Proc. Nat. Acad. Sci. USA 71 (1974)

Correction. In the article "Peptide Mapping of Amino-acyl-tRNA Synthetases: Evidence for Internal SequenceHomology in Escherichia coli Leucyl-tRNA Synthetase" byRobert M. Waterson and William H. Konigsberg, whichappeared in the February 1974 issue of the Proc. Nat. Acad.Sci. USA 71, 376-380, Fig. 2 on page 378 was inverted duringthe publication process. The correctly oriented figure and itslegend are reprinted below.

.f1

i^ *r

.kXi

.80 X....... .*.

....

*:::: : fun.''§.

.:

0

To00

00 0

0

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00 ° 0cD

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0

0

0

0ac:=C

FIG. 2. Autoradiograms of the lysine-containing peptides of achymotryptic digest of [14C]succinyl-S-carboxymethylleucyl-tRNA synthetase. Peptide mapping on cellulose or silica-gelthin-layer plates (upper and lower panels, respectively), using10-15 ,ug of protein, was done as described in Methods.

Correction 2151

.t. W,:.... .I

Proc. Nat. Acad. Sci. USAVol. 71, No. 2, pp. 376-380, February 1974

Peptide Mapping of Aminoacyl-tRNA Synthetases: Evidence for InternalSequence Homology in Escherichia coli Leucyl-tRNA Synthetase

(amino acid-activating enzymes/thin-layer electrophoresis/gene duplication)

ROBERT M. WATERSON AND WILLIAM H. KONIGSBERG

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Communicated by Aaron B. Lerner, September 17, 1973

ABSTRACT Most aminoacyl-tRNA synthetases con-tain polypeptide chains of about either 50,000 or 100,000daltons. Peptide mapping of tryptic, chymotryptic, orStaphylococcus aureus acid protease digests of seryl-tRNAsynthetase (100,000, dimer) and leucyl-tRNA synthetase(100,000, monomer) from E. coli was done after selectivemodification of lysine residues with j14Cjsuccinic anhydrideor of methionine residues with [14Cliodoacetate. By use ofthin-layer electrophoresis and chromatography on silica-gel or cellulose plates followed by radioautography it waspossible, depending upon the specific activity of the re-agent used, to detect radioactive peptides obtained fromas little as 1 ,ug of protein.Seryl-tRNA synthetase gave the correct number of

tryptic peptides expected for a dimer of identical subunits.Leucyl-tRNA synthetase, on the other hand, gave roughlyhalf the number of radioactive tryptic, chymotryptic, andacid protease peptides expected from the lysine, arginine,and methionine content of the 100,000 monomer. We haveinterpreted these results as indicating that extensive in-ternal homology exists among lysine- and methionine-containing peptides within the leucyl-tRNA synthetase.The simplest conclusion that can be drawn from theseobservations is that the NH2- and COOH-terminal halvesof leucyl-tRNA synthetase and perhaps other synthetasesof 100,000 molecular weight may have evolved through aprocess of gene duplication and fusion, followed by limiteddiversification by way of amino-acid substitutions accu-mulating during evolution.

The polypeptide chains of aminoacvl-tRNA synthetase fromvarious organisms, with few exceptions, are composed of abouteither 450 or 900 amino-acid residues per chain. They areusually isolated as monomeric proteins (al) of roughly 100,000daltons, or as dimers (a2) or tetramers (a4 or a2#2) of about50,000 daltons per subunits (1-3).The existence of two such distinct polypeptide chain lengths

for enzymes of common function suggested that the chains withmolecular weights of 100,000 might have resulted from partialgene duplication. If this were the case, a considerable amountof internal homology would be predicted. This possibility hasbeen examined by peptide mapping of an Escherichia colisynthetase of each type: seryl-tRNA synthetase (two 50,000subunits) and leucyl-tRNA (one 100,000 polypeptide chain).Tryptic digests of seryl-tRNA synthetase were examined byconventional two-dimensional peptide mapping procedures,but these methods proved inadequate for the leucyl-tRNAsynthetase. To study peptide digests of this enzyme, a thin-layer mapping procedure was devised, involving selectivemodification of lysine and methionine residues with 4C-labeledreagents before proteolytic digestion. This method requiredonly small amounts of protein and may have general applica-

376

bility. We report the results of these studies and discuss theirimplications concerning this class of enzymes.

MATERIALS A-ND METIFODS[1-14C]Iodoacetic acid (13.4 Ci/mol) and [1 ,4-14C]succinicanhydride (4.7 Ci/mol) were products of New EnglandNuclear Corp. N-Tosyl-L-phenylalanyJchloromethyl ketone(TPCK)-trypsin and N-tosyl-i.lysylchloromethyl ketone(TLCK)-chymotrypsin were obtained from Worthington,while the acid protease from Staphylococcus aureus (4)was a gift from Dr. Klaus Weber. Purified guanidine hydro-chloride was obtained from Heico and used without additionalrecrystallization. "Chromagram" silica-gel thin-layer plateswere purchased from Eastman and "Avicell (22)" microcrystal-line cellulose plates from Brinkman. All other compoundswere reagent grade and were used without further purification.

Seryl- and leucyl-tRNA synthetases from E. coli KL126were prepared in homogeneous form by described methods(5, 6). Polyacrylamide gel electrophoresis revealed each to be asingle component using Tris-glycine (7) and Na dodecyl sul-fate (8) gel systems. Molecular weights of 50,000 (seryl-tRNA synthetase) and about 100,000 (leucyl-tRNA synthe-tase) were determined from their relative mobilities on Nadodecyl sulfate-polyacrylamide gels, in agreement with pub-lished values (9, 6, 10).

Samples of both proteins were reduced with 2-mercapto-ethanol (0.1 M) in 7 M guanidine hydrochloride, 0.5 M Tris-HCl, pH 8.0, and reacted with iodoacetate (0.11 M) for 20min at 250 in the dark. The reactions were then terminated bya second addition of 2-mercaptoethanol, and the carboxy-methylated protein samples were then exhaustively dialyzedagainst deionized water and lyophilized. [14C]Succinyl-S-carboxymethylleucyl-tRNA synthetase was prepared in thefollowing manner (11): S-carboxymethylleucvl-tRNA syn-thetase (5 mg of lyophilized powder) was dissolved in 0.25 mlof 0.4 M sodium phosphate, pH 9.0, containing 6 M guanidinehydrochloride and mixed with 1.1 mg of ['4C]succinic an-hydride (4.7 Ci/mol). After 20 min at 250, an additional ali-quot of (unlabeled) succinic anhydride (5 mg in 0.1 ml ofbuffer) was added and the mixturewas incubated for another 30min at 250, dialyzed exhaustively against 0.05 M ammoniumbicarbonate, pH 8.5, and then Iyophilized. The product wastaken up in a minimal amount of water and lyophilized twicemore. The specific activity of the final product was 1.0 iCi/mg.

Methionine residues of S-carboxymethylleucyl-tRNA syn-thetase were labeled by treatment of the protein with [14C]-iodoacetate (13.45 Ci/mol) for 30 hr at 370 in 5% formic acid

E. coli Leucyl-tRNA Synthetase 377

as described (12). The specific activity of the final product was0.5-0.6 MCi/mg.

Tryptic or chymotryptic digests of the modified proteinswere prepared by incubation of the proteins (5-10 mg/ml in0.1 M ammonium bicarbonate, pH 8.5) with 3-6%. (w/w) ofeither proteolytic enzyme for 16-20 hr at 250. In another in-stance, a tryptic digest of ["4C]succinyl-S-carboxymethyl-leucyl-tRNA synthetase was redigested further at 370 for 18hr with S. aureus acid protease at pH 8.0 as described (4).

Two-dimensional peptide mapping, with either silica-gel or

cellulose thin-layer plates (20 X 20 cm), was done with stan-dard chromatography tanks and a DESAGA-Brinkman cold-plate electrophoresis unit. Aliquots (1-10 Ml) of each digestwere spotted on a plate with the aid of intermittent forced-air drying. Standard chromatography solvents such as bu-tanol-.acetic acid-water (BAW) (3: 1: 1; v/v) or pyridine-butanol-acetic acid-water (PBAW) (10: 15:6:24; v/v) andvolatile electrophoresis buffers at pH 3.6 or 6.5 (13) were usedin the following combinations: I, BAW, pH 6.5, II, BAW, pH3.6; HI, PBAW, pH 6.5; IV, PBAW, pH 3.6. Mapping on

silica-gel plates usually involved electrophoresis in one dimen-sion followed by ascending chromatography in the second di-mension (14). When cellulose plates were used, satisfactory re-

sults were obtained only when samples were first chromato-graphed parallel to the direction in which the cellulose platewas spread (i.e., parallel to the uncovered glass edges) andthen subjected to electrophoresis in the second dimension,perpendicular to the grain of the plate. The tryptic peptidesof S-carboxymethylseryl-tRNA synthetase were observed bymeans of standard ninhydrin stain (15), while the "4C-labeledpeptides of leucyl-tRNA synthetase digests were detected byradioautography (24- to 48-hour exposure times) using KodakRP/S x-ray film.

RESULTS AND DISCUSSION

To see if the 100,000, single-chain class of aminoacyl-tRNAsynthetases could have arisen by duplication and fusion ofgenes coding for polypeptide chains of 50,000 daltons, we

looked for evidence of internal sequence duplication in E. colileucyl-tRNA synthetase. The number of lysine- and methio-nine-containing peptides obtained from enzymatic digests ofthis enzyme was determined and compared to that expectedfrom the amino-acid composition of the enzyme. As a control,we also counted the number of tryptic peptides obtained fromseryl-tRNA synthetase, which is a dimer of two 50,000-daltonsubunits (9).

Two-dimensional peptide mapping on thin-layer platescoated with cellulose or silica gel was the method of choicebecause only small amounts (100-250 ug) of protein are re-

quired (14). When this technique was used with the trypticdigest of S-carboxymethylseryl-tRNA synthetase, between46 and 52 ninhydrin-positive spots were observed (Fig. 1).The best patterns were obtained with cellulose rather thansilica-gel plates. All of the tryptic peptides were soluble in thepH 6.5 pyridine-acetate buffer used for electrophoresis, andvery little ninhydrin-positive material remained at the origin.Based upon the lysine and arginine content reported earlierfor the seryl-tRNA synthetase dimer, a maximum of 108tryptic peptides would be expected if the subunits were totallydifferent (9). Since about half the maximum possible numberof tryptic peptides are observed, we conclude that the sub-

units of seryl-tRNA synthetase have very similar or identicalsequences. These findings confirm the designation of the

0I..I I. I

00

000

0

0

0 0 c1o~~

o oU(9

0

0

0o

1000

0

0

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0

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0

00

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0

FIG. 1. Peptide map of the tryptic peptides of S-carboxy-methylseryl-tRNA synthetase. A 10-,A aliquot (200 Mug) of thedigest was spotted (®, origin) on a cellulose thin-layer plate andsubjected to ascending chromatography in butanol-acetic acid-water followed by electrophoresis at pH 6.5. In this plate thecathode is at the right.

enzyme as an a2 synthetase, a conclusion initially based uponobservations that the subunits possessed identical size andelectrophoretic mobility (9) and that the molecule containedtwo noninteracting binding sites for each of its substrates (5).When the same conventional mapping procedure as was

used for the seryl-tRNA synthetase was applied to trypticdigests of the leucyl-tRNA synthetase, the maps showed con-siderable trailing, due to either limited solubility or the largesize of the peptides, thus making meaningful interpretationimpossible. For this reason we decided to treat the proteinwith radioactive reagents that react specifically with certainamino-acid residues. We chose ["4CJsuccinic anhydride, whichwill react with the amino-terminal residue and the E-aminogroups of lysine (11), and ["4C]iodoacetic acid, which can beused to selectively alkylate methionine residues under acidconditions (12). The introduction of radioactivity offersseveral potential advantages. Since radioautography is thesole method of detection, only those peptides containing the"4C-modified residues are seen. This permits proteases withbroader specificity than trypsin to be used without producingan overly complex map. Thus, digestion with chymotrypsin,acid protease, or possibly, thermolysin yields smaller pep-tides, which have more favorable chromatographic and elec-trophoretic properties than the larger tryptic peptides. Inaddition, it was possible to use much smaller samples thanrequired for conventional (14) mapping techniques. Depend-ing on the specific activity of the modifying reagents and theexposure time, less than 1 ,g of protein could be used.WhenS-carboxymethvl-[I4C]succinylated leucyl-tRNA syn-

thetase was digested with chymotrypsin and the peptideswere submitted to thin-layer electrophoresis and chroma-tography, a radioautogram of the plate gave the patternshown in Fig. 2. About 30 radioactive spots could be counted.This number is slightly over one-half of the 57 succinylated

Proc. Nat. Acad. Sci. USA 71 (1974)

378 Biochemistry: Waterson and Konigsberg

0'

00

0C0

i0

) C,:

o

C:2 o

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0o

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0 0

00A

° o00 0.°0,00 D

00

0

0

0

0

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. - . ..... ... ...........:._ < ._ . .. -r .....,; B

*.'' .,..e

FIG. 2. Autoradiograms of the lysine-containing peptides of achymotryptic digest of [14C]succinyl-S-carboxymethylleucyl-tRNA synthetase. Peptide mapping on cellulose or silica-gelthin-layer plates (upper and lower panels, respectively), using10-15 ,g of protein, was done as described in Methods.

peptides expected from the amino-acid composition of leucyl-tRNA synthetase (6). Additional two-dimensional maps of thechymotryptic digest gave 28-35 lysine-labeled peptides. Allthe radioactive peptides were soluble in the buffer used toapply the sample to the plate, and almost no radioactivityappeared at the origin; thus the number of spots on theradioautogram provides an estimate of the maximum numberof lysine-containing peptides in the digest. It might be ex-pected that a particular lysine residue, would appear in severaloverlapping peptides due to the variability in the rates ofchymotryptic cleavage at different residues. We tried tominimize this difficulty by digesting the leucyl-tRNA syn-

thetase with large amounts of chymotrypsin for extendedperiods. If incomplete digestion had occurred we would haveobserved more peptides than were actually found. Since onlyhalf the expected number of lysine-containing peptides wereobtained, we interpret this as evidence for extensive internalhomology in the enzyme. As an independent check of thisresult, we attempted to map a tryptic digest of the ["4C]-succinylated leucyl-tRNA synthetase. Despite the fact thateven larger peptides are obtained by treatment of the suc-cinylated rather than the unmodified enzyme with trypsin,the presence of so many negatively charged groups and thegreatly reduced sample loads permitted adequate resolutionof the lysine-blocked tryptic peptides. A representative map(Fig. 3) shows about 26 peptides or somewhat over half thenumber (43) expected from the arginine content of the en-zyme. While the relatively high background prevented anunambiguous interpretation, the results are consistent withthose obtained from the chymotryptic digest. Further con-firmation of internal sequence homology was obtained bytreatment of the tryptic digest with the acid protease of S.aureus, which splits at glutamic and aspartic acid residues(4). A map of this digest (Fig. 4) indicated the presence ofabout 35 lysine-containing peptides, or somewhat over halfthe number expected.When leucyl-tRNA synthetase, labeled with ['4Cliodo-

acetate on methionine residues, was digested with chymotryp-sin and submitted to thin-layer peptide mapping, the patternshown in Fig. 5 was obtained. The presence of 18 spots, com-pared to the expected 30, provides further evidence for in-ternal sequence homology and demonstrates that homologyis not confined to lysine-containing peptides. Taken together,the results of the various peptide maps of leucyl-tRNA syn-thetase point to extensive sequence repetition within theenzyme. The most probable explanation is that leucyl-tRNAsynthetase and perhaps other single-chain synthetases of100,000 daltons are products of duplication and fusion of genescoding for a 50,000-dalton polypeptide with aminoacylationactivity. Clearly, these results are preliminary and requireconfirmation. This confirmation is especially important inview of the fact that the spots on the radioautograms vary inintensity. While some of this variation may be due to thepresence of more than one lysine in a given peptide, it ismore likely that the more intense spots represent at leasttwo copies of a given peptide.

0

0o

C

0 C~0O:= °: '. CO°C

.

0

't~~~~~~.

FIG. 3. Autoradiographic peptide map of the tryptic peptides of [14C]succinyl-S-carboxymethylleucyl-tRNA synthetase. A sample(15-20 j&g) was applied to a silica-gel plate, subjected to electrophoresis at pH 6.5, and then chromatographed in pyridine-butanol-acetic acid-water.

Proc. Nat. Acad. Sci. USA 71 (1974)

a

4

E. coli Leucyl-tRNA Synthetase 379

O C

ClQrco C 5C° C:> #--

->(:o C ~)

0

0O 0

II.

FIG. 4. Peptide map of the [14C]lysine-containing peptides of modified leucyl-tRNA synthetase after digestion with trypsin andS. aureus acid protease. About 25 sg of the digest was applied to a silica-gel plate, subjected to electrophoresis at pH 3.6 in the first dimen-sion (the cathode is to the left), and chromatographed, in the second dimension, in butanol-acetic acid-water.

It is generally accepted that gene duplication, followed byindependent evolution of one (gene) of the subsequent pair,is probably responsible for generating new proteins possessingmore specialized functions necessary for the continuing evolu-tion of the species (16). Evidence for complete or partialgene duplication followed by gene fusion has been inferredfrom studies of proteins such as immunoglobulins (17), phos-phofructokinase (18), haptoglobin (19), tryptophan synthetase(20), and ferredoxin (21).For haptoglobin, the fusion event must have occurred

relatively recently since some populations do not have hapto-globin 2a and the sequences of the fused chains are nearlyidentical (19). The tryptophan synthetases of Neurosporacrassa and yeast also provide interesting examples of genefusion in that they each appear to contain a single polypep-tide chain which possesses both types of activities found inthe separable A and B chains of E. coli tryptophan synthetase(20). With the immunoglobulins, the internal sequence ho-mology is convincing enough in the constant regions of heavychains to infer that these regions of the immunoglobulinsarose by gene duplication and fusion but that subsequentamino-acid substitutions in the constant regions permitteddifferent functions to be assumed by each constant-regiondomain (17).Even though the single-chain, 100,000-dalton aminoacyl-

tRNA synthetases appear to have considerable internal se-

L4.s . , .. . . ... . ... :

ML~'i* . t. ... ;. ... 'A quence repetition they exhibit only a single binding site for each

substrate, whereas the multimeric synthetases contain an av-erage of one binding site for each 50,000-dalton subunit (1-3).Several explanations are possible. It may be, for instance,that the monomeric enzymes have so-called "half-site reac-tivity" (22). This is considered unlikely, however, sincedetailed kinetic and binding studies (3) with isoleucyl-tRNAsynthetase (100,000-dalton single chain) gave no evidence foradditional substrate-binding sites. A more plausible reason forsingle binding sites per 100,000-dalton monomer could be thathalf of each of the proteins diverged sufficiently to assume anew, and as yet unknown, function in the cell. In this respect,another role suggested for the synthetases is possible involve-ment in regulating the biosynthesis of their amino-acid sub-strates, or in their controlling the synthesis of their own poly-peptide chains (23). Explanations such as these could accountfor preservation of that part of the polypeptide chain that nolonger has an active site.

It is of interest that parts of the polypeptide chains ofcertain synthetases are not required for aminoacylation. Forinstance, significant portions of tryptophanyl-tRNA syn-thetase (beef pancreas) (24) methionyl-tRNA synthetase(E. coli) (25), and also possibly leucyl-tRNA synthetase(E. coli) (10, 26) can be removed by proteolysis with completeor partial retention of enzyme activity. Under conditionsthat inactivate the a2 (50,000 daltons) synthetases, proteo-

C) &

0~~~~ AnFIG. 5. Autoradiogram of the [14Cjmethionine-labeled chymotryptic peptides of leucyl-tRNA synthetase. About 10 ug of the chymo-

tryptic digest, prepared as described in Methods, was chromatographed in pyridine-butanol-acetic acid-water and subjected to electro-phoresis at pH 3.6.

Proc. Nat. Acad. Sci. USA 71 (1974)

380 Biochemistry: Waterson and Konigsberg

lytic cleavage of 100,000-dalton single-chain synthetases inE. coli extracts does not result in loss of activity, an observa-tion consistent with the hypothesis of the dual function forthe single-chain synthetases (27).The fact that leucine, valine, and isoleucine share a common

biosynthetic pathway and that their tRNA synthetases areall single-chain 100,000-dalton enzymes, coupled with otherobservations such as that isoleucyl-tRNA synthetase canactivate valine (28-30) and that proteolytically modifiedleucyl-tRNA synthetase can activate isoleucine and valinealmost as well as leucine (26), may be significant in terms ofthe evolutionary origin of these synthetases and the likeli-hood of their participation in some aspect of regulation.The possibility that all 20 of the aminoacyl-tRNA syn-

thetases have evolved from a common ancestral gene is con-sistent with several observations. Studies of various tRNAshave suggested that the structures of the recognition regionsof tRNAs have been strongly conserved, e.g., the heterolo-gous charging of transfer RNAs from E. coli and from a blue-green alga, a species separated from E. coli by three billionyears of independent evolution (31). In this respect it isplausible, although by no means proven, that constraintson the evolution of the aminoacyl-tRNA synthetases havebeen imposed by the requirements for tRNA recognition.Other observations of common features possessed by theseenzymes include: (a) virtually identical Michaelis constantsfor each of the three types of substrate (2); (b) very similarbinding affinities for tRNA suggestive of common thermo-dynamic features or at least basic similarities in the geometryof the enzyme-tRNA interactions (32) and; (c) the groupinginto two distinct molecular weight classes. All of these char-acteristics are consistent with the proposal that gene duplica-tion, fusion, and limited divergence occurred after establish-ment of a structure capable of catalyzing aminoacylation.

It is obvious that additional information concerning thestructures of the aminoacyl-tRNA synthetases is requiredbefore aspects such as the evolution of these enzymes andtheir structure-function relationships can be fully under-stood. Undoubtedly, numerous relationships among otherclasses of enzymes as well as between seemingly unrelated(33) proteins will become apparent when improved techniquesare applied to their study. It is quite likely that the radio-active mapping procedure used to study the leucyl-tRNAsynthetase can be used for similar studies of other high-molecular-weight proteins, especially those obtainable only inlimited quantity.

Note Added in Proof. Since submission of the manuscript,two reports have come to our attention that discuss internalsequence repetition in aminoacyl-tRNA synthetases. Theyare: Hartley, B. S. (1973) Ninth International Congress ofBiochemistry, Stockholm, p. 199 and Kula, M. R. (1973) FEBSLetters, 35, 299. Using different preparations of ["4C]succinicanhydride, we have obtained samples with 10- to 20-foldhigher specific gravity. Improved incorporation of label wasalso obtained when the "4C-labeled anhydride was taken upin dioxane and added in increments followed by addition ofthe unlabeled anhydride. When dealing with limited amountsof material, the use of unlabeled, succinylated carrier pro-tein is also suggested.

We thank Dr. Joel H. Shaper and Dr. Debdutta D. Roy ofthis department and Dr. Thomas C. Vanaman of Duke Uni-versity for their advice and suggestions concerning several aspectsof the mapping technique. We also thank Mr. Andrew Guttermanfor his excellent technical assistance and Dr. Joan A. Steitz forcritical reading of the manuscript. R.M.W. is a PostdoctoralFellow of the National Institutes of Health. This work wassupported by USPHS Research Grant (GM12607).

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Proc. Nat. Acad. Sci. USA 71 (1974)