p-azidophenylalanyl21,22 groups have been linked to amino

Analogs of methionyl-tRNA synthetase substrates containing photolabile groups

Ronald Wetzel and Dieter S611

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520,USA

Received 17 January 1977

ABSTRACTThree photolabile analogs of substrates of methionyl-tRNA synthetase

were synthesized. In one, the 4-thiouridine at the 8 position of E. colitRNAfMet was alkylated with [ 4C]p-azidobromoacetanilide. In the second,[l4C]p-azidobenzoic acid hydrazide was condensed with the 3'-terminaldialdehyde of periodate-oxidized Escherichia coli tRNAfMet. The modifiedtRNAs could be purified by chromatography on benzoylated DEAE-cellulose.The third photolabile compound was [3H]methioninyl-8-azido-adenosine5'-phosphate, an analog of the methionyl adenylate intermediate in theaminoacylation reaction. Irradiation of each of these compounds in thepresence of equimolar amounts of E. coli methionyl-tRNA synthetase at 1Mconcentrations gave 5-15% crosslinking.

INTRODUCTIONPhotoaffinity labeling has proven to be a very powerful tool for prob-

ing the interactions between ligands and biological macromolecules1'2. In

the case of nucleic acid "ligands" the studies have involved mononucleotidesand derivatives, oligonucleotides, RNA, DNA and tRNA2,3

The many reports of the preparation and use of modified tRNAs in affin-ity labeling studies can be divided into two groups, one in which chemical

probes have been attached to the heterocyclic bases of tRNA, and the otherin which the probe is bound through an amide linkage to the amino acid es-

terified to the 3'-end of tRNA. In the first category there are only two

cases, both involving the modification of the 4-thiouridine residue at

position 8 of E. coli tRNAs with an azidophenyl group47. The second groupis highly diverse; by reacting an activated carboxyl derivative with amino-acyl-tRNA, p-(2-nitro-4-azidophenoxy)-phenylacetyl 8, N-(2-nitro-4-azido-phenyl )glycyl9, ethyl-2-diazomalonyl 10 , p-azido-o-nitrobenzoyl 11, bromo-acetyl 11216, p-nitrophenylcarbamyl 17,18, chlorambucilyl 19,20, and N-(t-butoxycarbonyl)-p-azidophenylalanyl21,22 groups have been linked to amino-

acyl-tRNA through its amino acid. Slightly different analogs were recently

1681C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

Nucleic Acids ResearchVolume 4 Number 5 1977

Nucleic Acids Research

described23, in which mercuriacetamido or bromoacetamido groups were intro-duced at the 3'-position of the terminal cytidine of truncated tRNAPhe.Most of the modified tRNAs have been applied to studies of ribosome:tRNAinteractions613,18'2022 In this way useful information has been obtainedby identification of the labeled ribosomal nucleic acid or of the ribosomalproteins.

Some of the modified tRNAs described above, as well as analogs of theother substrates of the enzymes, have been employed in affinity labelingstudies of aminoacyl-tRNA synthetases. Chemical affinity labeling has beenattempted with an isoleucine analog24 and with modified isoleucyl-tRNA15'l6methionyl -tRNA17 and phenylalanyl-tRNA1 ,19. Photoaffinity labeling studies

have been conducted with ATP analogs (using phenylalanyl-25 and leucyl-tRNA26 Pe4,5synthetases ),and also with tRNAPhe modified at the 4-thiouridine4. In

addition, photo-induced crosslinking of unmodified tRNAIle to isoleucyl-tRNAsynthetase has been reported27'28, but so far only the contact sites on thetRNA chain have been located with this technique. While affinity labelinghas been satisfactorily demonstrated in several of the above studies, inonly two casesl7,24 have the labeled peptide fragments been isolated andcharacteri zed.

Our earlier work on the chemical modification of tRNA provided biologi-cally active tRNA species modified at different positions in the tRNA se-

29quence with fluorescent groups . We decided to search for photolabilegroups with similar reactivity and base specificity; this would allow thepreparation of several modified tRNA samples suitable for photoaffinitylabeling studies. The tRNA of our choice was E. coZi tRNAfMet. On the one

hand, this molecule has been used successfully in our chemical modification29experiments . On the other hand, there is a wealth of information about

its cognate aminoacyl-tRNA synthetase. The reaction mechanism of E. coZimethionyl-tRNA synthetase is well studied30, its amino acid sequence isalmost completely determined31, and work on the crystal structure of this

32protein is in progress . Thus, there is the expectation that any resultsof photoaffinity labeling studies of the substrate binding sites could soon

be interpreted with the knowledge of the detailed structure of this enzyme.

In this paper we describe the synthesis, purification and characteri-zation of two samples of tRNAfMet modified with photolabile groups at dif-ferent points in the molecule and a photolabile analog of methionyl ad-enylate, an intermediate in the enzyme-catalyzed aminoacylation reaction.In addition, we present some preliminary results of photoaffinity labeling

1682


experiments.

MATERIALS AND METHODS

General. Uniformly labeled [14C]methionine with a specific activityof 225 mCi/mmol was obtained commercially. [1-14C]bromoacetic acid, p-

amino[carboxy-14C]benzoic acid, and [3H]sodium borohydride, obtained commer-

cially, were diluted to specific activities of 3.2, 2.5 and 25 mCi/mmol, re-spectively. Polyethyleneimine (PEI) cellulose coated plastic sheets (Poly-gram Cell 300 PEI) were obtained from Brinkmann. BD-cellulose was a productof Boehringer-Mannheim. Nitrocellulose membrane filters (BA 85) were fromSchleicher-Schuell Co. Purified E. coZi tRNAfMet (specific activity 1.5nmoles/A260 unit) was kindly provided by Dr. A.D. Kelmers from the Oak RidgeNational Laboratory. Pure methionyl-tRNA synthetase was a gift of Dr. C.J. Bruton, Imperial College of Science and Technology, London.

Chromatography. Solvent systems used for paper chromatography andcellulose thin layer chromatography (tic) were: (A) 2-propanol-concentratedNH40H-water, 7:1:2; (B) 2-propanol-concentrated HCl-water, 170:41:39. T2RNase digests of tRNA were routinely characterized by PEI tic in 1.0 M LiCland by thin layer electrophoresis (on cellulose) run in 20 mM potassiumphosphate (pH 7.6).

Assay for Amino Acid Acceptor Activity. The incubation mixture con-tained per ml: 100 umol of sodium cacodylate (pH 7.2), 10 imol of magnesiumacetate, 10 imol of potassium chloride, 2 pmol of ATP, 0.05 A260 units oftRNA, 4 nmol of radioactive amino acid, and methionyl-tRNA synthetase.After incubation at 37°, aliquots were removed and the acid-insoluble radio-activity was determined by the filter paper technique.

AnaZysis of tRNA and of tRNA:Aminoacyl-tRNA Complexes. Enzymatic di-gestions of tRNA were performed with T2 RNase as described by Barrell33.The amount of 4-thiouridine present in the tRNA preparations was determinedby the photodimerization assay described by Ofengand et al.34. Dissociationconstants of complexes were determined by fluorescence quenching accordingto the method of Blanquet et al.35. Protein concentration was determinedby the membrane filter method of Schaffner and Weissmann36 with bovineplasma albumin as standard.

Affinity labeling. Photolysis experiments were conducted in 0.03 M

sodium acetate (pH 6) - 4 mM magnesium chloride in pyrex tubes using eithera 254 nm or 366 nm low pressure lamp at 40, at a distance of 5 cm. Covalentattachment of radioactivity to protein was assayed by acid-precipitation

1683


and membrane filtration of the photolysis mixture. An unirradiated samplewas used as background control. When [14C] photolabile tRNA was used, theRNA was digested with T2 RNase after photolysis prior to acid precipitation.

All work with light sensitive compounds was conducted in as light-freean environment as conveniently possible. Reaction mixtures were wrapped inaluminum foil and column chromatography was conducted in a darkened fumehood or in columns wrapped in foil. Flow-through UV-monitors of columneluates were not used.

Synthesis of Radioactive Reagents. The structures of the radioactivereagents are shown in Figure 1.

N3 N3 NH2

CH3

NH c-o 1H2I ~ ~ IC-O N H N~~~M-H2-CH270-P-P6

CH2Br NH2 09HO OH

ABA ABH met-ol-8-azido-AMP

Figure 1. Structures of photolabile compounds used in this study.

[14C]p-azido-bromoacetanilide (ABA) was prepared following the general4 -azidoaniline~~37procedure outlined by Budker et al.4. To a solution of p-azidoaniline

(52.5 mg; 0.39 mmol) and of [TC]bromoacetic acid (44.6 mg; 0.32 mmol) intetrahydrofuran (3 ml) was added dicyclohexylcarbodiimide (70.5 mg; 0.34mmol). After stirring for 4 hr at 250 the reaction mixture was filteredand the insoluble residue was washed with tetrahydrofuran (2 x 3 ml). Thecombined filtrates were added to ether (30 ml) and washed with 0.5 M HCO(5 ml), and then with water (5-10 ml). The organic phase was dried withmagnesium sulfate, filtered, and reduced to dryness. The residue was chro-matographed on a silica gel column (30 x 1 cm) by elution with dioxane-hexane(1:3, v/v). Product fractions, free of dicyclohexyl urea and p-azidoanilineas analyzed by tlc (dioxane-hexane, 1:3, v/v, Rf = 0.55) were pooled and re-duced to dryness. The product was dissolved and stored as a solution indioxane. The yield, based on absorbance at 276 nm (e = 13,600), was 96%.

38Specific activity was calculated to be 2.80 mCi/mmol

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[14C]p-azido-benzoic acid hydrazide (ABH) was prepared following the

procedure of [Carboxyl C]p-aminobenzoic acid (0.4 mmol) wasconverted to p-azidobenzoic acid40. Without further purification, thiscompound was esterified with N-hydroxysuccinimide (one equivalent) usingdicyclohexylcarbodiimide (one equivalent) in dioxane (2 ml). The filteredsolution of the activated ester was reacted with hydrazine (1.5 equivalents).After silica gel chromatography (ethanol-ethylacetate, 1:9, v/v), [14C]ABHwas obtained in 56% overall yield (225 pmol). The specific activity was

38calculated to be 1.7 mCi/mmol . The product was characterized by comparisonof its UV-spectral properties and melting point with literature values40.

[3H]L-methioninol was prepared by reduction of O-ethyl-L-methionine41'42with [3H]NaBH442. To O-ethyl-L-methionine (356 mg, 2 mmol) in a solution(6 ml) of ethanol-water (3:1, v/v) was added 1.0 M HC1 (0.01 ml) at 40, fol-lowed by dropwise addition of a solution (4 ml) of [3H]NaBH4 (151 mg, 4.0mnol) in 2.5 mM NaOH in ethanol-water (3:1, v/v). The mixture was stirredat 50 in a high-draft hood for 48 hr. At this time water was added (2 ml)and the reaction mixture was extracted with chloroform (3 x 10 ml). The com-bined chloroform extracts were washed with water (2 ml), dried with magnesiumsulfate and then reduced by rotary evaporation to an oil (139 mg, 1.03 mmol),which was pure methioninol as judged by tlc (silica gel, ethyl acetate-ethanol, 2:3, v/v; Rf = 0.48). It's Rf value was identical to that of [1H]methioninol prepared similarly, which had a melting range of 35-38° (lit.4328-300).

t-butoxycarbonyl-[3H]methioninol. [3H]methioninol (139 mg, 1.03 mmol)was dried by evaporation from anhydrous pyridine and dissolved in pyridine(1.5 ml) and ethyl acetate (2 ml). To this was added t-butoxycarbonylazide(270 0l, 1.95 mmol). The reaction mixture was stirred for 2 hr at 250 andthen reduced to an oil by rotary evaporation at 350. The oil was dissolvedin ethyl acetate (15 ml) and extracted at O° with cold 0.5 M HC1 (3 ml) andsaturated aqueous NaCl. The ethyl acetate layer was dried giving an impureoil which was partially purified by dissolving in petroleum ether, in whichthe impurities are essentially insoluble. After evaporation of the petroleumether, the purified oil weighed 104 mg (0.44 mmol, 44% yield). This wasidentical chromatographically to similarly prepared[lH]material which had a

melting range of 49-510 (lit.43 50°).8-Azido-adenosine 5'-phosphate was prepared by a modification of the

published procedure44 in which dimethyl sulfoxide is used as solvent so thatthe sodium salts of the reactants (8-Br-AMP and azide) could be used. Rf

1685


values in tic on PEI-cellulose in 1.0 M LiCl were: AMP, 0.46; 8-Br-AMP, 0.29;8-N3-AMP, 0.47. The triacetyl derivative was prepared by reacting the tri-n-octylammonium salt of the 8-azido-adenosine 5'-phosphate for four days in

45pyridine-acetic anhydride[3H]Methioninyl-8-azido-adenosine 5'-phosphate (met-ol-8-azido-AMP) was

prepared by a modification of a published procedure46 from t-butoxycarbonyl-

L3H]methioninol and 6, 0, 0 -triacetyl-8-azido-adenosine 5'-phosphate.The reaction mixture was not resolved until all the protecting groups hadbeen removed. Preparative paper chromatography (solvent A) gave the de-sired adenylate (Rf = 0.57) in 12% yield (specific activity = 3 mCi/mmol)38and one minor impurity (2%, Rf = 0.68), probably the corresponding sulfoxide.The main product had a UV-spectrum consistent with 8-azido-adenosine and wasof >85% radiopurity. Its Rf (cellulose, tlc, solvent B) is 0.43 compared toa value of 0.27 for methioninol-adenosine 5'-phosphate. Upon treatment withsnake venom phosphodiesterase, 80% of the radioactivity in this preparationco-chromatographed with L-methioninol on silica gel in ethanol-ethyl acetate,3:2, v/v. Another 15% remained at the origin and may be undegraded adenylate.

CovaZent Attachment of [14C]ABA to tRNA. tRNAfMet was modified at its4-thiouridine with [14C]ABA according to the procedure of Yang and Soll47.To E. coZi tRNAfMet (180 A260 unit, 0.3 jmol) in 0.3 phosphate buffer, pH8.5, (0.8 ml) and redistilled dimethylsulfoxide (7.2 ml) was added [14C]ABA(22.5 imol) in ethyl acetate (0.2 ml). After 15 min at 250 acetone wasadded and the precipitate collected by centrifugation. The tRNA was dis-solved in water, dialyzed well against water at 40, and then precipitatedfrom 0.5 M NaCl with 2.5 volumes of ethanol. The pellet was dissolved inthe starting buffer of the BD-cellulose chromatography.

Covalent Attachment of [4 C]ABH to tRNA. tRNAfMet modified at the3'-end with [14C]ABH was prepared as shown in Figure 2 according to publish-

29 feed procedures . To periodate oxidized tRNAfMet (78 A260 unit, 0.13 }imol)in 0.1 M sodium acetate, pH 4.5, (0.5 ml) was added [14C]ABH (2.5 pmol) indioxane (0.08 ml). After 2 hr at 370 the reaction mixture was precipitatedwith ethanol three times. The pellet was dissolved in the starting bufferof the BD-cellulose chromatography.

Purification of the Modified tRNA. The tRNA recovered after reaction(up to 160 A260 units) was applied to a BD-cellulose column (0.8 x 10 ml).The column was eluted at room temperature with a total volume of 50 ml foreach gradient. The salt gradient was from 0.4 to 1.5 M NaCl in 10 mM magnes-ium chloride - 10 mM sodium acetate (pH 4.5). The ethanol gradient was from

1686


Adenine Adenine Adenine

tRNA\ ZON-I tRNA\ ,0 tRNA 0~

Hc C 4 C c RNH-NH2 C \

\ I_/; H\ /H HH -c\HH/C-H H-C HO\C c/ H

II~~~~~~~~~~~~~~~~~~~~OH OH 0 0O XNoO

NHN3 R

R s<R

C=O

Figure 2. Scheme of hydrazone formation at the 3'-end of tRNA.

0 to 40% ethanol in 1.5 M NaCl - 10 mM magnesium chloride - 10 mM sodiumacetate (pH 4.5). Fractions of 3 ml were collected every 20 min.

Synthesis of Reference Compounds. Adduct of 4-thiouridine 3'-phosphateand ABA (ABA-4Sp). This was prepared by adding unlabeled ABA (0.75 mg) dis-solved in methanol (0.02 ml) to a solution of 4-thiouridine 3'-phosphate(2.5 iimol) in 50% aqueous methanol (0.04 ml). After seven hours at 250 thereaction mixture was subjected to paper chromatography (solvent A). Theproduct band was eluted to give a light-sensitive compound (6 A260 units).The Rf values (solvent A) were 4Sp, 0.29; ABA-4Sp, 0.69; ABA, 0.95.

Adduct of pseudouridine 3'-phosphate and ABA (ABA-*pp). This was pre-pared by reacting ABA (4 jmol) and pseudouridine 3'-phosphate (4 wmol) in asolvent system of water-dimethylsulfoxide-methanol (50:20:30) containing50 mM potassium phosphate (pH 8.6) for 5.5 hr at 500. Paper chromatography(solvent A) gave a photosensitive product (5 A270 units). Rf values (sol-vent A) were pp, 0.14; ABA-pp, 0.38; ABA, 0.95.

Hydrazono adduct of periodate-oxidized adenosine and ABH. This wasprepared as follows: to a solution of adenosine (132 imol) in 25% aqueousethanol (8 ml) was added a solution of NaIO4 (1 mmol) in water (2 ml).After 15 minutes at 250 the reaction was cooled to 0° and the periodate pre,cipitated by addition of 3 M KCI solution (0.4 ml). After 5 minutes at 5°the solution was rapidly filtered. Ethanol (5 ml) and ABH (20 mg) was addedand the reaction was stirred for 45 minutes at 250 and then reduced to dry-ness. The residue was dissolved in ethanol, filtered, and the filtratepurified by preparative thin layer chromatography on silica gel in ethyl

1687


acetate-ethanol (3:1, v/v). Upon photolysis, the UV spectrum of the product(Rf = 0.38) collapsed from a composite of those of adenosine and ABH to thatof adenosine alone. Two by-products (Rf = 0.48, 0.21) isolated from thechromatographic separation also showed this behavior upon photolysis andwere thus judged to be other ABH-adenosine adducts. Rechromatography of theisolated main product gave these minor products as well, which are presumedto be isomeric hydrazono-adenosines. Electrophoresis of the isolated mainproduct also showed evidence of these by-products; in this case the mixtureran as an unresolved streak toward the anode.

RESULTS

Preparation of tRNAfMet Modified with Photolabile Groups. In our earli-

er studies47 we had shown that alkylating reagents can react specificallyfMetwith 4-thiouridine and with pseudouridine in E. coli tRNA . Since 4-

thiouridine in tRNA (position 8) undergoes a facile addition reaction48 tocytidine (position 13) and thus is unavailable for alkylation, we had to

34determine the amount of free 4-thiouridine in our tRNA preparation3. Wefound that 77% of tRNA molecules still contained 4-thiouridine. Reactionof this tRNA preparation with [14C]ABA gave a preparation containi'ng 0.91moles of ABA per mole of tRNA. After digestion with T2 RNase of this mate-rial to mononucleotides a chromatographic analysis showed that 75% of theradioactivity is [14C]ABA-4Sp . The other [14C]ABA nucleotides are derivedby modification of pseudouridine and of other as yet unidentified nucleo-sides. This mixture of modified tRNAs could be purified using BD-cellulose.As shown in Figure 3, unmodified tRNA (presumably with 4S8-C13 crosslink)and 4S-[14C]ABA-tRNA eluted in the salt gradient. The ethanol gradient con-tained tRNA species containing two or more moles of [14C]ABA per mole of

20 gOj Figure 3. 4A[4C0~~~~~~~~~~~Purification ofo

20 |X.I ABA-tRNAfMet. The tRNAi,t\ i *' recovered after reaction

10!,o- |0 I-s J \ _ lo (about 160 A260 units)

4l \ was chromatographe. on.qiI \ g BD-cellulose. For de-

tails see Materials and0Z"OD NO Methods. Fractions 12-

20 (peak I) and 26-32FRACTIONUMER(peak II) were pooled.

1688


tRNA. Analysis of T2 RNase hydrolysates of the nucleotide material in peakI gave at least 96% of the radioactivity in ABA-4Sp. Digestion of tRNAfrom peak II yields [14C]ABA-*p, and other [14C]ABA nucleotides.

An attempt was made to modify tRNAfMet specifically at its only pseudo-uridine with [14C]ABA. First, its reactive 4-thiouridine was converted to

49uridine by cyanogen bromide treatment followed by base hydrolysis4. ThistRNA was then treated with [14C]ABA under a variety of conditions. Unfor-tunately we did not succeed in preparing *-[l4C]ABA-tRNAfMt. Upon T2RNase hydrolysis and chromatography always two or more additional radio-active products were obtained, presumably due to alkylation of other hetero-cyclic bases. Unlike in the preparation of 4S-ABA-tRNAfMet, BD-cellulosechromatography did not separate *-ABA-tRNAfMet from the undesired side pro-ducts.

Reaction of a 20-fold excess of [ 4C]ABH with periodate-oxidizedtRNAfMet gives (after ethanol precipitation) a preparation containing 1.25moles reagent per mole of tRNA. When this is subjected to BD-cellulosechromatography (Figure 4) a small amount of unreacted tRNA is released inthe salt gradient. The desired product is eluted with the ethanol gradient,

, Figure 4.+oX ~~~Purification Q '

B _ - 5 ^ _ 20 [EY4C]ABH-tRNAe.6X\"The tRNA recovered after4 \ reaction (about 73 A260

x~~~~~~~~0 X units) was chromato-t 3 1 ? graphed on BD-cellulose.lo >. 2 For details see Materials

0

10 20 30 40FRACTION NUMBER

and, after this purification, contains 0.99 mole reagent per mole of tRNA.All [14C]ABH is covalently bound to tRNA. No radioactivity could be inducedto separate from undigested tRNA by tlc or electrophoresis, so it is likelythat ABH is present only in the hydrazono-form at the 3'-terminus of tRNA.This was confirmed by exchanging [14c]ABH bound to tRNA with hydrazine.After incubation of 3I-E14C]ABH-tRNAfMet with hydrazine (10% aqueous solu-tion, 10 min at 370) 95% of the radioactivity migrates with the referenceABH marker upon electrophoresis. Finally, all the radioactivity of a T2

lea9


RNase digest of this tRNA preparation ran toward the anode, in a streaksimilar in mobility to that of the reference hydrazono-adenosine.

Preparation of Methioninyl-8-Azido-Adenosine 5'-Phosphate. [3H]Methi-oninol was readily condensed with 8-azido-AMP to give [3H]met-ol-8-azido-AMP in 12% yield. The product, purified by paper chromatography, was homo-geneous in two tlc systems when detected by UV-absorbance, and greater than85% radiopure. It was characterized by its UV sDectrum, incorporation ofradioactivity, and tlc chromatographic similarity to the met-ol-AMP and itsability to act as a competitive inhibitor in the methionyl-tRNA synthetasereaction (see below).

Do the Photo labile Analogs Still Bind to Methionyl-tRNA Synthetase? Inorder to assess the usefulness of the photolabile analogs their binding tomethionyl-tRNA synthetase was tested. Dissociation constants (at 25°) weredetermined for native tRNAfMet and the modified tRNAs by fluorescence quench-inc35 at conditions similar to those used in the photolysis experiments.The Kd values for native tRNAfMet, 4S-[14C]ABA-tRNAfMet and 3'-[14C]ABH-tRNAfMet were 7 x 10-8 M, 11 x 10-8 M, and 4 x 10-8 M, respectively. Inaddition, 4S-[14C]ABA-tRNAfMet could be aminoacylated to 90% of the levelof unmodified tRNAfMet by pure methionyl-tRNA synthetase. The same testwas not possible for 3'-ABH-tRNAfMet since its 3'-terminus was modified. Asexpected met-ol-8-azido-AMP proved to be a competitive inhibitor of methi-onine in the aminoacylation reaction, with a Kj value of 1.7 x 10 8 M.Methioninyl-AMP has a K of 0.9 x 10-8 M.46

These studies indicate that the three substrate analogs bind properlyto the enzyme.

Photoaffinity LabeZing of MethionyZ-tRNA Synthetase. A 15 min photo-lysis with pyrex filtered light from a 366 nm source of an equimolar mix-ture (at lIM concentration) of 4S-[14C]ABA-tRNAfMet and the enzyme gave 5-10%incorporation of radioactivity into protein after digestion of the tRNA withT2 RNase (see Materials and Methods). The yield of photo-labeled enzyme waspH-dependent (maximum around pH 5.5-6.0), and was reduced to 0% in high saltconcentrations (0.3 M NaCl or higher). Other buffers (e.g., 2[N-morpholino]-ethane sulfonic acid) at the same pH gave less crosslinking. The presenceof 5 mM dithiothreitol reduced the yield. This may be due to reactionssimilar to the reduction of 8-azido-adenosine by thiol compounds to 8-amino-adenosine.50 The same amount of crosslinking was obtained using a pyrex-filtered 254 nm light source. Under the same conditions 3'-[14C]ABH-tRNAfMetalso gave 5-10% crosslinking.

1690


Since the yield of crosslinking the tRNA to the enzyme was low it isdesirable to separate the unreacted enzyme from the covalently linked com-

plex. Such a separation is possible by DEAE-cellulose chromatography (Fig-ure 5). The unreacted enzyme is eluted at lower salt concentration (aroundfraction 10) while the tRNA and the non-dissociable complex (as assayed byenzyme bound radioactivity) is released later (around fraction 20). In aphotolysis experiment with 5 mg of enzyme 125 pg of radioactively labeledenzyme could be recovered, an isolated yield of 2.5%. The enzyme elutingin the first peak retained 50% of its original activity; it could be reusedwith fresh 4S-[14C]ABA-tRNAlfet in photolysis experiments.

When an equimolar mixture of [3H]met-ol-8-azido-AMP and methionyl-tRNAsynthetase (at pM concentration) was photolyzed under the conditions des-cribed in Materials and Methods 5-15% of the radioactivity became irrever-sibly bound to the enzyme. Again, the yield of crosslinking was influencedby the nature of the buffer and was dependent on the absence of thiol re-

agents.48

II Figure 5.2 Separation of crosslinked

0-0 A enzyme:tRNA comolex froms \ eunreacted enzyme. A [email protected] ff \ \ TQ. ,,, action mixture containing_I ffi methionyl-tRNA synthetase

l6 12 9 (5 mg) and a stoichiome-*E\-tric amount of 4S-[14C]-.4-E4Y-qr A = , F ° ABA-tRNAfMet was irradia-

-9 0

/ \ ted under the usual con-2 .2: Ax &4 < ditions and applied to a

IL DEAE-cellulose column (0.8x 4 cm) after adjusting

,0 20 3o0 the ionic strength to thatFRACTION NUMBER of starting buffer with

NaCl. Elution was carriedout at 50 with a linear

salt gradient of 0.05 - 0.80 M NaCl in 0.02 M Hepes (pH 7.5) - 0.001 Mdithiothreitol. Radioactivity covalently linked to protein was determinedas described in Materials and Methods. Fractions of 1 ml were collectedevery 3 min.

DISCUSSIONfMet

E. coZi tRNA contains one 4-thiouridine and one pseudouridine resi-

lue. From our earlier experience29 they were the only bases in that tRNAexpected to react with alkylating reagents. Unfortunately, ABA turned outto be less specific than the fluorescent compound 4-bromomethyl-7-methoxy-2-oxo-2H-benzopyran. Thus, in the reaction with 4-thiouridine also some

1691


pseudouridine became alkylated. Fortunately, the differently modified tRNAs

could be separated by BD-cellulose chromatography. However, under the moredrastic conditions for modification using tRNA fMet, in which 4S had been

converted to U, other bases in the tRNA, presumably purines, were reactive.

Apart from reaction at the 3'-terminus hydrazine derivatives like ABHcan be introduced into other positions of tRNAfet. Substitutions of di-hydrouridine 29and also of 7-methylguanosine51 should be possible. Thus,additional analogs of tRNAfMet with photolabile groups can be prepared.

Photoaffinity labeling offers several advantages over chemical affinity

labeling 92, but it is not without potential pitfalls. One potential diffi-culty, especially with simple azides such as those used in this study, is

the possibility that a nitrene, even when generated at the binding site,might be so long-lived that the reactive molecule could dissociate andeventually react elsewhere on the protein. As shown above the analogs usedin this study bind very tightly to the enzyme. This is no guarantee thattrue photoaffinity labeling will occur, but does increase the likelihoodthat labeling, by whatever mechanism, will be specific for the binding site.

Our first results suggest that the observed crosslinking is associatedwith normal enzyme-substrate interactions. Each analog undergoes photo-catalyzed reaction with protein at low concentrations when present inamounts equirnolar with enzyme subunits. The pH dependence of the extent

of crosslinking of 4s-[14jC]ABA-tRNA gives a profile consistent with the pH-

dependence of tRNA binding to aminoacyl-tRNA synthetases. Most efficient

crosslinking occurs at pH 5.5-6.0, which is the pH range in which many

aminoacyl-tRNA synthetases52, including, at least in one buffer system, the

methionyl enzyme35, form their strongest complexes with their cognate tRNAs.High ionic strength, which destabilizes enzyme-tRNA complexes35, completely

inhibits crosslinking. Only further experiments can tell us whether "true

affinity labeling" occurred. However these results encourage our hopes

that these photolabile analogs may be useful in determining the substrate

binding sites of methionyl-tRNA synthetase.

ACKNOWLEDGEMENTS

We are indebted to Dr. C.J. Bruton for his gift of methionyl-tRNA syn-

thetase. We thank Dr. Friedrich Hansske for communicating portions of histhesis research prior to publication. This work was supported by grants

from the National Institutes of Health (GM 22854 and HD 09167).

1692


REFERENCES

*This paper is dedicated to the memory of Jerome Vinograd.

1. Knowles, J.R. (1972) Accounts Chem. Res. 5, 155-160.2. Cooperman, B.S. in "Aging, Carcinogenesis, and Radiation Biology",

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