Effect of formylation on the chromatographic behavior of methionyl transfer ribonucleic acid

ANALYTICAL

47, 244-252

BIOCHEMISTRY

Effect

of Formylation of Methionyl CHARLES

Department

(1972)

on the

Chromatographic

Transfer

Ribonucleic

E. SAMUEL

of Biochemktry,

University Received

JESSE

AND

Acid

C. RABINOWITZ

of Cnlifonrin,

September

Behavior

Berkeley,

California

94720

22. 1971

Widespread interest in the structure and funct’ion of the methionyl tRNA species has been accompanied by the development of several chromatographic methods which permit the separation of tRNAfMPt and tRNAmM”.’ Column chromatography utilizing BD-cellulose (1,2) has been used to resolve the isoaccepting tRNAMF’ species present in bulk tRNA prepared from various prokaryotic and eukaryotic organisms including Escherichia coli (3-5), Mycoplasrnn laidlawii (6,7), bakers’ yeast (8,9), rabbit reticulocytes (101, and mouse liver and Krebs II mouse ascites tumor cells (9). In all these chromatographic separations obtained with BD-cellulose (3-10)) the apparent initiator species (tRNAfMet) was observed to elute before the species which is believed to function in peptide chain elongation (tRNA,,P1’t) (9,ll). During the course of study on the multiplicity and specificity of Streptococcus faecalis R methionyl tRNA, we observed that enzymic formylation of Met,-tRNArMet had a profound effect on the elution position of the tRNAfMet species when chromatographed on BD-cellulose which altered the elution order of tRNAfMPt with respect to tRNA,,,&Iet. We believe that this observation may be useful in the separation and isolation of the two species of tRNA1let. MATERIALS

AND

METHODS

[14C]Sodium formate (53.5 mCi/mmole) and L- [“Hlmethionine (2.6 Ci/mmole) were obtained from Schwarz BioResearch. L- [‘“Cl methio1 Abbreviations and trivial names : DEAE-cellulose, diethglaminoethylcellulose. BD-cellulose, benzoylated diethylaminoethylcellulose. ODU, amount of tRNA which when dissolved in 1 ml solvent has an absorbance of 1.00 at 260 nm. tRNA”“‘, methionine-accepting tRNA which cannot methionine-accepting tRNA. tRNA,“‘“‘, be enzymically formylated. Met-t,RNA,“‘let, aminoacylated form to tRNA,““t. tRIYAi41et methionine-accepting tRNA which can be enzymically formylated. Met-tRNk”ef , aminoacylated form of tRNAffil”‘. fMet-tRNAf”“‘, formylmethionyl ester of tRNAF. 244 @ 1972 by

Academic

Press,

Inc.

tRNA””

CHROMATOGRAPHY

245

nine (13.6 mCi/mmole) was purchased from New England Nuclear Corporation. DEAE-cellulose (lot 2178) was purchased from Schleicher and Schuell. lo-Formyltetrahydrofolate and lo- [‘“Cl formyltetrahydrofolatc were prcparecl cnzymically wit11 crystalline Clostridizm cyEinchsporu)H formyltetrahydrofolate synthetase (12) as previously described (13). Unfractionated tRNA from S. fnecnlis R grown in folate-sufficient, medium (14) was prepared by t’hc method of Silbcrt et al. (14,15), and was deacylated according to the method of Sarin and Zamecnik (16). The source of the mcthionyl tRNAhret synthetase and transformylase enzymes was a crude K. fnecc&s R S-100 supernatant fraction (17) which had been subjected to ammonium sulfate fractionation, dialyzed, and chromatographed on DEAE-cellulose (18 1. BD-cellulose was prepared according t’o the procedure of Gillam et al. (1)) and had an average of 2.5 benzoyl rcsiducx per mole anhydroglucose (1,19j. BD-cellulose columns were packed and operated at, room temperature as described by Gillam et al. (1). The tRNA applied to BD-columns was elutcd with a positive sodium chloride gradient and standard buffer composed of 0.01 )W magnesium chloride and 0.01 dl sodium acet’ate, pH 4.5. When uncharged tRNARrrt was chromatographecl, the tRNA contained in column fractions was recovered by ethanol precipitation and assayed for methioninc and formyl acceptance in a single incubation mixture (50 ~1) containing the following: Tris-chloride buffer, pH 7.4, 0.1 &l; magnesium chloride, 15 rnM; ammonium acetate, 10 mM; 2-mercaptoethanol, 20 rnilf; ATP, 5 mM; L- [“H]methionine, 0.100 mM; 10-[‘4C]formyltetrahydrofolate, 0.025 m,W; tRNA fraction; and enzyme. Incubation was for 15 min at 37”. The reaction was stopped by addition of cold 10% trichloroacetic acid and the precipitate collected on a glass fiber filter (Whatman GF/C, 2.4 cm) prewashed with [*‘C]methionine. The assay tube was then rinsed with 5% trichloroacetic acid and the washes were decanted onto the filter, which was subsequently counted for radioactivity in 10 ml Bray’s solution (20) with a Nuclear-Chicago Mark I liquid scintillat.ion counter. When combinations of charged and formylated tRNAMPt were to be chromatographed, the fractionated tRNAMEt species were aminoacylatcd with methionine and, where indicated, formylated, as described in the standard assay except for the following modifications. Resolved tRNA,,,Mrt or tRNAfMet, radioactively labeled or [Wlmethionine, and [‘“Cl or [“Cl formyltetrahydrofolate were present in the incubation mixture as indicated under the respective figure descriptions. The fMet-tRNAfMet, Met-tRNAfMet, and Met-tRNA,,,“” prepared for BD-cellulose chromatography were isolated from the charging and formylation reaction mix-

246

SAMUEL

AND

RABINOWITZ

tures by phenol extraction and repeated ethanol precipitation before application to a BD-cellulose column. tRNAfire’ contained in column fractions which was labeled with either a radioactive methionyl residue or radioactive formyl group was precipitated directly with trichloroacetic acid and analyzed for radioactivity as outlined above. RESULTS

AND

DISCUSSION

The chromatographic fractionation on BD-cellulose of X. fa.ecalis R bulk tRNA stripped of aminoacyl esters yields two separate isoaccepting methionine tRNA species (Fig. 1). The peak of methionine acceptance activity which elutes first is capable of accepting formyl groups from IO-formyltetrahydrofolate after aminoacylation with methionine, whereas the second peak of methionine tRNA act’ivity cannot be enzymitally formylated under these conditions. The order in which the unacylated S. faecalis R methionyl tRNA species elute, tRNAfMet before tRNA,,,‘Mrt, is similar to that observed for the methionyl tRNA species prepared from several organisms (3-10). Aminoacylation with methionine does not alter the relative elution order of the two tRNAMet species (6,211. However, when S. faecalis R tRNAfMet aminoacylated with methioI

I

I

100

150

200

0

FRACTION

250 NUMBER

FIG. 1. Chromatography of uncharged bulk 8. fnecnlis R tRSA on BD-cellulose. tRNS (approximately 80 ODU) was loaded on a 1.0 X 25.5 cm column of BDcellulose equilibrated with 0.4 M sodium chloride. 0.01 M sodium acetate (pH 4.5), and 0.01 M magnesium chloride. After washing with 20 ml of the same buffer, the tRn’A was eluted with a linear sodium chloride gradient formed from 150 ml each of 0.4 M and 1.0 M sodium chloride, each solution containing 0.01 M sodium acetate (pH 4.5) and 0.01 M magnesium chloride. 1.0 ml fractions were collected and the flow rate was 10.2 ml/hr,

tRNA”*“’

CHROM.\TOGRAPHT

247

FRACTION NUMBER

FIG. 2. Chromatography of L”HImrthiongl-tRNAr~‘et nnd [“Clformvl-[“ClmPthionyl-tRNAf”“t on BD-wllulosc. A sample containing tRNAr”” chnr&tl with L’Hlmethionine but not formylatcd, tRNAr”let charged with [“Clmethioninr and formylated with the 10-~“‘C1formy1tctr:~hydrofo1ate as the formyl donor, and unchargrd unfractionated carrier tRNA was applied to a 1.0 X 25.0 cm column of BD-cellulose rquilihratrd and developed as described under Fig. 1. rxwpt that 1.3 ml fractions wwc collect4 and ihr flow rate was 12.0 ml/lx.

nine is also formylated, its elution position on BD-cellulose is dramatically changed relative to that of the unformylated aminoacylated species (Fig. 2). Formylation causes a marked shift in the elution position of Met-tRNA fnret to a higher salt concentration. This was verified by cochromatography of [“HI methionyl-tRNALMV” and [ *%I formyl[ lzC] methionyl-tRNA,nf”t (Fig. 2). In order to establish definitively the effect of formylation of MettRNAflLlet on the elution order with respect’ to Met-tRNA,,,Rx’t, lmformylated [3H]metl~ionyl-tRNAfMrt and 1W] formyl- [ “H]methionyltRNAfMp+ were cochromatographed with [‘4C]nletl~iollyl-tRNA,,,R”“. As shown in Fig. 3, the retardation of Met-tRNA,““’ upon formylation causes it to elute slightly after, rather than before, t’he hlet-t’RNA,,,“Vt species. Thus, upon enaymic formylation of the Met-t’RNAfXfrt species, tbe elution order of the isoaccepting methionyl-tRNA species is reversed from the elution order commonly observed for unacylated tRNAcMFt and tRNA,,“-’ on BD-cellulose (3-10) (Fig. 1). Aminoacylation with methioninc in itself does not generate the altered elution order under the chromatographic conditions described in this investigation since [3H]methionyl-t’RNAf11rt and [ “Cl nlethionyl-tRNA,,,R”’ (Fig. 3) elute in the same order as unaminoacylated tRNAf%lrt and tRNA,,,nf\l’t (Fig. I ) : the formylatable tRNAfnIrt species elute:: before the nonformylat-

248

FRACTION NUMBER FIG. 3. Chromatography of 13Hlmethionyl-tRNA1’*\“t, l”Clformyl-[3Hlmethionyl-tRNAr’U”, and [“Cl mcthionyl-tRNA,,l”” on BD-cellulose. A sample containing tRNAr”” charged with [“Hlmethionine hut not formylated, tRNAi”rpt charged with 13H1methionine and formylated with lo-[“Clformyltetrahydrofolate as the formyl donor, tRNAmJret charged with [“Clmethionine, and uncharged carrier t.RNA was applied to a 1.0 X 36.0 cm BD-cellulose column equilibrated and developed as described under Fig. 1, except that 1.0 ml fractions were collected and the flow rate was 12.5 ml/hr.

able tRNA,,&ICt species. However, slightly higher salt concentrations are required to elute the aminoacylatecl tRNAfhTet and tRNA,,,Met species (Fig. 3) from BD-cellulose than the respective unacylated methionyl tRNA species (Fig. 1) under comparable conditions. The ionic strength required to elute the various forms of tRNAMet from BD-cellulose under the conditions employed in this study are summarized in Table 1. These values differ somewhat from those reported by other investigators because the salt concentration required

El&ion

Position

TABLE 1 of tRNA Met Species

Species

chloride

BD-cellulose

NaCl

concn.n M 0.58 0.65 0.79 0.67 0.76

t KNAfn’et Met-tIiNArsret fMet-tRNArMet tRNA,“ct Me&tRNA,$ret a In 0.01 M magnesium

from

and

0.01 M sodium

acetate,

pH

4.5.

tRNA”“t

CHROMATOGRAPHI

249

for elution of a given tRNA species varies with the temperature of column operation, buffer conditions, and degree of benzoyl substitution of the BD-cellulose used (1,21:). Henes et al. (21) previously found that on BD-cellulose at 4”, but not at 22”, the phenoxyacetyl methionyl derivatives of E. coli tRNA elute in a reversed order characterized by the tRNA,,,“” derivative eluting first, and suggested that S-acylation of the methionyl residue induced a differential conformation change in the two tRNAhret species (21). In agreement with the results reported here for S. faecalis R methionyl tRNA species, aminoacylation alone was not sufficient to invert the elution order of E. coli tRNArfiTVt 121). In recent studies carried out by Feldmann and Falter (6) with ~11. laid'laulii A tRNL4, formylation caused the tRYA, hIzt species to be retarded on BD-cellulose; however, the clution order was not reversed with respect to tRNA,,,“*“. Experiments carried out with two reverse-phase columns (RPC-2 and RPC-3) and methylatecl albumin silicic acid (MASA) likewire have established that the chromatographic property of formylated methionyltRNA is somewhat different than that of unformylatcd methionyl-tRNA. E. coli tRNA,&liet and tRNA,,,>pc’ are not resolved on MASA when chromatographed in either the unacylatcd or aminoacylated state; however, formylmethionyl-tRNAR”” can bc separated from methionyl-tRNA”” (22-24). Multiple methionine isoacceptor species can be resolved from uncharged, bulk :\-eurospora crassa mitochondrial tRNA on RPC-2 (25) and from E. coli tRNA on RPC-3 (126,271. A shift in the elution position of both K. crassa, mitochondrial Met-t,RNAcMrt 125) and E. coli Met-tRNA,“c’ (26,271 is observed upon formylation, but t’he shift does not alter the elution order based on the elution position of McttRNA,,R’Vt. Evidence from circular dichroism (28) , chromatography 124 1, magnetic resonance (29)) oligonuclcotidc binding (30), and sedimentation 131) experiments has suggested that a conformational difference may exist between unacylated and aminoacylatecl tRNA (24,28-31). Although the difference in elution position of uncharged and charged methionyltR.NA species observed in our investigation of S. fnecnlis R tRNA may be the result of such a conformational difference, an increased interaction of the aminoacylated tR?rTA”r’t specks with BD-cellulose due to the hydrophobic nature of the methioninc side chain cannot be excluded as a contributing source responsible for the difference in elution position. Whether or not the difference in elution position 1)etween formylated and unformylatcd tRNA, Mrt is due to a conformational change of physiological significance is still an open qu&ion in view of recent findings concerning the possibility that polypcptidc chain initiation proceeds without formylation of the initiat,or tRNA in the cytoplasm of eukaryotic

250

SAMUEL

AND

RABIKOWITZ

organisms (9,10,3%34) and in certain la&c ac.id bacteria (13,35), and the apparent role that magnesium ions play in asserting tRNA conformation (36,37). The change in elution position of tRNACMrt on BD-cellulose resulting from formylation of Met-tRNAfMrt might serve as an efficient step in the purification of the tRNAffilICt species. Perhaps more important, however, in view of the methods which have already been developed for purification of the methionine tRNA species (3,21,38), is the inherent danger of using aminoacyl-tRNA synthetase preparations to aminoacylate tRn‘A with methionine for analytical BD-cellulose chromatography analysis which have not been adequately treated to remove endogenous folate cofactors. The presence in such enzyme preparations of endogenous folate cofactors readily convertible to lo-formyltetrahydrofolate, which has been established as the formyl donor in the methionyl-tRNA,Mc’ transformylase reaction (39,401, can lead to addit.ional tRKA”?’ activity peaks when bulk tRNA precharged with radioactive methionine is analyzed for Met-tRNAMet multiplicity on BD-cellulose.

The effect of formylation on the chromatographic behavior of iLlet-tRT\JA,“et on BD-cellulose has been investigat,ed. Under conditions comparable to those routinely employed in analytical BD-cellulose chromatography, formylated RIet-tRNA,“r’t was observed to flute at a significantly higher salt concentration than unformylated iL!let-tRNArnlCt. Unformylated Met-tRNA,*l”‘t elutes well before Rlet-tRNA,,,“et, whereas fMet-tRNAfbret elutes slightly after Met-tRNA,,,“Iet; thus the net effect of formylation is an apparent inversion of the elution order of the isoaccepting methionyl tRNA species, tRNACMrt and tRNA,,,Efet. Although aminoacylated tRNArRIrt and t.RNA,,,Met elute slightly later than their respective unacylated forms, aminoacylation alone does not produce the inverted elution order observed upon formylat’ion of Met-tRNAIMet. ACKSOWLEDGMENTS The expert technical assistance of Mrs. Cheryl Murray is gratefully acknowledged. The authors also wish to acknowledge the invaluable support of the National Institute of Arthritis and Metabolic Diseases, U. 8. Public Health F&rice (Grant A-2109) ; nutI one of us (C.E.S.) was supported by a U. S. Public Health Service Training grant (5 TO1 GM 31-13). REFERENCES 1. (;II.L.IM, I., MILLWARU. S., BLEW, D., TIGNICR! G. M., Biochemistry 6, 3043

VON TIGERSTROM, (1967).

M..

W’IMMF.R,

E..

.AND

tRNAR”’

CHROMATOGRBPHY

I., BLEW, D., WARRINGTON, R. C., G. M. Biochemistry 7, 3459 (196~).

2. GILLAM, 3. SENO,

T.,

(1968). 4. ROY, K.,

KOBA~ASHI,

KISHIM~X.\.

Biophys.

Ljk~l~im.

Actn

T~SNEH, 169,

SO

14.

SAMUEL,

i’.

8. 9.

10. 11.

Biochim.

S..

AND

12. 13.

6.

AND

S., ASD

D.,

AXD

M.,

TIGERSTROM,

Acta 161, 572 (1968). Biochem. 12, 177 (1970). FELDMAN, H., H., Ew’. J. Biochem. 18, 573 (1971). HliyasIr1, H., FISHER, H., ASD SGLL, D., Biochemislry 8, 3650 (1969). TAICEISHI, k-., ITKITA, T., AND KIBHIMURA, S.. J. Biol. Chem. 243, 5761 (1968). SMITH, A. E., END MARTKER, Ei. A., Nature 226, 607 (1970). HUNTER. A. R., AND JACTKSON, Ii. J., Eur. J. Biochem. 19, 316 (1971). MARCKER, Ii. A., CLARK, B. F. C., AND ANDERSOS, J S., Colt1 Spring Harbo? Symp. &ant. Biol. 31, 279 (1966). RABISOWITZ, J. C., .~ND PRICER, W. E., JR., J. Biol. Chem. 237, 2898 (1962). SAML-EL: C. E.. D’ARI, L., ASD R~BINOWITZ, J. C., J. Biol. Chem. 245, 5115

5. CORY,

SBLL,

M.,

VON

MARCKER, K. AND FALTER,

A.,

Biophys. Eur. J.

(1970). C.

E.,

MURRAY,

C.

L..

AND

RABINOWITZ,

J.

C.,

J.

Biol.

Chem.

(in

preparation). 15.

SILBERT,

16. SARIN,

17.

D. F., FINK, G. R., P. S., AND ZAMECNIK,

NIRENBERG,

Kaplan, 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30.

31. 32. 33. 34. 35. 36.

AMES, B. N., J. hfol. Biol. 22, 335 P. C., Biochim. Biophys. Acfn 91, 653

AND

M. W. in “Methods in Enzymology” (S. P. Colowick eds.), Vol. VI, p. 17. Academic Press, New York, 1963.

(1966).

(1964). and N. D.

P., U’EBSTER, R. E., AND ZINDER, N. D., J. hlol. Biol. 43, 177 (1969). H. E., AND LAMB. R. w., J. Amer. Chem. Sot. 74, 3759 (1952). BR.~Y, G. A., Anal. Biochem. 1, 279 (1960). HENES, C., KRAUSKOPF, M., AND OFENGAND, J., Biochemistry 8, 3024 (1969). LEDER, P., AND BURSZTYN. H., Proc. Nat. Acad. Xci U. S. 56, 1579 (1966). STERN, R.. ZUTRA, L. E., ASD LITTAUER, U. Z., Biochemistry 8, 313 (1969). LITTATTER, U. Z., AND STERN, R., Fed. Eur. Biochem. Sot., 4th Meeting, Oslo 3, 93 (1967). EPLER, J. L., SHUGART, L. R., AND BARSETT, m”. E., Biochemistry 9, 3575 (1970). SHUGART, I,.. CHASTAIN, B., AND NOVELLI, G. D., Biochem. Biophys. Res. Commun. 37, 305 (1969). SHUGART, L., CHASTAIS: B.? .~ND XOVELLI, G. D., Biochim. Biophys. Acts 186, 384 (1969). SARIN, P. S., AND ZAMECNIK, P. C., Biochem. Biophys. Res. Commurr. 29, 400 ( 1965). COHN, M., DANCHIN, A., AND GRUNBERG-MANAGO, M., J. ~1fol. Biol. 39, 199 (1969). DANCHIN, A., AND GRUNBERG-MANAGO, M., FEBS Lett. 9, 327 (1970). CHATTERJEE, S. Ii.. AND K.4~1, H., Biochim. Biophys. Acta 224, 88 (1970). MARCUS, A., WEEKS, D. P.. LEIS, J. P., AND KELLER, E. B., Proc. Nnt. Acad. Sci. U. S. 67, 1681 (1970). HOUHMAN, D., JACOBS-LOREN.4, M., RAJBHANDARY, U. L., ASD LODISH! H. Ii., Nature 227, 913 (1970). WIGLE, D. T., AND DIXON, G. H., Nature 227, 676 (1970). PINE, M. J., GORDON, B., AND SARIMO, S. S.. Biochim. Biophys. Acta 179, 439 ( 1969) REEVES, R. H., CANTOR, C. R., AND CHAMBERS, R. IV., Biochemistry 9, 3993 ( 1970) . MODEL,

CNGNADE,

252

SAMUEL

37.

N’ILLICK,

38.

WEISS,

39.

DICKERMAN,

40.

MARCKER,

J.

G.

J. F., Biol.

E..

AND

KAY,

PEARSON,

R.

<‘. I,..

Ah-D

M., AND

HARINOWITZ

~~iochemisl,y I~ELMERS,

H. TV.. STEERS, E., JR., Chem. 242, 1522 (1967). K., J. Mol. Biol. 14, 63 (1965).

10, 2216 (1971). A. D.. BiochemisLyy

REDFIELD,

H.

G..

AND

7, 3479 WEISSBACH,

(1968) H