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Effect of selective chemical modification of 4-thiouridine of phenylalanine transfer ribonucleic acid on enzyme recognition
ARCHIVES
OF
BIOCHEMISTRY
Effect
.4ND
BIOPHYSICS
of Selective Phenylalanine
148, 488-495 (1972)
Chemical
Modification
Transfer Enzyme
Ri bonucleic
of 4-Thiouridine Acid
of
on
Recognition’
LEE SHUGART Biology
Division,
Oak Ridge National
Laboratory,
Oak Ridge, Tennessee $7830
Received September 22, 1971; accepted November 9, 1971 Transformation of 4-thiouridine residues in Escherichia coli transfer ribonucleic acids is achieved under conditions which leave the major bases and the primary structure unaffected. The modifications of 4-thiouridine involve either alteration with N-ethylmaleimide, cyanogen bromide, or hydrogen peroxide, or a photochemical transformation effected by irradiation at 330 nm of tRNA in an organic solvent. These selective modifications were made on unfractionated species (Phe, Leu, fMet, Tyr, andVa1) and purified species (Phe, fMet, andVa1) of E. coli tRNA withlittle or noloss in their capacities to be aminoacylated. Of the tRNA species tested, subsequent treatment of 4-thiouridineless-tRNA with sodium borohydride affects only the capacity of tRNAPhe to be aminoacylated. These observations are consistent with the proposal that the cognate ligase recognition site on tRNAPhe is situated in the nonhydrogenbonded dihydrouridine loop area of the molecule.
An important, but as yet unsolved question in molecular biology is how an aminoacyl-tRNA ligase recognizes its cognate transfer RNA2 and discriminates against all others (1). The significance of this recognition phenomenon becomesevident when one realizes that this interaction is probably the r This research was jointly sponsored by the National Institute of General Medical Sciences of the National Institutes of Health and by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2 The abbreviations used are: tRNA, transfer ribonucleic acid; tRNAne, tRNALeu, tRNALya, tRNAnet, tRNAPhe, tRNATYr, tRNAvar, transfer ribonucleic acids specific for isoleucine, leucine, lysine, methionine, phenylalanine, tyrosine, and valine respectively; tRNA,N”$ transfer ribonucleic acid specific for methionine and capable of being formylated; Leu-tRNA, Lys-tRNA, Met-tRNA, Phe-tRNA, Tyr-tRNA, Val-tRNA, transfer ribonucleic acid aminoacylated with its respective formyl-methionylamino acid; fMet-tRNA, tRNA; NEM, N-ethylmaleimide; Aliquat 336, tricaprylylmethylammonium chloride; Freon 214, tetrachlorotetrafluoropropane. 488 Copyright
@ 1972 by Academic
Press,
Inc.
most critical step in maintaining the fidelity of protein synthesis. A previous study (2) in this laboratory had tentatively identified the enzyme recognition site of Eschmichia coli phenylalanine tRNA to be that area situated in the last one-third of the molecule near the 5’ terminus. Subsequently, it was shown that the nonhydrogenbonded dihydrouridine loop probably contained the information for ligase recognition because selective chemical modification of the minor nucleosides dihydrouridine and 4-thiouridine in E. coli tRNAPhe with NaBH4 resulted in complete loss of the ability of tRNAPh” to be aminoacylated (3). At the time of that investigation it was thought that 4-thiouridine was situated in the dihydrouridine loop of tRNAPhe (4). Burton (5) has since reported that 4-thiouridine is in a region of the molecule adjacent to this area. His findings are compatible with the structure of tRNAPhe proposed by Barrell and Sanger (6). Therefore, the study in this paper was undertaken in an effort to further pinpoint
4-THIOURIDINE
AND AMINOACYLATION
the recognition site by determining whether the inactivation of NaBHI-treated E. coli tRNAPh” was the consequence of a modification to the 4-thiouridine, to the dihydrouridines, or to both. It was decided to determine the amino acid-accepting activities of E. coli tRNA after modification of the 4-thiouridine residue by several different procedures. The reactions employed for the chemical modification of 4-thiouridine by NEM (7), CNBr (8, 9)) and Hz02 (10) have been described previously, as has photochemical transformation by irradiakion (11). Since two of these alterations were performed in a nonaqueous medium, a method is presented whereby these modifications can be accomplished after tRNA is selectively partitioned into a water-immiscible organic phase containing a quarternary ammonium compound (12, 13). The results reported here show that specific modification to 4-thiouridine in unfractionated and purified E. coli tRNAs by these several methods does not result in a concomitant loss of aminoacyl-tRNA formation. However, of the tRNAs tested only Phe-tRNA formation is destroyed upon subsequent treatrnent with NaBH4 . EXPERIMENTAL
PROCEDURES
Materials Unfractionated E. coli tRNAs were prepared as previously described (14) and had the following amino acid-accepting activities (expressed as picomoles of amino acid accepted per Azso ,,-unit3 of tRNA): Leu-tRNA, 163; Lys-tRNA, 66; MettRNA, 65; Phe-tRNA, 46; Tyr-tRNA, 58; and Val-tRNA, 142. Purified individual species of tRNA were obtained from A. D. Kelmers (15) and possessed the following amino acid-accepting activities: Met-tRNAti , 1,469; Met-tRNAfz , 1,373; Phe-tRNA, 1,366; and Val-tRNA, 1,149. With the purified species, amino acid acceptance is approximately equivalent to the 3’ adenosine content of the tRNA. All preparations had an Ate0 nm to AGO nm ratio of approximately 2.0 in 1 mM MgCl, . Aliquat 336 was obtained from General Mills, Inc., and Freon 214 from E. I. duPont de Nemours 3 The relation between A260 n,,,-unit and the amount of tRNA in solution has been detailed previously (16-18). One absorbance unit is that amount of tRNA in 1 ml of 1 mM MgC12 possessing an absorbance of 1.0, measured with a l-cm optical path at a wavelength of 260 nm.
OF tRNA
489
and Co. “C-labeled amino acids were obtained from New England Nuclear Corporation and had the following specific activities (Ci/mole): Leu, 263; Lys, 248; Met, 217; Phe, 375; Tyr, 379; and Val, 208. A-6 cation exchanger resin was obtained from BioRad. Other chemicals were of reagent grade.
Methods tRNA concentration. This was expressed as AHO rim-units/ml. Spectrophotometric measure-
ments of tRNA in solution were made at room temperature with a Cary Model 14 spectrophotometer or with a Beckman DU monochromater equipped with a Gilford Model 220 optical density converter. Amino acid-accepting activity. Approximately 0.5 AsCOrim-units of unfractionated tRNA, or 0.02 A~60 rim-units of purified species were aminoacylated in a reaction mixture containing 25 Etmoles of Tris-HCl buffer, pH 8.0,2.5 rmoles magnesium acetate, 1.25 pmoles KCl, 0.5 rmoles ATP, 1 pmole 2-mercaptoethanol, 2 nmoles (‘Q-1 amino acid, and 0.1 mg of enzyme protein in a final volume of 0.25 ml. The reaction was initiated by the addition of enzyme (14) and allowed to continue at 30” until aminoacylation was obtained (usually 30 min). The procedure for determining the amount of aminoacyl-tRNA formed was carried out essentially as outlined by Kelmers et al. (14). The determination of fMet-tRNA was performed as described by Shugart et al. (19). Selective partitioning of tRNA. (i) Organic phase. A 5% solution (w/v) of Aliquat, 336 in Freon 214 was prepared and washed as outlined by Weiss and Kelmers (20) and stored at 4’ in the dark. (ii) Extraction (13). tRNA (about 30 A26,, ,,-units in one ml of 1 mM MgCls) was diluted to 3 ml with 10 mM Tris-HCI buffer, pH 7.5, containing 10 rnM MgC12, 1 mM EDTA, and 0.1 M NaCl in a IO-ml screwcap centrifuge tube. An equal volume of organic phase was added and after 10 min of mixing the phases were separated by centrifugation (at room temperature) and the aqueous phase was removed. The tRNA was recovered from the organic phase by repeating the above extraction procedure with 5 ml of the above Tris buffer containing 1.0 M NaCI. The tRNA was removed from the aqueous phase by ethanol precipitation followed by filtration on a Millipore filter (21). Modijication
to tRNA.(i)
N-Ethylmaleimide
(7).
To 3 ml of organic phase containing about 30 Azso ,,-units of tRNA was added 3.4 pmoles of NEM dissolved in 20 ~1of hexane. The mixture was incubated at 37” for 1 hr, and the tRNA was recovered from the organic phase as described above. (ii) Cyanogen bromide. The treatment of tRNA with CNBr was performed as outlined by Sane-
490
SHUGART
yoshi and Nishimura (8). (iii) Irradiation (11). Three milliliters of organic phase containing 30 A260.m-units of tRNA were pipetted into a glass-stoppered Beckman cuvette. The solution was stirred constantly by means of a small magnetic stirring bar and irradiated at 330 nm with a Bausch and Lomb 0.5-m grating monochromator. The flux rate, determined with a Kettering model 65 Radiometer, was 6000 ergs/mm/ml incident on the organic phase. After irradiation the tRNA was recovered from the organic phase as described above. (iv) Hydrogen peroxide. The treatment of tRNA with Hz02 was performed as outlined by Scheit (10) for t,he conversion of 4-thiouridylyl(3’-5’)-4-thiouridine to uridylyl-(3’-5’)-uridine. Ten microliters of 3070 Hz02 were mixed with 1 ml of 0.1 M Tris-HCl buffer, pH 8.0 containing about 26 At60 rim-units of tRNA, and the reaction was allowed to proceed at room temperature. The tRNA was recovered by ethanol precipitation (21). (v) Sodium borohydride. The treatment of tRNA with NaB& was performed as outlined by Shugart and Stulberg (21). Nucleoside determination. Approximately 25 A260 ,,-units of tRNA were hydrolyzed enzymatitally as previously described by Uziel (22) to the nucleoside level with snake venom phosphodiesterase (Worthington) and alkaline phosphatase (Worthington) in 0.2 M ammonium acetate buffer, pH 8.8, containing 23 mM magnesium acetate. The mixture was incubat.ed for 4 hr at 45” and the nucleoside content was determined quantitatively by cation column chromatography (22). RESULTS
Efect of modification of 4-thiouridine absorption spectrum of E. coli tRNA.
on the
The presence of 4-thiouridine in tRNA can be monitored by its characteristic absorption maximum near 335 nm (23). Figure 1 shows the effect on the absorption profile between 300 and 400 nm of E. coli tRNA as a result of the modification of 4-thiouridine by any of the four methods outlined. Curve 1 is the absorption profile before treatment and curve 2 is the absorption profile that results after treatment of the tRNA preparations. Part A shows the results obtained on unfractionated tRNA, and Part B shows the results obtained using several purified tRNA’s which contain 4-thiouridine. It is possible that not all 4-thiouridine residues in the tRNA were modified since after treatment there is a residual absorption at 340 nm. This is attributed to the tail from the
320
360
400 320 WAVELENGTH (IX,,)
360
4C
FIG. 1. Effect of selective chemical modification to 4thiouridine on the UV-absorption spectrum between 309 and 400 nm of (A) unfractionated E. coli tRNA and (B) purified E. coli tRNAs. (All curves normalized to 20 A260 ,,,,,-units of tRNA in 1 ml of 1 mM MgClz .) Curve 1, typical spectrum of tRNA before treatment; curve 2, typical spectrum after treatment of tRNA with CNBr for 10 min, with Hz02 for 90 min (unfractionated) and 246 min (purified), with NEM for 60 min, and with irradiation for 20 min.
260-nm absorption peak of the tRNA (18), because a nucleoside analysis (22) of the treated-tRNAs showed only a trace of 4thiouridine and no new or unidentifiable nucleosides. These data show the modification to 4thiouridine (under the conditions specified) to be essentially complete. Effect of modi$ication of 4-thiouridine aminoacylation of E. coli tRNAs.
on
(i) N-ethylmaleimide. Carbon and David (7) showed that this sulfhydryl reagent selectively reacts with 4-thiouridine residues in unfractionated E. coli tRNA with only a small reduction in total amino acid-accepting capacity of certain tRNAs. The problem of the rapid decomposition of the reagent in aqueous alkaline solutions can be circumvented by carrying out the reaction with tRNA in the organic phase (see “Methods”). The data in Table I show that treatment of unfractionated tRNAs and purified tRNAPhe with NEM does not result in an appreciable
4-THIOURIDINE
AND AMINOACYLATION
TABLE I EFFECT OF S~~LECTIVECHEMICAL OF 6THIOURIDINE UNFRACTIONATED SPECIES
MODIFICATION ON THE AMINOACYLATION OF AND PURIFIED E. coli TRNA
Chemical modiicationD tRNA preparation
unfractionated: Phe Leu LYS fMet Tyr Val purified : Phe fMet Val
NEM CNBr Irrad. Hech IO/Jaminoacylation remaining/
96 93
65 95 34 loo 85
91 90
104 93 83
92 102
90
78
85
75 966 102
a 4-thiouridine in tRNA was modified as outlined in Methods Section with times of modification the same as detailed in Fig. 1. Percent aminoacylation based on control samples that were treated under identical conditions except that the modifying reagent was omitted. 6 Formylation (19) of HlOt-treated tRNAyet was equivalent to methionylation.
loss of amino acid-accepting activity for tRNAPhe or tRNALeU (ii) Cyanogen bromide. CNBr reacts with 4-thiouridine in E. co& and under appropriate conditions the intermediate thiocyanato derivative is converted to uridine (8,9). This modification of 4-thiouridine and its subsequent conversion to uridine does not result in. an appreciable loss of amino acid-accepting capacity for purified or unfractionated tRNAPhe or for several other unfractionated tRNA activities (Table I). Saneyoshi and Nishimura (8) have stated that the minor component X (6) in E. coli tRNAPhe is modified by reaction with CNBr. Retention of Phe-tRNA formation after CNBr treatment eliminates component X from the ligase recognition site in tRNAPhe. The significant inactivation of tRNALya by this procedure is consistent with an oxidative modification to the molecule as previously reported (24, 25). No attempt was made to restore the inactive tRNALy8 by treatment with a reducing agent (24). The
OF tRNA
491
data of Reid (26) argue that 4-thiouridine is involved in the inactivation of Lys tRNA formation, This is not supported, however, by the results of Harris et al. (27). (iii) PhotochemicaZ transformation: Pleiss and Cerutti (11) have described a reaction that transforms the 4-thiouridine of unfractionated E. coli tRNAs to uridine under extremely mild photochemical conditions (irradiation at 330 nm in tert-butyl alcohol). It should be noted that a different modification occurs to 4-thiouridine when tRNA is irradiated in aqueous solutions, presumably because the secondary structure of tRNA is maintained (28). The photochemical transformation of E. coli tRNAs in this study was accomplished in a 5 % solution of Aliquat 336 in Freon 214. The advantages of partitioning the tRNA into this organic phase for irradiation are: (1) It provides an environment in which the tRNA is less structured4; (2) it simplifies the manipulation of small amounts of tRNA, and; (3) it alleviates the interference of water (11). Unfractionated E. coli tRNA was irradiated as described under experimental procedures and the photochemical transformation of 4-thiouridine, as measured by the decrease in 340-nm absorption, was found to be complete in 20 min (Fig. 1A). During this period there was less than a 10 % loss in the capacity of tRNALeu, tRNAPhe, and tRNATYP to be aminoacylated (Table I). Under similar conditions, a purified sample of tRNAPhe that had been irradiated for 10 min showed a 50 % decrease in its 340-nm absorption, and a 15 % loss in its capacity to be aminoacylated. Irradiation of the tRNAPhe for an additional 10 min resulted in negligible loss of amino acid acceptance (Table I>. Irradiation of 4-thiouridine in the organic phase at a concentration equivalent to that in the tRNA experiments results in an initial rapid oxidation of the 4-thiouridine to 4-thiouridine disulfide followed by the appearance of a stoichiometric amount of 4 A decrease in ordered structure of tRNA while in the organic phase is indicated by a bathochromic shift of the maximum of its ultraviolet absorption spectrum.
492
SHUGART
uridine in 45 min. None of the four major nucleosides, nor 7-methyl guanosine, Nmethyl l-methyl guanosine, guanosine, dihydrouridinc, or l-methyl adenosine is affected by irradiation in the organic phase, .as determined by their characteristic UVabsorption spectra. (iv) Hydrogen peroxide. Scheit (10) has ,described a procedure for the mild hydrolysis of 4-thiouridylyl-(3’-5’)-4-thiouridine with HzOz at pH 8. This method has been adapted hcrc to selectively modify 4-thiouridine in l!C. coli tRNA. Figure 2 shows that after 90min exposure of unfractionated tRNA to HzOz, only a small residual absorption at 340 nm remains. The HzOz-treated tRNA is recoverable by ethanol precipitation, and its chromntographic profile upon Sephadex G-100 column chromatography, as outlined by Egan et al. (29), is similar to that of native tRNA. A nucleoside analysis gave no detectable 4-thiouridine. In addition the HzOp-treated sample showed no appreciable loss of amino acid acceptance for the four aminos acid tested (Table I). The 4-thiouridine in purified species of E. coli tRNA on the other hand are more resistant to alteration by this reagent. Additional exposure time was required to bring about a loss of 340-nm absorption in the purified tRNAs equivalent to that observed in equal A2G0,,-units of the unfractionated
tRNA (Fig. 3A). The data in Table I also show that there is no significant loss of amino acid acceptance (and no loss of the ability of tRNAyt to be formylated) after H202 treatment of the purified species of tRNA. An interesting observation that resulted from this study is given in Panel B of Fig. 3, which shows that the characteristic loss of absorption at 340 nm does not occur when E. coli tRNAp (30) is treated with HzOz. This tRNA species is an artifact that arises from the major naturally occurring formylatable species (tRNAy) in E. coli as a result of an unknown modification of its 4-thiouridine during isolation (31). Because of the pronounced skew to the absorption spectrum between 300 and 400 nm, the modification is believed to be similar to that reported for tRNAV”’ (28). Subsequent e$ect of NaBH4 modi$cation on aminoacylation of Q-thiouridineless E. wli tRNA. As pointed out in the introduction, the purpose of this study waa to determine whether the inactivation of NaBH&-treated E. coli tRNAPhe (3) was a consequence of a
B
A
0.5 (
I I
L
I 403
WAVELENGTH hn)
2. Effect of HZ02 treatment on the UVabsorption spectrum between 300 and 400 nm of 19 A*eOrim-units of unfractionated E. coli tRNA in 1 ml of Tris buffer. Curves represent the following exposure times (min.) to Hz02 : 1, 1.6; 2, 2.9; 3, 8.7; 4, 16.1; 5, 25.4; 6, 66.2; 7, 90.8. FIG.
1 1 4c
340 WAVELENGTH
InIn)
FIG. 3. Effect of Ha02 treatment on the UVabsorption spectrum between 300 and 400 nm of 17 Azso rim-units each of (A) purified tRNAFt [31]; and (B) purified tRNA2t [31, 321. Curve 1, zero time, curve 2, after 90 min; curve 3, after 24.0min exposure to Hz02 . Panel A is also representative of the effect of Hz02 treatment on purified tRNAPhe and tRNAva1 samples (see Fig. 1 and Table I).
4-THIOURIDINE
AND AMINOACYLATION TABLE
EFFECT
OF SODIUM
TRNA
BOROHYDRIDE AND PURIFIED
tRNA preparaticm
Unfractionated:
II
IZEDUCTION
ON THE AMINOXYLATION OF UNE.R.WTIONITED TRNA SPECIES AFTER SELECTIVE MODIFICATION TO 6THIOURIDINE
E. coli
Chemical modification” NEM
+ +
493
OF tRNA
Irrad
Hz02
CNBr
+ +
+ +
Purified :
NaBHp
+ +
+ +
+ +
E. coli
Aminoacylation
1% remaining\
Phe 100 96 8 100 85 18 Phe 100 75 19 100 92 12
Leu 100 93 74 100 90 84 Val 100 76 64 100 71 71
0 4-thiouridine in tRNA was modified as outlined in Methods Section. Control samples (-) were treated under identical conditions except that the modifying reagent was omitted. Amino acid acceptor activities were determined upon recovery of tRNA after treatment with NEM for 60 min, irradiation for 30 min, Hz02 for 4 hr, CNBr for 10 min, and NaB& for 4 hr.
modification to its 4-thiouridine or dihydrouridines. The results reported thus far lend strong support, to the argument that the 4-thiouridine residue of E. coli tRNAPhe is not involved in ligase recognition, since its modification by four different methods does not result in an appreciable loss of PhetRNA formation (or several other aminoacyl-tRNA formations). Thus, indirectly, it would appear that selective loss of acceptor activity by E. coli tRNAPhe upon treatment with NaBH4 was the result of a modification to the dihydrouridines. This was tested directly by determining the acceptor activity of 4-thiouridinelesstRNA upon subsequent treatment with NaBH4. Table II details the results of this experiment. Phe-tRNA formation in unfractionated, as well as in purified E. coli tRNA was severely curtailed when NaBH4 treatment followed 4-thiouridine modification. Aminoacylation of other tRNAs was not similarly affected. The possibility does exist that a modification of E. coli tRNA by any of the methods detailed could result in an alteration of the rate at which aminoacyl-tRNA formation occurs, with no effect on the total amount of
amino acid accepted. This observation has been reported (28). No attempt was made in this investigation to study the kinetics of tRNA-ligase interaction. DISCUSSION
Several lines of experimental evidence suggest that the presence of the minor nucleoside 4-thiouridine is not necessary for amino acid acceptor activity. First, 4thiouridine is not ubiquitously distributed, but is found almost exclusively in those tRNAs of bacterial origin. In this connection, there are numerous examples of the heterologous aminoacylation of tRNA species from higher organisms with bacterial ligases (32). Second, modifications that are highly specific toward 4-thiouridine (7-9, 11, 28, 33-36) often result in no appreciable loss of amino acid acceptance in t,he tRNAs tested. Third, Harris et al. (27) have shown that sulfur-deficient tRNA obtained from a cysteine-requiring, “relaxed” Wain of E. coli was devoid of 4-thiouridine, but had amino acid acceptances that, for most, of the amino acids tested, were not drastically different from those of normal tRNAs isolated from the same organism. Fourth, Johnson et al.
494
SHUGART
(37) have shown that aminoacylation of tRNAI’e isolated from Mycoplasma sp. (Kid) proceeds nearly to the theoretical level even though the tRNA is deficient in 4thiouridine. We have previously performed certain specified modifications on tRNAPhe in an effort to identify and characterize an enzyme recognition site of the molecule (2, 3). The possible importance of the minor nucleosides dihydrouridine and 4-thiouridine in this recognition phenomenon was implied from our studies with NaBHd-treated tRNAPhe. The data presented in this paper support the general supposition that 4-thiouridine is not necessary for the aminoacylation of E. coli tRNAs. In addition, the data further suggest that the area in E. coli tRNAPhe that contains the information for ligase recognition is probably dependent upon the presence of unmodified dihydrouridine residues. Since the dihydrouridine residues are confined to the nonhydrogen-bonded 5’ loop area of the molecule, this region and its integrity becomes more suspect as the ligase recognition sit,e. It should be emphasized that the selective reduction of dihydrouridines in tRNAPhe by NaBH4 and the concomitant loss of amiioacylation appears to be unique to the E. coli tRNAPhe-E. coli ligase system. As previously indicated (3), the response of individual t,RNAs to specific treatment with this reagent varies, and may depend on the source and purity of the tRNA, the specific tRNA species tested, and the location of susceptible bases. Admittedly, ligase-tRNA interaction is a highly complicated phenomenon with the possible involvement of several independent but common binding sites on the tRNA molecule in addition to a specific recognition .&e(s) (38). This aspect of the problem is currently being investigated through studies on complex formation (39) between modified tRNAPhe and its ligase. ACKNOWLEDGMENTS I wish to acknowledge the technical expertise of Barbara Chastain and Kenneth Isham, who assisted in the experiments reported here. I want to thank Drs. J. W. Longworth and It. Rahn for their assistance with the phot,ochemical experiments, and Drs. B. Pal, M. Uziel and M. P. Stulberg for
their helpful investigation.
suggestions
during
t,he course of this
REFERENCES 1. ZAMECNIK, P., “Cold Spring Harbor Sympos. on Qua&. Biol.“, 34, p. 1 (1969). 2. STULBERG, M. P., AND ISHAM, K. R., Proc. Nat. Acad. U.S.A. 67, 1310 (1967). 3. SHUGART, L. R. AND STULBERG, M. P., J. Biol. Chem. 244, 2806 (1969). 4. UZIEL, M., AND GLASSEN, H., Riochemistry 8, 1643 (1969). 5. BURTON, K., Fed. Eur. Biochem. Sot. Lett. 6, 77 (1970). 6. BARREL, B. G., AND SANGER, F., Fed. Eur. Biochem. Sot. Lett. 3, 275 (1969). 7, 7. CARBON, J., AND DAVID, H., Biochemistry 3851 (1968). 8. SANEYOSHI, M., AND NISHIMURA, S., Biochim. Biophys. Acta 204, 389 (1970). 9. WALKER, R. T., AND RAJBHANDARY, Biochem. Biophys. Res. Commun. 36, 907 (1970). Biophys. Acta 166, 285 10. SCHEIT, K. H., B&him. (1968). 11. PLEISS, M., AND CERUTTI, P. A., Biochemistry 10, 3093 (1971). 12. KHYM, J. X., J. Biot. Chem. 241.4529 (1966). 13. EGAN, B. Z., RHEAR, R. W., AND KELMERS, A. D., Anal. Biochem. 36,139 (1970). 14. KELMERS, A. D., NOVELLI, G. D., AND STULBERG, M. P., J. Biol. Chem. 240,3979 (1965). 15. KELMERS, A. D., AND STULBERG, M. P., Science 167, 238 (1970). 16. VON EHRENSTEIN, G., Methods Enzymol. l% 588 (1967). 17. LINDAKL, T., AND FRESCO, J. R., Methods Enzymol. 12, 601 (1967). 6, 18. ZIFF, E. B., AND FRESCO, J. R., Biochemistry 3242 (1969). 19. SHUGART, L. R., CHASTAIN, B., AND NOVELLI, G. D., Biochim. Biophys. Acta 186, 384 (1969). 20. WEISS, J. F., AND KELMERS, A. D., Biochemistry 6, 2507 (1967). 21. SHUGART, L. R., AND STULBERG, M. P., J. Biol. Chem. 244, 2806 (1969). 22. UZIEL, M., KOH, C. K., AND COHN, W. E., Anal. Biochem. 26, 77 (1968). 23. LIPSETT, M. N., J. Biol. Chem. 240.3975 (1965). 24. CARBON, J., HUNG, L., AND JONES, D., Proc. Nat. Acad. Sci. U.S.A. 63 (1965). 25. SANEYOSHI, M., AND NISHIMURA, S., Biochim. Biophys. Acta 145, 208 (1967). 26. REID, B., Biochem. Biophys. Res. Commun. 33, 627 (1968). 27. HARRIS, C. L., TITCHENER, E. B., AND CLINE, A. L., J. Bacteriology 100, 1322 (1969). as. FAVRE, A., YANIV, M., AND MICHELSON, A. M.,
4-THIOURIDINE
29. 30.
31.
32.
33.
AND AMINOACYLATION
Biochem. Biophys. Res. Commun. 37, 266 (1969). EQAN, B. Z., RHEAR, R. W., AND KELMERS, A. D., Biochim. Biophys. Acta 174, 23 (1969). WEISS, J. F., PEARSON, R. L., AND KELMERS, A. D., Biochemistry 7, 3479 (1968). SHUGART, L. II., CHASTAIN, B., AND NOVELLI, G. D., Biochem. Biophys. Res. Commun. 37, 305 (1969). JACOBSON, K. B., in “Progress in Nucleic Acid Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.) Vol. 11, p. 461. Academic Press, New York, 1971. HARA, H., HC)RIUCHI, T., SANEYOSHI, M., AND NISHIMURA,
S.,
Biochem.
Commun. 38, 305 (1970).
Biophys.
Res.
OF tRNA
495
34. SENO, T., KOBAYASHI, I., FUKAHARA, M., AND NISHIMURA, S., Fed. EUT. Biochem. Sot. Lett. 7, 343 (1970). 35. FAULKNER, R., AND UZIF,L, M., Biochim. Biophys. Acta 238, 464 (1971). 36. SANEYOSHI, M. AND NISHIMURA, S., Biochim. Biophys. Acta 246, 123 (1971). 37. JOHNSON, L., HAYASHI, H., AND SILL, D., Biochemistry 9, 2823 (1970). 38. CHAMBERS, R. W., in “Progress in Nucleic
Acid Research andMolecular Biology” (J. N. Davidson and W. E. Cohn, eds.) Vol. 11, p. 489. Academic Press, New York, 1971. 39. FARRELLY, J. G., LONGWORTH, J. W., AND STULBERG, M. P., J. Riol. Chem. 246, 1266 (1971).
OF
BIOCHEMISTRY
Effect
.4ND
BIOPHYSICS
of Selective Phenylalanine
148, 488-495 (1972)
Chemical
Modification
Transfer Enzyme
Ri bonucleic
of 4-Thiouridine Acid
of
on
Recognition’
LEE SHUGART Biology
Division,
Oak Ridge National
Laboratory,
Oak Ridge, Tennessee $7830
Received September 22, 1971; accepted November 9, 1971 Transformation of 4-thiouridine residues in Escherichia coli transfer ribonucleic acids is achieved under conditions which leave the major bases and the primary structure unaffected. The modifications of 4-thiouridine involve either alteration with N-ethylmaleimide, cyanogen bromide, or hydrogen peroxide, or a photochemical transformation effected by irradiation at 330 nm of tRNA in an organic solvent. These selective modifications were made on unfractionated species (Phe, Leu, fMet, Tyr, andVa1) and purified species (Phe, fMet, andVa1) of E. coli tRNA withlittle or noloss in their capacities to be aminoacylated. Of the tRNA species tested, subsequent treatment of 4-thiouridineless-tRNA with sodium borohydride affects only the capacity of tRNAPhe to be aminoacylated. These observations are consistent with the proposal that the cognate ligase recognition site on tRNAPhe is situated in the nonhydrogenbonded dihydrouridine loop area of the molecule.
An important, but as yet unsolved question in molecular biology is how an aminoacyl-tRNA ligase recognizes its cognate transfer RNA2 and discriminates against all others (1). The significance of this recognition phenomenon becomesevident when one realizes that this interaction is probably the r This research was jointly sponsored by the National Institute of General Medical Sciences of the National Institutes of Health and by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2 The abbreviations used are: tRNA, transfer ribonucleic acid; tRNAne, tRNALeu, tRNALya, tRNAnet, tRNAPhe, tRNATYr, tRNAvar, transfer ribonucleic acids specific for isoleucine, leucine, lysine, methionine, phenylalanine, tyrosine, and valine respectively; tRNA,N”$ transfer ribonucleic acid specific for methionine and capable of being formylated; Leu-tRNA, Lys-tRNA, Met-tRNA, Phe-tRNA, Tyr-tRNA, Val-tRNA, transfer ribonucleic acid aminoacylated with its respective formyl-methionylamino acid; fMet-tRNA, tRNA; NEM, N-ethylmaleimide; Aliquat 336, tricaprylylmethylammonium chloride; Freon 214, tetrachlorotetrafluoropropane. 488 Copyright
@ 1972 by Academic
Press,
Inc.
most critical step in maintaining the fidelity of protein synthesis. A previous study (2) in this laboratory had tentatively identified the enzyme recognition site of Eschmichia coli phenylalanine tRNA to be that area situated in the last one-third of the molecule near the 5’ terminus. Subsequently, it was shown that the nonhydrogenbonded dihydrouridine loop probably contained the information for ligase recognition because selective chemical modification of the minor nucleosides dihydrouridine and 4-thiouridine in E. coli tRNAPhe with NaBH4 resulted in complete loss of the ability of tRNAPh” to be aminoacylated (3). At the time of that investigation it was thought that 4-thiouridine was situated in the dihydrouridine loop of tRNAPhe (4). Burton (5) has since reported that 4-thiouridine is in a region of the molecule adjacent to this area. His findings are compatible with the structure of tRNAPhe proposed by Barrell and Sanger (6). Therefore, the study in this paper was undertaken in an effort to further pinpoint
4-THIOURIDINE
AND AMINOACYLATION
the recognition site by determining whether the inactivation of NaBHI-treated E. coli tRNAPh” was the consequence of a modification to the 4-thiouridine, to the dihydrouridines, or to both. It was decided to determine the amino acid-accepting activities of E. coli tRNA after modification of the 4-thiouridine residue by several different procedures. The reactions employed for the chemical modification of 4-thiouridine by NEM (7), CNBr (8, 9)) and Hz02 (10) have been described previously, as has photochemical transformation by irradiakion (11). Since two of these alterations were performed in a nonaqueous medium, a method is presented whereby these modifications can be accomplished after tRNA is selectively partitioned into a water-immiscible organic phase containing a quarternary ammonium compound (12, 13). The results reported here show that specific modification to 4-thiouridine in unfractionated and purified E. coli tRNAs by these several methods does not result in a concomitant loss of aminoacyl-tRNA formation. However, of the tRNAs tested only Phe-tRNA formation is destroyed upon subsequent treatrnent with NaBH4 . EXPERIMENTAL
PROCEDURES
Materials Unfractionated E. coli tRNAs were prepared as previously described (14) and had the following amino acid-accepting activities (expressed as picomoles of amino acid accepted per Azso ,,-unit3 of tRNA): Leu-tRNA, 163; Lys-tRNA, 66; MettRNA, 65; Phe-tRNA, 46; Tyr-tRNA, 58; and Val-tRNA, 142. Purified individual species of tRNA were obtained from A. D. Kelmers (15) and possessed the following amino acid-accepting activities: Met-tRNAti , 1,469; Met-tRNAfz , 1,373; Phe-tRNA, 1,366; and Val-tRNA, 1,149. With the purified species, amino acid acceptance is approximately equivalent to the 3’ adenosine content of the tRNA. All preparations had an Ate0 nm to AGO nm ratio of approximately 2.0 in 1 mM MgCl, . Aliquat 336 was obtained from General Mills, Inc., and Freon 214 from E. I. duPont de Nemours 3 The relation between A260 n,,,-unit and the amount of tRNA in solution has been detailed previously (16-18). One absorbance unit is that amount of tRNA in 1 ml of 1 mM MgC12 possessing an absorbance of 1.0, measured with a l-cm optical path at a wavelength of 260 nm.
OF tRNA
489
and Co. “C-labeled amino acids were obtained from New England Nuclear Corporation and had the following specific activities (Ci/mole): Leu, 263; Lys, 248; Met, 217; Phe, 375; Tyr, 379; and Val, 208. A-6 cation exchanger resin was obtained from BioRad. Other chemicals were of reagent grade.
Methods tRNA concentration. This was expressed as AHO rim-units/ml. Spectrophotometric measure-
ments of tRNA in solution were made at room temperature with a Cary Model 14 spectrophotometer or with a Beckman DU monochromater equipped with a Gilford Model 220 optical density converter. Amino acid-accepting activity. Approximately 0.5 AsCOrim-units of unfractionated tRNA, or 0.02 A~60 rim-units of purified species were aminoacylated in a reaction mixture containing 25 Etmoles of Tris-HCl buffer, pH 8.0,2.5 rmoles magnesium acetate, 1.25 pmoles KCl, 0.5 rmoles ATP, 1 pmole 2-mercaptoethanol, 2 nmoles (‘Q-1 amino acid, and 0.1 mg of enzyme protein in a final volume of 0.25 ml. The reaction was initiated by the addition of enzyme (14) and allowed to continue at 30” until aminoacylation was obtained (usually 30 min). The procedure for determining the amount of aminoacyl-tRNA formed was carried out essentially as outlined by Kelmers et al. (14). The determination of fMet-tRNA was performed as described by Shugart et al. (19). Selective partitioning of tRNA. (i) Organic phase. A 5% solution (w/v) of Aliquat, 336 in Freon 214 was prepared and washed as outlined by Weiss and Kelmers (20) and stored at 4’ in the dark. (ii) Extraction (13). tRNA (about 30 A26,, ,,-units in one ml of 1 mM MgCls) was diluted to 3 ml with 10 mM Tris-HCI buffer, pH 7.5, containing 10 rnM MgC12, 1 mM EDTA, and 0.1 M NaCl in a IO-ml screwcap centrifuge tube. An equal volume of organic phase was added and after 10 min of mixing the phases were separated by centrifugation (at room temperature) and the aqueous phase was removed. The tRNA was recovered from the organic phase by repeating the above extraction procedure with 5 ml of the above Tris buffer containing 1.0 M NaCI. The tRNA was removed from the aqueous phase by ethanol precipitation followed by filtration on a Millipore filter (21). Modijication
to tRNA.(i)
N-Ethylmaleimide
(7).
To 3 ml of organic phase containing about 30 Azso ,,-units of tRNA was added 3.4 pmoles of NEM dissolved in 20 ~1of hexane. The mixture was incubated at 37” for 1 hr, and the tRNA was recovered from the organic phase as described above. (ii) Cyanogen bromide. The treatment of tRNA with CNBr was performed as outlined by Sane-
490
SHUGART
yoshi and Nishimura (8). (iii) Irradiation (11). Three milliliters of organic phase containing 30 A260.m-units of tRNA were pipetted into a glass-stoppered Beckman cuvette. The solution was stirred constantly by means of a small magnetic stirring bar and irradiated at 330 nm with a Bausch and Lomb 0.5-m grating monochromator. The flux rate, determined with a Kettering model 65 Radiometer, was 6000 ergs/mm/ml incident on the organic phase. After irradiation the tRNA was recovered from the organic phase as described above. (iv) Hydrogen peroxide. The treatment of tRNA with Hz02 was performed as outlined by Scheit (10) for t,he conversion of 4-thiouridylyl(3’-5’)-4-thiouridine to uridylyl-(3’-5’)-uridine. Ten microliters of 3070 Hz02 were mixed with 1 ml of 0.1 M Tris-HCl buffer, pH 8.0 containing about 26 At60 rim-units of tRNA, and the reaction was allowed to proceed at room temperature. The tRNA was recovered by ethanol precipitation (21). (v) Sodium borohydride. The treatment of tRNA with NaB& was performed as outlined by Shugart and Stulberg (21). Nucleoside determination. Approximately 25 A260 ,,-units of tRNA were hydrolyzed enzymatitally as previously described by Uziel (22) to the nucleoside level with snake venom phosphodiesterase (Worthington) and alkaline phosphatase (Worthington) in 0.2 M ammonium acetate buffer, pH 8.8, containing 23 mM magnesium acetate. The mixture was incubat.ed for 4 hr at 45” and the nucleoside content was determined quantitatively by cation column chromatography (22). RESULTS
Efect of modification of 4-thiouridine absorption spectrum of E. coli tRNA.
on the
The presence of 4-thiouridine in tRNA can be monitored by its characteristic absorption maximum near 335 nm (23). Figure 1 shows the effect on the absorption profile between 300 and 400 nm of E. coli tRNA as a result of the modification of 4-thiouridine by any of the four methods outlined. Curve 1 is the absorption profile before treatment and curve 2 is the absorption profile that results after treatment of the tRNA preparations. Part A shows the results obtained on unfractionated tRNA, and Part B shows the results obtained using several purified tRNA’s which contain 4-thiouridine. It is possible that not all 4-thiouridine residues in the tRNA were modified since after treatment there is a residual absorption at 340 nm. This is attributed to the tail from the
320
360
400 320 WAVELENGTH (IX,,)
360
4C
FIG. 1. Effect of selective chemical modification to 4thiouridine on the UV-absorption spectrum between 309 and 400 nm of (A) unfractionated E. coli tRNA and (B) purified E. coli tRNAs. (All curves normalized to 20 A260 ,,,,,-units of tRNA in 1 ml of 1 mM MgClz .) Curve 1, typical spectrum of tRNA before treatment; curve 2, typical spectrum after treatment of tRNA with CNBr for 10 min, with Hz02 for 90 min (unfractionated) and 246 min (purified), with NEM for 60 min, and with irradiation for 20 min.
260-nm absorption peak of the tRNA (18), because a nucleoside analysis (22) of the treated-tRNAs showed only a trace of 4thiouridine and no new or unidentifiable nucleosides. These data show the modification to 4thiouridine (under the conditions specified) to be essentially complete. Effect of modi$ication of 4-thiouridine aminoacylation of E. coli tRNAs.
on
(i) N-ethylmaleimide. Carbon and David (7) showed that this sulfhydryl reagent selectively reacts with 4-thiouridine residues in unfractionated E. coli tRNA with only a small reduction in total amino acid-accepting capacity of certain tRNAs. The problem of the rapid decomposition of the reagent in aqueous alkaline solutions can be circumvented by carrying out the reaction with tRNA in the organic phase (see “Methods”). The data in Table I show that treatment of unfractionated tRNAs and purified tRNAPhe with NEM does not result in an appreciable
4-THIOURIDINE
AND AMINOACYLATION
TABLE I EFFECT OF S~~LECTIVECHEMICAL OF 6THIOURIDINE UNFRACTIONATED SPECIES
MODIFICATION ON THE AMINOACYLATION OF AND PURIFIED E. coli TRNA
Chemical modiicationD tRNA preparation
unfractionated: Phe Leu LYS fMet Tyr Val purified : Phe fMet Val
NEM CNBr Irrad. Hech IO/Jaminoacylation remaining/
96 93
65 95 34 loo 85
91 90
104 93 83
92 102
90
78
85
75 966 102
a 4-thiouridine in tRNA was modified as outlined in Methods Section with times of modification the same as detailed in Fig. 1. Percent aminoacylation based on control samples that were treated under identical conditions except that the modifying reagent was omitted. 6 Formylation (19) of HlOt-treated tRNAyet was equivalent to methionylation.
loss of amino acid-accepting activity for tRNAPhe or tRNALeU (ii) Cyanogen bromide. CNBr reacts with 4-thiouridine in E. co& and under appropriate conditions the intermediate thiocyanato derivative is converted to uridine (8,9). This modification of 4-thiouridine and its subsequent conversion to uridine does not result in. an appreciable loss of amino acid-accepting capacity for purified or unfractionated tRNAPhe or for several other unfractionated tRNA activities (Table I). Saneyoshi and Nishimura (8) have stated that the minor component X (6) in E. coli tRNAPhe is modified by reaction with CNBr. Retention of Phe-tRNA formation after CNBr treatment eliminates component X from the ligase recognition site in tRNAPhe. The significant inactivation of tRNALya by this procedure is consistent with an oxidative modification to the molecule as previously reported (24, 25). No attempt was made to restore the inactive tRNALy8 by treatment with a reducing agent (24). The
OF tRNA
491
data of Reid (26) argue that 4-thiouridine is involved in the inactivation of Lys tRNA formation, This is not supported, however, by the results of Harris et al. (27). (iii) PhotochemicaZ transformation: Pleiss and Cerutti (11) have described a reaction that transforms the 4-thiouridine of unfractionated E. coli tRNAs to uridine under extremely mild photochemical conditions (irradiation at 330 nm in tert-butyl alcohol). It should be noted that a different modification occurs to 4-thiouridine when tRNA is irradiated in aqueous solutions, presumably because the secondary structure of tRNA is maintained (28). The photochemical transformation of E. coli tRNAs in this study was accomplished in a 5 % solution of Aliquat 336 in Freon 214. The advantages of partitioning the tRNA into this organic phase for irradiation are: (1) It provides an environment in which the tRNA is less structured4; (2) it simplifies the manipulation of small amounts of tRNA, and; (3) it alleviates the interference of water (11). Unfractionated E. coli tRNA was irradiated as described under experimental procedures and the photochemical transformation of 4-thiouridine, as measured by the decrease in 340-nm absorption, was found to be complete in 20 min (Fig. 1A). During this period there was less than a 10 % loss in the capacity of tRNALeu, tRNAPhe, and tRNATYP to be aminoacylated (Table I). Under similar conditions, a purified sample of tRNAPhe that had been irradiated for 10 min showed a 50 % decrease in its 340-nm absorption, and a 15 % loss in its capacity to be aminoacylated. Irradiation of the tRNAPhe for an additional 10 min resulted in negligible loss of amino acid acceptance (Table I>. Irradiation of 4-thiouridine in the organic phase at a concentration equivalent to that in the tRNA experiments results in an initial rapid oxidation of the 4-thiouridine to 4-thiouridine disulfide followed by the appearance of a stoichiometric amount of 4 A decrease in ordered structure of tRNA while in the organic phase is indicated by a bathochromic shift of the maximum of its ultraviolet absorption spectrum.
492
SHUGART
uridine in 45 min. None of the four major nucleosides, nor 7-methyl guanosine, Nmethyl l-methyl guanosine, guanosine, dihydrouridinc, or l-methyl adenosine is affected by irradiation in the organic phase, .as determined by their characteristic UVabsorption spectra. (iv) Hydrogen peroxide. Scheit (10) has ,described a procedure for the mild hydrolysis of 4-thiouridylyl-(3’-5’)-4-thiouridine with HzOz at pH 8. This method has been adapted hcrc to selectively modify 4-thiouridine in l!C. coli tRNA. Figure 2 shows that after 90min exposure of unfractionated tRNA to HzOz, only a small residual absorption at 340 nm remains. The HzOz-treated tRNA is recoverable by ethanol precipitation, and its chromntographic profile upon Sephadex G-100 column chromatography, as outlined by Egan et al. (29), is similar to that of native tRNA. A nucleoside analysis gave no detectable 4-thiouridine. In addition the HzOp-treated sample showed no appreciable loss of amino acid acceptance for the four aminos acid tested (Table I). The 4-thiouridine in purified species of E. coli tRNA on the other hand are more resistant to alteration by this reagent. Additional exposure time was required to bring about a loss of 340-nm absorption in the purified tRNAs equivalent to that observed in equal A2G0,,-units of the unfractionated
tRNA (Fig. 3A). The data in Table I also show that there is no significant loss of amino acid acceptance (and no loss of the ability of tRNAyt to be formylated) after H202 treatment of the purified species of tRNA. An interesting observation that resulted from this study is given in Panel B of Fig. 3, which shows that the characteristic loss of absorption at 340 nm does not occur when E. coli tRNAp (30) is treated with HzOz. This tRNA species is an artifact that arises from the major naturally occurring formylatable species (tRNAy) in E. coli as a result of an unknown modification of its 4-thiouridine during isolation (31). Because of the pronounced skew to the absorption spectrum between 300 and 400 nm, the modification is believed to be similar to that reported for tRNAV”’ (28). Subsequent e$ect of NaBH4 modi$cation on aminoacylation of Q-thiouridineless E. wli tRNA. As pointed out in the introduction, the purpose of this study waa to determine whether the inactivation of NaBH&-treated E. coli tRNAPhe (3) was a consequence of a
B
A
0.5 (
I I
L
I 403
WAVELENGTH hn)
2. Effect of HZ02 treatment on the UVabsorption spectrum between 300 and 400 nm of 19 A*eOrim-units of unfractionated E. coli tRNA in 1 ml of Tris buffer. Curves represent the following exposure times (min.) to Hz02 : 1, 1.6; 2, 2.9; 3, 8.7; 4, 16.1; 5, 25.4; 6, 66.2; 7, 90.8. FIG.
1 1 4c
340 WAVELENGTH
InIn)
FIG. 3. Effect of Ha02 treatment on the UVabsorption spectrum between 300 and 400 nm of 17 Azso rim-units each of (A) purified tRNAFt [31]; and (B) purified tRNA2t [31, 321. Curve 1, zero time, curve 2, after 90 min; curve 3, after 24.0min exposure to Hz02 . Panel A is also representative of the effect of Hz02 treatment on purified tRNAPhe and tRNAva1 samples (see Fig. 1 and Table I).
4-THIOURIDINE
AND AMINOACYLATION TABLE
EFFECT
OF SODIUM
TRNA
BOROHYDRIDE AND PURIFIED
tRNA preparaticm
Unfractionated:
II
IZEDUCTION
ON THE AMINOXYLATION OF UNE.R.WTIONITED TRNA SPECIES AFTER SELECTIVE MODIFICATION TO 6THIOURIDINE
E. coli
Chemical modification” NEM
+ +
493
OF tRNA
Irrad
Hz02
CNBr
+ +
+ +
Purified :
NaBHp
+ +
+ +
+ +
E. coli
Aminoacylation
1% remaining\
Phe 100 96 8 100 85 18 Phe 100 75 19 100 92 12
Leu 100 93 74 100 90 84 Val 100 76 64 100 71 71
0 4-thiouridine in tRNA was modified as outlined in Methods Section. Control samples (-) were treated under identical conditions except that the modifying reagent was omitted. Amino acid acceptor activities were determined upon recovery of tRNA after treatment with NEM for 60 min, irradiation for 30 min, Hz02 for 4 hr, CNBr for 10 min, and NaB& for 4 hr.
modification to its 4-thiouridine or dihydrouridines. The results reported thus far lend strong support, to the argument that the 4-thiouridine residue of E. coli tRNAPhe is not involved in ligase recognition, since its modification by four different methods does not result in an appreciable loss of PhetRNA formation (or several other aminoacyl-tRNA formations). Thus, indirectly, it would appear that selective loss of acceptor activity by E. coli tRNAPhe upon treatment with NaBH4 was the result of a modification to the dihydrouridines. This was tested directly by determining the acceptor activity of 4-thiouridinelesstRNA upon subsequent treatment with NaBH4. Table II details the results of this experiment. Phe-tRNA formation in unfractionated, as well as in purified E. coli tRNA was severely curtailed when NaBH4 treatment followed 4-thiouridine modification. Aminoacylation of other tRNAs was not similarly affected. The possibility does exist that a modification of E. coli tRNA by any of the methods detailed could result in an alteration of the rate at which aminoacyl-tRNA formation occurs, with no effect on the total amount of
amino acid accepted. This observation has been reported (28). No attempt was made in this investigation to study the kinetics of tRNA-ligase interaction. DISCUSSION
Several lines of experimental evidence suggest that the presence of the minor nucleoside 4-thiouridine is not necessary for amino acid acceptor activity. First, 4thiouridine is not ubiquitously distributed, but is found almost exclusively in those tRNAs of bacterial origin. In this connection, there are numerous examples of the heterologous aminoacylation of tRNA species from higher organisms with bacterial ligases (32). Second, modifications that are highly specific toward 4-thiouridine (7-9, 11, 28, 33-36) often result in no appreciable loss of amino acid acceptance in t,he tRNAs tested. Third, Harris et al. (27) have shown that sulfur-deficient tRNA obtained from a cysteine-requiring, “relaxed” Wain of E. coli was devoid of 4-thiouridine, but had amino acid acceptances that, for most, of the amino acids tested, were not drastically different from those of normal tRNAs isolated from the same organism. Fourth, Johnson et al.
494
SHUGART
(37) have shown that aminoacylation of tRNAI’e isolated from Mycoplasma sp. (Kid) proceeds nearly to the theoretical level even though the tRNA is deficient in 4thiouridine. We have previously performed certain specified modifications on tRNAPhe in an effort to identify and characterize an enzyme recognition site of the molecule (2, 3). The possible importance of the minor nucleosides dihydrouridine and 4-thiouridine in this recognition phenomenon was implied from our studies with NaBHd-treated tRNAPhe. The data presented in this paper support the general supposition that 4-thiouridine is not necessary for the aminoacylation of E. coli tRNAs. In addition, the data further suggest that the area in E. coli tRNAPhe that contains the information for ligase recognition is probably dependent upon the presence of unmodified dihydrouridine residues. Since the dihydrouridine residues are confined to the nonhydrogen-bonded 5’ loop area of the molecule, this region and its integrity becomes more suspect as the ligase recognition sit,e. It should be emphasized that the selective reduction of dihydrouridines in tRNAPhe by NaBH4 and the concomitant loss of amiioacylation appears to be unique to the E. coli tRNAPhe-E. coli ligase system. As previously indicated (3), the response of individual t,RNAs to specific treatment with this reagent varies, and may depend on the source and purity of the tRNA, the specific tRNA species tested, and the location of susceptible bases. Admittedly, ligase-tRNA interaction is a highly complicated phenomenon with the possible involvement of several independent but common binding sites on the tRNA molecule in addition to a specific recognition .&e(s) (38). This aspect of the problem is currently being investigated through studies on complex formation (39) between modified tRNAPhe and its ligase. ACKNOWLEDGMENTS I wish to acknowledge the technical expertise of Barbara Chastain and Kenneth Isham, who assisted in the experiments reported here. I want to thank Drs. J. W. Longworth and It. Rahn for their assistance with the phot,ochemical experiments, and Drs. B. Pal, M. Uziel and M. P. Stulberg for
their helpful investigation.
suggestions
during
t,he course of this
REFERENCES 1. ZAMECNIK, P., “Cold Spring Harbor Sympos. on Qua&. Biol.“, 34, p. 1 (1969). 2. STULBERG, M. P., AND ISHAM, K. R., Proc. Nat. Acad. U.S.A. 67, 1310 (1967). 3. SHUGART, L. R. AND STULBERG, M. P., J. Biol. Chem. 244, 2806 (1969). 4. UZIEL, M., AND GLASSEN, H., Riochemistry 8, 1643 (1969). 5. BURTON, K., Fed. Eur. Biochem. Sot. Lett. 6, 77 (1970). 6. BARREL, B. G., AND SANGER, F., Fed. Eur. Biochem. Sot. Lett. 3, 275 (1969). 7, 7. CARBON, J., AND DAVID, H., Biochemistry 3851 (1968). 8. SANEYOSHI, M., AND NISHIMURA, S., Biochim. Biophys. Acta 204, 389 (1970). 9. WALKER, R. T., AND RAJBHANDARY, Biochem. Biophys. Res. Commun. 36, 907 (1970). Biophys. Acta 166, 285 10. SCHEIT, K. H., B&him. (1968). 11. PLEISS, M., AND CERUTTI, P. A., Biochemistry 10, 3093 (1971). 12. KHYM, J. X., J. Biot. Chem. 241.4529 (1966). 13. EGAN, B. Z., RHEAR, R. W., AND KELMERS, A. D., Anal. Biochem. 36,139 (1970). 14. KELMERS, A. D., NOVELLI, G. D., AND STULBERG, M. P., J. Biol. Chem. 240,3979 (1965). 15. KELMERS, A. D., AND STULBERG, M. P., Science 167, 238 (1970). 16. VON EHRENSTEIN, G., Methods Enzymol. l% 588 (1967). 17. LINDAKL, T., AND FRESCO, J. R., Methods Enzymol. 12, 601 (1967). 6, 18. ZIFF, E. B., AND FRESCO, J. R., Biochemistry 3242 (1969). 19. SHUGART, L. R., CHASTAIN, B., AND NOVELLI, G. D., Biochim. Biophys. Acta 186, 384 (1969). 20. WEISS, J. F., AND KELMERS, A. D., Biochemistry 6, 2507 (1967). 21. SHUGART, L. R., AND STULBERG, M. P., J. Biol. Chem. 244, 2806 (1969). 22. UZIEL, M., KOH, C. K., AND COHN, W. E., Anal. Biochem. 26, 77 (1968). 23. LIPSETT, M. N., J. Biol. Chem. 240.3975 (1965). 24. CARBON, J., HUNG, L., AND JONES, D., Proc. Nat. Acad. Sci. U.S.A. 63 (1965). 25. SANEYOSHI, M., AND NISHIMURA, S., Biochim. Biophys. Acta 145, 208 (1967). 26. REID, B., Biochem. Biophys. Res. Commun. 33, 627 (1968). 27. HARRIS, C. L., TITCHENER, E. B., AND CLINE, A. L., J. Bacteriology 100, 1322 (1969). as. FAVRE, A., YANIV, M., AND MICHELSON, A. M.,
4-THIOURIDINE
29. 30.
31.
32.
33.
AND AMINOACYLATION
Biochem. Biophys. Res. Commun. 37, 266 (1969). EQAN, B. Z., RHEAR, R. W., AND KELMERS, A. D., Biochim. Biophys. Acta 174, 23 (1969). WEISS, J. F., PEARSON, R. L., AND KELMERS, A. D., Biochemistry 7, 3479 (1968). SHUGART, L. II., CHASTAIN, B., AND NOVELLI, G. D., Biochem. Biophys. Res. Commun. 37, 305 (1969). JACOBSON, K. B., in “Progress in Nucleic Acid Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.) Vol. 11, p. 461. Academic Press, New York, 1971. HARA, H., HC)RIUCHI, T., SANEYOSHI, M., AND NISHIMURA,
S.,
Biochem.
Commun. 38, 305 (1970).
Biophys.
Res.
OF tRNA
495
34. SENO, T., KOBAYASHI, I., FUKAHARA, M., AND NISHIMURA, S., Fed. EUT. Biochem. Sot. Lett. 7, 343 (1970). 35. FAULKNER, R., AND UZIF,L, M., Biochim. Biophys. Acta 238, 464 (1971). 36. SANEYOSHI, M. AND NISHIMURA, S., Biochim. Biophys. Acta 246, 123 (1971). 37. JOHNSON, L., HAYASHI, H., AND SILL, D., Biochemistry 9, 2823 (1970). 38. CHAMBERS, R. W., in “Progress in Nucleic
Acid Research andMolecular Biology” (J. N. Davidson and W. E. Cohn, eds.) Vol. 11, p. 489. Academic Press, New York, 1971. 39. FARRELLY, J. G., LONGWORTH, J. W., AND STULBERG, M. P., J. Riol. Chem. 246, 1266 (1971).