . Treatment of puri$ed tRNAPhe with [W]pClHgBzOH. Purified tRNAPhe solution (148 A260 and 2.8 Aa units) was reacted with 0.25 ml of 1 M Tris buffer (pH 8) and 0.1 ml of [14C]p-ClHgBzOH (5 pmole) in 1 ml of 0.1 N NaOH. The reaction mixture was incubated for 1 hr at 45% and then dialyzed against water at room temperature. The specific activity of the dialyzate indicated incorporation of 1277 pmoles of reagent per As6,, unit of tRNAPhe. The modification of 4-thiouridine appears to be essentially quantitative when calculated on the basis of 1300 pmoles of tRNArhe per AMO unit. The reaction of p-HgBzOH-tRNAPbe with magnesium acetate. The p-HgBzOH-tRNArhe (9.08 A&ml) in 0.05 M Tris buffer, pH 8.2, was treated with 0.05 ml of 0.1 M magnesium acetate. The uv absorption spectra were recorded before and after the addition of magnesium acetate solution. Enzymatic hydrolysis of p-HgBzOH-tRNA. The p-HgBzOH-tRNA (50 A260 units in 0.55 ml) was treated with a solution containing 0.1 ml of 0.2 M ammonium acetate (pH 8.8), 0.125 ml of 0.3 M magnesium acetate, and 0.25 ml each of phosphodiesterase (95 mg/ml) and phosphatase (1 mg/ml). The reaction mixture was incubated at 46°C for 4 hr. Chromatographic isolation of the 4-[ (p-carboxyphenyl)mercurithio] &dine from the enzymic hydrolyzate of the p-HgBzOH-tRNAPhe. Chromatography of the nucleosides obtained from enzymitally hydrolyzed tRNA was performed at 5°C on a DEAE-cellulose column (Whatman DE-52,0.5 x 59 cm) previously equilibrated with 0.2 M triethylammonium bicarbonate solution (pH 7.5). Usually 25-40 A26a nm units of hydrolyzed tRNA in 1 ml were applied directly to the column and washed on with an equal volume of buffer. The sample was eluted with the same buffer at a flow rate of 0.5 ml/min, and the column eluant was monitored at 310 nm by a Beckman DB spectrophotometer equipped with a l-cm flow cell. Fractions (5 ml) were analyzed by the method of Stulberg et al. (20). The far uv spectrum of specified fractions were recorded at pH 7.5 after the fractions were concentrated by being evaporated to dryness and redissolved in water.
88
ET AL.
PAL
Aminoacylation of tRNA. The assay conditions and procedures for determining the total amount of phenylalanyl-tRNA formation were performed as outlined by Kelmers et al. (16). Unfractionated tRNA samples were assayed with a crude amino acid:tRNA ligase and the purified tRNAPhe samples with a purified phenylalanine: tRNA ligase (17). The uv absorption spectra were recorded in a Cary Recording Spectrophotometer Model 14 PM. RESULTS
AND
,O.1; FIG. methyl
1
DISCUSSION
Since neither the character nor the stoichiometry of the product of the reaction of 4-thiouridine with p-ClHgBzOH has been determined, we decided initially to carry out the reaction of the latter with the methyl analog of 4-thiouridine. The uv absorption spectrum of the reaction product at pH 7 showed a x,,, at 310 nm, as compared with aX max at 330 nm of l-methyll-thiouracil (Fig. 1). This compound has also been studied by X-ray crystallography, which shows that the mercury is covalently bonded to the sulfur (21). We also find that the compound readily undergoes thiolysis in the presence of fl-mercaptoethanol or dithiothreitol. The reaction of 4-thiouridine with the mercurial has been found to be very similar to that of its methyl analog.
290 WAVELENGTH
04
310 Inn,)
330
1. The uv-absorption spectrum - 4 - thiouracilyl - paramercuribenaoate phosphatebuffer at pH 7.
350
370
of
lin
-0.6
’
FIG. 2. The difference spectrum of tRNA before (A--A) and after treatment with p-ClHgBzOH. Details Experimental Section.
E. coli mixed (O---O) described
in
The uv absorption spectrum of mixed (E. coli) tRNA shows a small peak at 330 nm. At least two-thirds of this peak has been attributed to the 4-thiouridine moiety of the tRNA (G. Goldstein, 1970, personal communication). p-ClHgBaOH does not show any absorption at 330 nm, and an increase in A310and decreasein A,,o may be taken as indicative of the reaction of 4-thiouridine with the mercurial (Fig. 2), since the same spectral changes are obtained when 4-thiouridine or its methyl analog are treated with it. This agrees with observations made by Lipsett and Doctor in their studies on tRNATyr (12). The mixed (E. coli) tRNA after treatment with the mercurial was dialyzed against 0.001 M Mg2+ to remove excess reagent. Upon spectroscopic examination, the dialyzed tRNA unexpectedly revealed the regeneration of the peak at 330 nm, indicating that the p-HgBzOH had dissociated from the tRNA. We next attempted to confirm this observation with mononucleoside reactions. Neither p-ClHgBzOH-treated 4thiouridine nor its methyl analog (0.05 mM) showed any spectral change in the presence
MODIFICATION
OF
tRNAPtle
WITH
of as much as 0.5 mM magnesium chloride. Thus, it is apparent that 4-thiouridine dissociates readily from p-HgBzOH when it is a component of tRNA, as compared to its reactivity as a mononucleoside in solution. It is quite possible, however, that Mg2+ is altering the dissociation constant only slightly, such that a very small fraction of the mercurial is dissociated. Dialysis would, of course, disturb the equilibrium and eventually result in complete removal of the mercurial. To rule out this possibility, we studied the effect of addition of Mg2+ on the ultraviolet absorption spectrum of p-HgBzOH-tRNAPhe. The A310/A940 changed from 2.32 to 1.41 indicating the release of paramercuribenzoate from the modified tRNAPhe. This somewhat parallels the experience of Seno et al. (22), who found that Mg2+ does not affect the uv spectrum of 4-thiouridine but does affect profoundly the melting profile of dif200
T
160
7 P ;
120
F :: FE \ E 0 so. 7 .!.
40-
0 .A! 0
5 10 FRACTION NO.
15
FIG. 3. Isolation of the 4-([‘%](p-carboxyphenyl)mercurithio) uridine complex from after enzymic hydrolysis. Approximately nm units of onzymically hydrolyzed tionated tRNA which was pretreated with ClHgBzOH was chromatographed on a cellulose column. 2.5 X Los cpm applied covered.
tRNA 39 -4260 unfrac[XJ]pDEAEand re-
89
PARACHLOROMERCURIBENZOATE
I r r 300 WAVELENGTH
3
/ I 350 (nm)
I
I
, 41
1
FIG. 4. Far uv spectrum of the 4-([14C](pcarboxyphenyl)mercurithio) uridine complex recovered from enzymically hydrolyzed tRNA. Fractions No. 10-13 of Fig. 3 were pooled and concentrated, and the spectrum was recorded at pH 7.5.
ferent tRNA’s containing 4-thiouridine as followed by variation of A335with temperature. However, when we carried out the dialysis of the treated tRNA against water alone, the uv spectrum remained unaltered. The p-HgBzOH-tRNA thus obtained waa hydrolyzed to nucleosidesusing a mixture of venom phosphodiesterase and phosphatase, and the modified 4-thiouridine was then isolated by chromatography on DEAE-cellulose and identified spectrophotometrically. We then repeated the experiment with the (E. colz. tRNAPhe using [14C]p-CIHgBzOH. Isolation and characterization of the modified 4-thiouridine are shown in Figs. 3 and 4. All the radioactivity was associatedwith the peak whose position (Fig. 3) corresponds to that of modified 4-thiouridine. Furthermore, the spectrum of the material of this peak demonstrated the characteristic appearance of a X,,, at 310 nm and a decrease of the absorbance at 330 nm (Fig. 4). We attempted to assay the treated tRNAPhe for amino acid acceptance. Unfortunately the minimum amount of Mg2+ (0.0025 M) necessary for supporting activity was enough to
90
PAL
dissociate the p-HgBzOH from the tRNAPhe as indicated by the loss of 14C radioactivity upon dialysis of the modified tRNA (that) had been exposed to Mg2+) against water and also by the immediate change in the uv absorption spectrum (Experimental Section) before dialysis. The extent of aminoacylation of the tRNA that was dissociated from the p-HgBzOH was found to be the same as untreated tRNAPhe. Among a number of divalent cations tested, only Mg2+ and Mn2+ support phenylalanyl-tRNA formation to a significant extent in E. coli (23). Our attempts to replace Mg2+ with Mn2+ were thwarted because Mn2+ causesprecipitation in the obligate pH range utilized for the assay. Although the role of Mg”f in the biological activity of the nucleic acids and in their secondary and tertiary structures has been recognized for a long time (24), nothing is known about its binding sites in the tRNA. The displacement of the p-HgBzOH from the p-HgBzOH-tRNAPhe in the presence of Mg2f suggests that 4-thiouridine may be a binding site for this ion in tRNA, or that Mg2+ perturbs the tRNA structure sticiently to favor hydrolysis of the PCMB group. The amount of radioactivity of [‘“Cllabeled p-HgBzOH-tRNAPhe indicates the incorporation of 1 mole of the reagent per mole of tRNAPhe. The use of p-CIHgBzOH for specifically labeling a 4-thiouridine moiety of tRNA for X-ray crystallography would be useful only under conditions whereby this derivative could be stabilized in the presence of Mg2+. REFERENCES 1. LIPSETT, M. N. (1965) J. Biol. Chem. 240, 3975. 2. CORY, S., AND MARCKER, K. A. (1970). Eur. J. Biochem. 12. 177.
ET
AL. 3. DUBE:, 8. K., MARCK~R, K. A., CL.~RE~, B. F. C., AND CORY, S. (1968). Nature London 218, 232. 4. BARRELL, B. G., .~ND SINGER, F. (1969). Fed. Eur. Biol. Sac. Letf. 3,275. 5. GOODMAN, H. M., ABELSON, J., L.\NDY, A., BRENNER, S., AND SMITH, J. D. (1968) Nature London 217, 1019. 6. GASSEN, H. G., AND UZIEL, M. (1969). Abstr. Fed. Eur. Biol. Sot. Madrid 128, Biochemistrv 8. 1643. 7. F&I&, K., FUMIO, H., AND NISHIMURA, S. (1971) Biochemistry 10, 3277. 8. HOLLEY, R. W. (1966). Sci. Amer. 214,30. 9. STULBERG, M. P., AND ISHAM, K. R. (1967) Proc. Nat. Acad. Sci. U.S.A. 67,131O. 10. SHUGART, L., AND STULBERG, M. P. (1969) J. Biol. Chem. 244, 2806. 11. BROWN, D. J. (1962) “The Pyrimidines”, p. 485. Interscience, New York. 12. LIPSETT, M. N., AND DOCTOR, B. P. 0967) J. Biol. Chem. 242,4072. 13. CARBON, J., AND DAVID, H. (1968). Biochemistry 7,385l. 14. SCHEIT, K. H. (1968) Biochim. Biophys. Acta 166, 285. 15. SHUGART, L. Arch. Biochem. Biophys. (in press). 16. KELMERS, A. D., NOVELLI, G. D., AND STULBERG, M. P. (1965) J. Biol. Chem. 240,3979. 17. STULBERG, M. P. (1967) J. Biol. Chem. 242, 1060. 18. BOYER, P. D. (1954) J. Amer. Chem. Sot. 76, 4331. 19. FOX, J. J., PRAG, D. V., VEMPEN, I., DOERR, I., CHEONG, L., KNOLL, J. E., EDINOFF, M. L., BENDICH, A.! AND BROWN, G. B. (1959) J. Amer. Chem. Sot. 81,178. 20. STULBERG, M. P., ISHAM, K. R., AND STEVENS, A. (1969) Biochim. Biophys. Acla 186,297. 21. HAWKINSON, S. W., PAL, B. C., AND EINSTEIN, J. R. (1970) Abstr. of the Winter Meeting of the American Crystallography Association, March 2-5 (1970), New Orleans, La. 22. SENO, T., KOBAYASHI, M., AND NISHIMUR.~, S. (1969) Biochim. Biophys. Acta 174,71. 23. ItWIN, I. B., KELMERS, A. D., AND GOLDSTEIN, G. (1967) Anal. Biochem. 20,533. 24. FELSENFELD, G., AND MILES, H. T. (1967) Ann. Rev. Biochem. 36,497.