Editing Errors Selection Amino Acids Protein Synthesis · errors in the selection of correct amino...

MICROBIOLOGICAL REVIEWS, Sept. 1992, p. 412-4290146-0749/92/030412-18$02.00/0Copyright X 1992, American Society for Microbiology

Editing of Errors in Selection of Amino Acidsfor Protein Synthesis

HIERONIM JAKUBOWSKI* AND EMANUEL GOLDMAN

Vol. 56, No. 3

Department of Microbiology and Molecular Genetics, New Jersey Medical School, University ofMedicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark New Jersey 07103

INTRODUCTION .............................................................................. 412THE PROBLEM.............................................................................. 413DISCOVERY OF EDITING .............................................................................. 413CONTRIBUTION OF EDITING TO SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES...........413EDITING PATHWAYS .............................................................................. 415

Misacylation-Deacylation Pathway .............................................................................. 416Deacylase Activity of Aminoacyl-tRNA Synthetases ...................................................................416Adenylate Pathway.............................................................................. 417Relative Contribution of Editing Pathways to Selectivity.............................................................417

EDITING IN LIVING CELLS .............................................................................. 418COST OF EDITING IN VIVO.............................................................................. 419RELATIONSHIP BETWEEN EDITING, SELECTIVITY, AND AMINO ACID BIOSYNTHETIC

PATHWAYS .............................................................................. 419Methionine Pathway and Homocysteine .............................................................................. 419Branched-Chain Amino Acid Pathway and Norleucine (Norvaline, a-Aminobutyrate)........................420Other Possible tRNA Misacylation Errors Due to Amino Acid Pool Imbalances....................... ....422Implications for Disorders of Amino Acid Metabolism in Humans ...................................... *.........422

MOLECULAR BASIS FOR SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES ......................423CONCLUSIONS .............................................................................. 425ACKNOWLEDGMENT .............................................................................. 425REFERENCES...............................................................................425

INTRODUCTION

The accurate processing of genetic information is funda-mental to the growth, development, and function of livingcells. As with any genetic trait, the accuracy of macromo-lecular synthesis is under evolutionary pressure. The levelsof accuracy observed in DNA replication, transcription, andtranslation are the result of a balance between the need, onthe one hand, to preserve the gene and its function and, onthe other hand, to be flexible enough to allow adaptation toa changing environment. Maximum accuracy, owing to itshigh energy cost, is never achieved by a living cell (34, 107,108, 120, 121). The existence of proofreading mutants withenhanced (10, 55, 61) or lowered (1, 2, 61) accuracy indicatesthat present-day cells operate at an optimal, i.e., intermedi-ate, level of accuracy (25) and have a reserve of proofreadingcapacity that can be selected for (84).The replication of DNA, central to the preservation of the

species, is the most accurate step in the flow of geneticinformation, with error rates in the range of 10-6 to 10-10(21, 60). These low error rates in DNA replication arepossible due to the existence of proofreading (14) and repairprocesses (115, 128). The accuracy of subsequent steps ofexpression of genetic information is several orders of mag-nitude lower than the accuracy of DNA replication. Theerror rate for transcription is about 10-4 (3, 116, 117, 135).An editing mechanism has been implicated in maintainingthe accuracy of RNA synthesis (87).The shape of the present-day genetic code is determined

* Corresponding author.

by two crucial steps of protein synthesis which are alsoimportant for accurate translation of genetic information.The first step involves the selection of a cognate amino acidand tRNA by an aminoacyl-tRNA synthetase to providecorrectly aminoacylated (also referred to as charged) tRNA.In the second step, a correct aminoacyl-tRNA is selected inthe codon-programmed ribosomal A site. The in vivo errorfrequencies in protein synthesis (substitution of one aminoacid for another) are in the range of 10-3 to 10' (seereferences 24, 90, 92, and 110 and references therein). Mosttranslational errors measured in vivo are due to mistakes inthe selection of correct aminoacyl-tRNAs by the ribosome(149). The in vitro measurements of the selectivity of ami-noacyl-tRNA synthetases indicate that the upper limit forerrors in the selection of correct amino acids for proteinsynthesis is in the range of 10' to 10-5 (66, 89, 147). Theselectivity of aminoacyl-tRNA synthetases toward tRNA iseven greater. The frequency of errors involving noncognatetRNA aminoacylation is in most cases 10-6 or lower (112,125, 126, 133, 149).The accuracy of protein synthesis depends not only on the

initial selectivity of aminoacyl-tRNA synthetases and of thecodon-programmed ribosomal A site, which are in manyinstances limited, but also on subsequent editing of errors inthe initial selection. The editing in amino acid selection forprotein synthesis by an aminoacyl-tRNA synthetase was thefirst proofreading process to be discovered in the flow ofgenetic information (5, 105). The proofreading of an aminoacid has recently been shown to be an important in vivoprocess which prevents incorporation of a wrong amino acidinto tRNA and protein in both Escherichia coli (75) andSaccharomyces cerevisiae (76). Editing was also postulated

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EDITING OF ERRORS IN AMINO ACID SELECTION 413

(65, 103) and eventually found (119, 138, 139) to occur duringribosomal selection of aminoacyl-tRNA. This selection isalso influenced by the relA locus in bacteria (reviewed inreferences 56 and 110; see also reference 74). There is alsoevidence for editing of an aberrant polypeptide on theribosome immediately after peptide bond formation, whenan incorrect aminoacyl-tRNA has donated the wrong aminoacid to the growing peptide chain (95). Since at least 95% ofmetabolic energy is consumed for protein synthesis in E. coli(69), and perhaps not much less in S. cerevisiae, the editingof errors in translation can be quite costly to the cell (75, 76).

This review will focus on the nature and magnitude oferrors made by aminoacyl-tRNA synthetases during aminoacid selection, alternative pathways for editing of theseerrors, the magnitude of the contribution of editing to theselectivity of aminoacyl-tRNA synthetases, the energy costof editing, and the in vivo significance of editing. We willalso examine the relationship between editing, selectivity,and amino acid biosynthetic pathways in living cells. Impli-cations of limited selectivity of synthetases for disorders ofamino acid metabolism in humans will be discussed. Selec-tivity of aminoacyl-tRNA synthetases will be examined atthe molecular level. For the purposes of this review, theterms "editing" and "proofreading" refer to the ability ofaminoacyl-tRNA synthetases to correct or prevent incorrectaminoacylation of tRNA.

THE PROBLEM

Aminoacylation of tRNA is a two-step reaction. In the firststep (equation 1), which is generally referred to as aminoacid activation, an amino acid (AA) is activated to formenzyme (E)-bound aminoacyl adenylate.

E + AA + ATP ; E AA-AMP + PPi (1)In the second step (equation 2), the amino acid is transferredfrom the adenylate to tRNA.

E AA-AMP + tRNA"-A E + AA-tRNA'` + AMP (2)The accuracy of these reactions depends on the ability of

an aminoacyl-tRNA synthetase to select 1 of 20 proteinamino acids (and a few nonprotein ones such as homocys-teine, homoserine, or ornithine, which are intermediates inamino acid biosynthetic pathways) and 1 cognate tRNAfamily out of 20 tRNA families. Selection of tRNAs is not amajor problem since these are relatively large moleculeswith adequate scope for distinctive structural variation. Forexample, the selectivity of E. coli isoleucyl-tRNA syn-thetase for tRNAI'le is 4 x 107 against tRNAP ie and 2 x 107against tRNAf"et (96, 148, 149), and many other synthetasesexhibit similar selectivities (23, 112, 126). The molecularbasis for such discrimination, aptly called tRNA identity, isnow known in considerable detail for several tRNAs (re-viewed in references 104, 122, 125, 133, and 150).

In contrast, amino acids are small molecules; some ofthem are so similar in structure that aminoacyl-tRNA syn-thetases cannot initially distinguish between them with ade-quate selectivity and often mistakenly activate them to formenzyme-bound noncognate aminoacyl adenylates. This isgenerally referred to as misactivation. The problem was firstrecognized by Pauling (111), well before the basic frameworkfor protein synthesis had been established. He calculated anerror rate of about 1 in 5 for glycine replacing alanine, valinereplacing isoleucine, and so on. Pauling's calculation wasbased on a value of about 1 kcal/mol (4.2 kJ/mol) for thehydrophobic binding energy of a methylene group, the figure

usually found by physical-chemical methods from partition-ing between hydrophilic and hydrophobic solvents. Al-though subsequent enzymatic measurements indicatedtighter binding to proteins than to hydrophobic solvents (3.4kcal/mol [14.2 kJ/mol] per methylene group [44]), the initialdiscrimination between amino acids differing just by onemethylene group in their structures cannot be better than afactor of -200, as found for many synthetases in equation 1.This is still not adequate to account for the ability ofaminoacyl-tRNA synthetases to distinguish such closelyrelated amino acids with an overall discrimination factor of104 to 105 (66, 147). In these cases, paradoxically, the overalltRNA aminoacylation reaction is more accurate than thepartial activation reaction.

DISCOVERY OF EDITING

The solution to the problem of how the overall reactioncan be more accurate than the partial reaction came fromstudies of enzyme-bound aminoacyl adenylates formed byisoleucyl-tRNA synthetase (IleRS) (5, 105). The enzymeforms relatively stable cognate E . Ile-AMP and noncog-nate EIe Val-AMP complexes in the presence of ATP andeither isoleucine or valine. Whereas the cognateEile. Ile-AMP complex reacts with tRNAile to form Ile-tRNAile, the noncognate EIle Val-AMP complex is quan-titatively hydrolyzed in the presence of tRNAile and noVal-tRNAile is formed. Thus, in the presence of tRNAI'e,ATP, and valine, the isoleucyl-tRNA synthetase acts as anATP pyrophosphatase, hydrolyzing ATP to AMP (5).

Theoretical analysis of the specificity problem in macro-molecular biosynthesis led to Hopfield's proposal of a ki-netic proofreading scheme in which intermediate complexeshave access to a rejection path in addition to the main pathleading to the final product (65, 147). A similar proofreadingscheme was independently proposed by Ninio (103). In thekinetic proofreading scheme, discrimination occurs at twosteps: first during initial binding and then during editing ofthe intermediate. A cognate amino acid will flow through themain path, and a noncognate amino acid will be discardedthrough an irreversible (driven by hydrolysis of ATP) editingpath. A diagnostic feature of this editing is ATP hydrolysis inthe presence of a noncognate amino acid.The experimental discovery of editing did not demonstrate

a specific mechanism. Also, the kinetic proofreading schemedid not propose any specific mechanism of editing (65), asemphasized by Yamane and Hopfield (147). Experimentalproof of specific pathways of editing came later (37, 41, 70,72, 77, 88, 89).

CONTRIBUTION OF EDITING TO SELECTIVITY OFAMINOACYL-tRNA SYNTHETASES

The initial recognition of amino acids by an aminoacyl-tRNA synthetase can be conveniently studied by measuringATP-PPi exchange in equation 1. Although the measure-ments are straightforward, they may lead to artifactualresults if care is not taken to quantitatively control the purityof amino acid and aminoacyl-tRNA synthetase preparations.In studying errors in amino acid activation, it is clearlyimportant to use amino acid preparations of extreme purityso that error rates as low as 10-5 can be unambiguouslydetected. Commercial samples of amino acids often containsignificant trace quantities of contaminants, which in somecases is as high as 0.5% (for tyrosine contamination inphenylalanine preparations) (88). Extremely poor discrimi-

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414 JAKUBOWSKI AND GOLDMAN

nation of valyl-tRNA synthetase against isoleucine and ofphenylalanyl-tRNA synthetase against tyrosine, which wasthought to implicate the need for an editing mechanism (68),turned out to be due to the presence of cognate amino acid asa contaminant in preparations of noncognate amino acids(40, 88). Apparent misactivation of isoleucine and valine bymethionyl-tRNA synthetase was in fact due to the presenceof 0.2% methionine in commercial isoleucine and valinepreparations (39). Fortunately, when recognized, theseproblems can be adequately controlled and will not obscuregenuine misactivations and requirements for editing mecha-nisms.

Editing of misactivated amino acids can be inferred frommeasurements of an apparent ATP pyrophosphatase activityof an aminoacyl-tRNA synthetase:E + AA + ATP + tRNA # E. AA-AMP. tRNA + PPj (3)

E. tRNA + AA + AMP

Although equation 3 implies participation of tRNA in theediting reaction, in many cases efficient editing occurs in theabsence oftRNA (77). This point will be developed further in

the section on editing pathways (below).The notion of limited specificity of aminoacyl-tRNA syn-

thetases in initial selection of amino acids and of essentiallyabsolute specificity in tRNA aminoacylation originated in1961 when Bergman et al. described misactivation of valineby E. coli isoleucyl-tRNA synthetase and of threonine by E.coli valyl-tRNA synthetase (7). The spectra of misactiva-tions by these two synthetases were considerably expandedin subsequent years (68, 72, 77), and new misactivationswere discovered for alanyl- (142), leucyl- (27), methionyl-(39, 109), and phenylalanyl- (68, 88) tRNA synthetases;valyl- and isoleucyl-tRNA synthetases hold the distinction ofbeing the most promiscuous among the family of syn-thetases. In all cases, the original notion of an extremelyhigh specificity of an aminoacyl-tRNA synthetase in thetRNA aminoacylation reaction has been confirmed. In gen-eral, most misactivations occur at a relatively high frequencyand require subsequent correction by editing. Some misac-tivations, although measurable, are so inefficient that noediting is needed to remove the very infrequent errors. Also,the editing function is not and does not have to be universal.Some aminoacyl-tRNA synthetases, such as cysteinyl- (38)and tyrosyl- (44) tRNA synthetase, are so selective in theinitial activation reaction that there is no need, and in fact noevidence, for an editing mechanism.The data on misactivation and editing of noncognate

amino acids by E. coli aminoacyl-tRNA synthetases, com-piled in Table 1, include only studies in which amino acidpurity has been documented and both misactivation andediting rates have been measured. Overall selectivitiesagainst noncognate amino acids are also given. The data aresummarized below.

Isoleucyl-tRNA synthetase activates, in addition to itscognate substrate isoleucine, seven other naturally occurringamino acids: isoleucine >> valine > homocysteine > cys-

teine ca-aminobutyrate - threonine > homoserine >>alanine. All of the misactivated amino acids are edited.Editing contributes at least a factor of 5 (for homoserine) up

to a factor of 100 (for cysteine) to the selectivity of isoleucyl-tRNA synthetase against noncognate amino acids.Given that valine is just one methylene group smaller than

isoleucine, it is not surprising that isoleucyl- and valyl-tRNAsynthetases share the same amino acid substrates. Thus,valyl-tRNA synthetase will activate, in addition to valine,

TABLE 1. Misactivation, editing, and selectivity of E. coliaminoacyl-tRNA synthetases against noncognate amino acids

Synthetase and Relative Relative rate of Selectamino acid rate of editin Slctviy

activation0 edimIsoleucyl-tRNA

synthetasedIsoleucine 1 1 (0.014 s-1) 1Valine 0.007 43 6,000wThreoninef 0.0002 23 115,000ca-Aminobutyratef 0.0003 24 80,000Cysteine 0.0003 100 330,000Homocysteine 0.0025 76 30,000Homoserinef 0.00004 5 125,000Alaninef 1 x 10-6 8.5 8.5 x 106

Valyl-tRNA synthetasedValine 1 1 (0.02 s-') 1Threonine 0.004 180 45,000wca-Aminobutyrate 0.005 58 12,000Cysteine 0.001 90 90,000Serine 0.00016 10 62,000Alanine 0.000i 48 480,000Homocysteine 0.0002Homoserinef 0.00014 1.6 11,000Isoleucineg 0.000017 1 60,000

Leucyl-tRNAsynthetaseh

Leucine 1 1 (0.08 s-1)i 1Homocysteine 0.0083 25 3,000

Methionyl-tRNAsynthetased

Methionine 1 1 (0.04 s-1)f 1Homocysteine 0.0054 60 11,000Norleucinef 0.005 5 1,000Ethioninef 0.035 7 200

Alanyl-tRNAsynthetase'

Alanine 1 1 (0.04 s-1) 1Glycine 0.004 11.5 2,900Serine 0.002 23 11,500Cysteine 0.00008 > 12,500a Relative values of kcat/Km in the ATP-PP1 exchange reaction. The recip-

rocal of these relative values is defined as the initial selectivity.6 Relative values of kcat in the ATP pyrophosphatase reaction. Absolute kC,tvalues for cognate reactions are given in parentheses.

c Ratio of the relative rate of editing to the relative rate of activation.d Reference 77.e Similar selectivities obtained by measurements of ATP consumption per

mol of noncognate aminoacyl-tRNA formed in the presence of elongationfactor Tu (66, 147).f Reference 76a.g Reference 40.h Reference 27.' Rate constant for enzymatic deacylation of Leu-tRNAJu (71).Reference 142.

eight other natural amino acids: valine > > o-aminobutyrate- threonine > cysteine > alanine - serine - homoserine -

homocysteine > isoleucine. Editing improves selectivityagainst noncognate amino acids which are the most effi-ciently activated (oa-aminobutyrate, threonine, cysteine) bytwo orders of magnitude. The initial selectivity of valyl-tRNA synthetase against isoleucine, homoserine, and homo-cysteine is adequate (-104), and there is no evidence fortheir editing; despite similar initial selectivities against ala-

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nine and serine, these two amino acids are neverthelessedited.Homocysteine, an immediate precursor of methionine in

all organisms from bacteria to humans, is the most fre-quently misactivated amino acid. Four aminoacyl-tRNAsynthetases misactivate homocysteine. These include, notsurprisingly, methionyl-tRNA synthetase (109), isoleucyl-and valyl-tRNA synthetases (77) (mentioned above), andleucyl-tRNA synthetase (27). Misactivated homocysteine istransformed into homocysteine thiolactone during editing bythe four synthetases in vitro (27, 77). However, homocys-teine has also been reported to be stably aminoacylated totRNAIeU and tRNAVal by leucyl- (27) and valyl- (28) tRNAsynthetases, respectively, although this has not been quan-titated. There is no mischarging of tRNAMet during editing ofhomocysteine by methionyl-tRNA synthetase (39). Misacti-vation and editing of homocysteine, as a result of its uniquechemistry (77), have also been studied in vivo (see Editing inLiving Cells, below).Alanyl-tRNA synthetase, in addition to alanine, activates

two other natural amino acids: glycine and, surprisingly,serine (142). The initial selectivity of 250 to 500 againstglycine and serine is further improved by a factor of 10 to 20by subsequent editing.The initial selectivity of yeast phenylalanyl-tRNA syn-

thetase against tyrosine is about 2,000 (88). Editing improvesthe selectivity of the enzyme by another factor of 10 (89).The following conclusions can be drawn from this survey.

(i) The selectivity of aminoacyl-tRNA synthetases is betterthan the error rate in protein synthesis. (ii) The initialselectivity against noncognate naturally occurring aminoacids may be anywhere from 120- to 10,000-fold or better andsets an upper limit for the overall selectivity. (iii) Editingcontributes at least a factor of 10 to 100 to the selectivity ofan aminoacyl-tRNA synthetase and sets a lower limit for theselectivity. The most efficient misactivations are also themost efficiently edited, and the least efficient misactivationsare the least efficiently edited. (iv) Finally, the figures givenin the last column of Table 1 show that selectivity, albeitvery high, is limited: the initial selectivity component ofoverall selectivity is variable and depends on concentrationsof cognate and noncognate amino acids. This may lead tolower selectivity during unbalanced conditions when oneamino acid is in great excess over another, which is observedin vivo (see Relationship between Editing, Selectivity, andAmino Acid Biosynthetic Pathways, below).There have been indirect investigations of the discrimina-

would be difficult to achieve even in the presence of elonga-tion factor Tu to protect putative mischarged tRNA againstdeacylation (66, 147). However, the authors do not presentevidence confirming that the radioactive material incorpo-rated into tRNA is in fact noncognate amino acid. Further,the description in their original Materials and Methods isconfusing because they state that no incorporation of non-cognate amino acids above background was observed (51).In many cases the reported Km values for noncognate aminoacids were equal to or lower than that for the cognate aminoacid. This implies that the synthetases would be severelyinhibited by noncognate amino acids, which is apparentlynot the case. Moreover, the authors appear to have mea-sured, under one set of experimental conditions, amino-acylation rates differing as much as 4 x 104-fold (for isoleu-cyl-tRNA synthetase) or 5 x 105-fold (for tyrosyl-tRNAsynthetase), which does not seem to be feasible.

In the absence of any supporting evidence, it is difficult tojustify quantitative conclusions made from the data obtainedwith modified tRNAs. Since chemical modifications of the 3'terminus of tRNA influence the mechanism of tRNA ami-noacylation (46), the kinetic data obtained with modifiedtRNAs have different meaning from data obtained withintact tRNAs. This could account for conflicting conclusionsreached by the same group with differently 3'-modifiedtRNAs (50, 51). Moreover, the discrimination factors calcu-lated from measurements with various 3'-modified tRNAsappear to be artifactual at least for E. coli isoleucyl-tRNAsynthetase. For example, the initial selectivities calculatedby Freist et al. (52) for valine, alanine, cysteine, and threo-nine are 53, 430, 18, and 85, respectively; however, theinitial selectivities obtained from direct measurements inwell-defined systems (see Table 1, footnote a) are 143 (Val[45, 77, 91]), >2 x 104 (Ala [45]), 3,330 (Cys [77]), and 5,000(Thr [76a]). Also, the initial selectivities of isoleucyl-tRNAsynthetase against leucine and glycine, calculated to be 20and 1,970, respectively, by Freist et al. (52), differ by at leastan order of magnitude from the values of 640 and >20,000,which can be calculated from classical direct physical-chemical measurements with the same enzyme (45).

EDITING PATHWAYS

There are two intermediates on the pathway to aminoacyl-tRNA. The first is an enzyme-bound aminoacyl adenylate,and the second is an enzyme-bound aminoacyl-tRNA (equa-tion 4).

E + AA + ATP + tRNA ; E AA-AMP. tRNA ; E. AA-tRNA z E + AA-tRNAa lb

E- tRNA + AA + AMP

tion against all 19 noncognate amino acids by yeast iso-leucyl- (51, 52), valyl- (47), tyrosyl- (48), and arginyl- (53)tRNA synthetases and by E. coli isoleucyl-tRNA synthetase(51, 52). The discrimination factors were calculated frommeasurements of aminoacylation of native and 3'-modifiedtRNA with 20 protein amino acids and from accompanyingAMP production. Remarkably, the authors appear to havemeasured, under standard conditions, the formation of mis-acylated tRNAs with all protein amino acids, a feat that

E. tRNA + AA

Both of these intermediates can be proofread, indicated byside reactions a and b in equation 4. Although it wasrecognized by Baldwin and Berg (5) that editing duringrejection of valine by the isoleucyl-tRNA synthetase couldoccur by the hydrolysis of EI' Val-AMP or could involvetransient formation of incorrect Val-tRNAIle followed by itshydrolysis by the enzyme, the mechanism proved to bedifficult to identify unequivocally (31). However, there areseveral clear-cut cases in which either the adenylate (side

(4)

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reaction a in equation 4) or the misacylation-deacylation(side reaction b in equation 4) pathway has been directlydemonstrated.

Misacylation-Deacylation PathwayFollowing the discovery of editing in tRNA aminoacyla-

tion, a substantial amount of circumstantial evidence sup-porting the misacylation-deacylation pathway for the rejec-tion of valine by isoleucyl-tRNA synthetase accumulated.For example, it was found that synthetases are weak deacy-lases toward cognate aminoacyl-tRNA but the deacylaseactivity is higher with some mischarged tRNAs (26, 124,148). Also, 3'-modified tRNAIl' can be aminoacylated withvaline by isoleucyl-tRNA synthetase (145). However, defin-itive proof was elusive and has never been obtained with thissystem. In fact, even fast kinetic approaches failed to detectVal-tRNAI'e in reaction mixtures containing substrateamounts of isoleucyl-tRNA synthetase, ATP, valine, andtRNAI'le (31). In addition, the kinetics of deacylation ofVal-tRNAI'le (which can be made under artificial conditions)by the enzyme were not consistent with the proposedmisacylation-deacylation pathway (31).The proof of the misacylation-deacylation pathway came

from studies of misactivation of threonine and a-aminobu-tyrate by valyl-tRNA synthetases from Bacillus stearother-mophilus, E. coli, S. cerevisiae, and lupin seeds by using arapid quenching apparatus to trap and observe the mis-charged tRNAVal (33, 37, 41, 77). Threonine is isosteric withvaline and is readily activated by valyl-tRNA synthetases toform the enzyme-bound threonyl adenylate. On mixingEVal Thr-AMP with tRNAVal in a rapid-quenching appara-tus, Thr-tRNAVal is detected transiently at a maximum of22% at 25 ms and then disappears after 150 ms (41).Using a similar fast kinetic approach with yeast phenyl-

alanyl-tRNA synthetase, Lin et al. (89) have shown that aminor fraction (4 to 5%) of misactivated tyrosine is trans-ferred to tRNAPhe but the resulting Tyr-tRNAPh, is veryrapidly deacylated. By monitoring the fate of Tyr-AMP,these authors also demonstrated that the major part of themisactivated tyrosine is edited by fast hydrolysis of enzyme-bound tyrosyl adenylate.

In an attempt to rationalize observed misactivations in onesimple framework, a "double-sieve" model was proposed(32, 33, 40). The experimental basis of the model came frommeasurements of editing of noncognate amino acids byvalyl-tRNA synthetase. The essence of the double-sievemodel is the proposal of the existence of two sites: onesynthetic site and one hydrolytic site. The synthetic siteexcludes amino acids larger than the cognate one by stericrepulsion. The smaller (and isosteric) amino acids are acti-vated by the enzyme at progressively lower rates as theirstructures differ more and more from that of the cognatesubstrate. The hydrolytic site edits the products of misacti-vation of smaller (and naturally occurring isosteric) sub-strates. The model was formulated at a time when a limitednumber of misactivations and only one editing pathway wereknown (32) and was later modified (33, 35, 40, 142). How-ever, the number of exceptions outweigh the number ofcases fitting the model. The double-sieve model breaks downfor MetRS, which misactivates and edits noncognate aminoacids that are either larger (e.g., ethionine) or smaller (e.g.,homocysteine) than the cognate substrate, methionine (Ta-ble 1). In addition, efficient editing of homocysteine byMetRS is achieved by just one active site and by a differentmechanism from the proposed double sieve (77, 142). Simi-

larly, one active site can account for editing of homocys-teine, cysteine (77), and possibly valine (31) by IleRS.Although editing of threonine and a-aminobutyrate byValRS fit the model by definition, editing of alanine by theenzyme may involve only one active site (31, 77). Editing oftyrosine (89) and serine (142) by PheRS and AlaRS, respec-tively, also does not obey the double-sieve mechanism sincethe noncognate amino acids are larger (by an -OH group)than the cognate substrate, yet they both are misactivatedand then efficiently edited. In addition, a multistep mecha-nism of editing of tyrosine by PheRS (88, 89) does not fit thesimple idea of a double sieve.

Deacylase Activity of Aminoacyl-tRNA Synthetases

The two aminoacyl-tRNA synthetases which edit noncog-nate amino acids through the misacylation-deacylation path-way also exhibit a weak deacylase activity toward theirrespective cognate aminoacyl-tRNAs. Moreover, tyrosyl-tRNA synthetase, which is extremely selective in the initialrecognition of its cognate amino acid substrate, tyrosine(68), and does not possess any editing mechanism (44), alsodoes not exhibit any deacylase activity toward Tyr-tRNATYr(99, 145). This may suggest that weak deacylase activity isindicative of the existence of a misacylation-deacylationpathway in amino acid selection by a synthetase. However,several other aminoacyl-tRNA synthetases which either areunlikely to require an editing function, such as seryl-tRNAsynthetase (145), or are known not to edit through themisacylation-deacylation pathway, such as methionyl-tRNAsynthetase (77), or not to edit at all, such as cysteinyl-tRNAsynthetase (38), still possess a weak deacylase activitytoward cognate aminoacyl-tRNA. Isoleucyl-tRNA synthe-tase exhibits a weak deacylase activity toward Ile-tRNAI1e(k = 0.8 min-1 [124]) and a strong deacylase activity towardVal-tRNAI'le (k = 10 s-1 [26, 31]), but Val-tRNAIle does notform during editing of valine by isoleucyl-tRNA synthetase(31). Valyl-tRNA synthetase possesses a weak deacylaseactivity toward Val-tRNAVal (k = 0.02 s-1 [Table 1]) andedits misactivated threonine via deacylation of Thr-tRNAVal(k = 40 s-1 [37]), yet it has been reported to form stablehomocysteinyl-tRNAVal (28). Leucyl-tRNA synthetase isknown to possess an efficient deacylase activity towardLeu-tRNAJU (k = 0.08 s-1 [71]), but nevertheless it formsmischarged homocysteinyl-tRNAIeu which does not seemto be deacylated by the enzyme (27). A half-life of 15 mingiven for deacylation of homocysteinyl-tRNAIeu in thepresence of leucyl-tRNA synthetase (27) is approximatelywhat can be expected for nonenzymatic deacylation of themischarged tRNA. Thus, although in some cases a weakdeacylase activity is associated with a specific editing func-tion, the general significance of this activity of aminoacyl-tRNA synthetases toward cognate aminoacyl-tRNA is notclear.A neighboring hydroxyl group of the terminal adenosine is

required for enzymatic deacylation of aminoacyl-tRNA.Chemically modified tRNAI'le whose terminal adenosine hasbeen replaced with 3'-deoxyadenosine still accepts aminoacids, but the aminoacyl-tRNAIle_C_C-3'dA is not de-acylated by isoleucyl-tRNA synthetase (145). This suggestedthat a 2'-OH is an acceptor site for amino acids and that a3'-OH is required for deacylation. The requirement for the3'-OH was explained by proposing that it activates a watermolecule that participates in hydrolysis of the adjacent bondof aminoacyl-tRNA (chemical proofreading [28, 67, 68,145]). Alternatively, the 3'-OH may be required so that the

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amino acid can migrate onto it and become accessible to thehydrolytic site (hydrolytic editing [41]). This notion of twosites was subsequently elaborated into the double-sievemodel (32, 40) (see preceding section). Although tRNA-C-C-2'dA was available to test those proposals, the controlexperiments were not done until 13 years later, when Freistand Sternbach showed that aminoacyl esters of tRNA"'e-C-C-2'dA are as resistant to enzymatic deacylation, as are theaminoacyl esters of tRNA'le-C-C-3'dA, implying that theonly requirement for deacylation is a free cis-OH, regardlessof the position of the aminoacylated hydroxyl group (49).

Adenylate PathwayInitial studies of editing in tRNA aminoacylation were

greatly influenced by the notion that tRNA directly contrib-utes to the specificity of an aminoacyl-tRNA synthetase bybeing first misacylated and then deacylated to remove anerror. This effectively delayed for several years the discov-ery of a much more effective and widespread editing path-way in which an enzyme-bound noncognate aminoacyl ade-nylate is rejected before it can misacylate tRNA; this isknown as the adenylate pathway.The evidence for the adenylate editing pathway came from

studies with valyl-tRNA synthetase. Valyl-tRNA synthetaseforms enzyme-bound adenylates with several noncognateamino acids. Enzyme-bound noncognate aminoacyl adenyl-ates can be isolated by nitrocellulose disk filtration (70, 72).In contrast to enzyme-bound valyl adenylate, which is verystable, enzyme-bound noncognate aminoacyl adenylates arerapidly hydrolyzed and disappear within less than 1 min (70).In effect, valyl-tRNA synthetase acts as an ATP pyrophos-phatase in the presence of threonine, ot-aminobutyrate,cysteine, alanine, and serine but in the absence of tRNA(72). Subsequently, very efficient tRNA-independent editingwas discovered with isoleucyl-tRNA synthetase (rejectingcysteine and homocysteine) and methionyl-tRNA synthetase(rejecting homocysteine) (77, 132). The unique feature of theediting of homocysteine is the formation of homocysteinethiolactone (equation 5) (77):

.+

NH3 NH3

=0

S

'AMPSH

+ AMP

There are three distinct mechanisms by which noncognateamino acids are edited through the adenylate pathway.Examples of each, as well as the relative importance ofmisacylation-deacylation and adenylate pathways, are dis-cussed in the following section.

Relative Contribution of Editing Pathways to SelectivityEditing of errors in amino acid selection by an aminoacyl-

tRNA synthetase can take place by the four routes shown inFig. 1.

(i) The first route is via k1, the dissociation of an enzyme-bound aminoacyl adenylate to give free aminoacyl adenylatewhich hydrolyzes in solution (70, 72, 77). This route maycontribute to specificity in cases in which the edited noncog-nate adenylate can be scavenged by its corresponding ami-noacyl-tRNA synthetase, which might happen in vivo. The

E *AA-AMP * tRNA

-tRNA

E + AA + ATP - PP, + E * AA-AMP AMP + E * AA-tRNA --protein

1k, jk2 k3 l k4

E + AA-AMP - E + AA* + AMP

FIG. 1. Possible routes for the editing of errors in amino acidselection for protein synthesis. Routes k1, k2, and k3 represent threedistinct adenylate pathways; k4 is the final step in the misacylation-deacylation pathway. Abbreviations: E, enzyme; AA, amino acid;AA*, edited amino acid.

best examples of the k1 route are isoleucyl-tRNA synthetaseediting cysteine (77) and phenylalanyl-tRNA synthetase ed-iting tyrosine, which is also edited by the k3 and k4 routes(89).

(ii) The second is via k2, the tRNA-independent deacyla-tion of an enzyme-bound aminoacyl adenylate (27, 70, 72,77). Homocysteine misactivated by isoleucyl- and methio-nyl-tRNA synthetases is efficiently edited by this route, withthe formation of homocysteine thiolactone (equation 5).tRNA does not affect the formation of the thiolactone duringediting (77), and there is also no evidence for transientmischarging of tRNAMet with homocysteine (39). The k2route has been shown to exist in vivo (see the followingsection).

(iii) The third is via k3, the tRNA-dependent hydrolysis ofan enzyme-bound aminoacyl adenylate without transientmischarging oftRNA (31, 72). The most publicized (althoughnot always correctly) textbook case of editing of misacti-vated valine by isoleucyl-tRNA synthetase is exclusively bythe k3 route. Incorrectly formed valyl adenylate is hydro-lyzed by isoleucyl-tRNA synthetase in the presence oftRNAI'le (5), but there is no transient formation of mis-charged tRNAI'e (31). There are two other well-documentedcases in which efficient editing requires tRNA but the majorfraction of the misactivated amino acid is edited by hydrol-ysis of noncognate aminoacyl adenylate with only a minorfraction (4%) of misacylated tRNA formed transiently: edit-ing of threonine by plant valyl-tRNA synthetase (72, 77) andediting of tyrosine by yeast phenylalanyl-tRNA synthetase(89). In general, tRNA dependence does not necessarilymean that editing involves transient misacylation of tRNA ashas frequently been assumed (35); an allosteric change in thesynthetase upon binding of tRNA could account for stimu-lation of the editing. In fact, in some cases enzymatichydrolysis of noncognate aminoacyl adenylates duirng edit-ing is weakly stimulated by tRNA which has been deprivedof its ability to accept amino acids by periodate oxidation(72).

(iv) The fourth is via k4, the deacylation of an enzyme-bound misacylated tRNA (26, 37, 41, 77, 89, 145, 148). Thereare only three examples thus far of editing through the k4route: editing of threonine and a-aminobutyrate by valyl-tRNA synthetases (reviewed in reference 35) and of tyrosineby phenylalanyl-tRNA synthetase (89). In some cases, 3'-modified tRNAs were shown to be stably misacylated (67,68, 145), which suggested but did not prove the misacylation-deacylation pathway. Although it was originally described asbeing edited exclusively through the k4 pathway, a signifi-

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cant fraction of threonine misactivated by valyl-tRNA syn-thetase may also be edited by the k3 pathway. In fact, Lin etal. (89) have shown that the data of Fersht and Kaethner (41)better fit a model in which a major fraction of Thr-AMP ishydrolyzed before transfer to tRNAVal.

There is one problem with the k4 pathway which mayaccount for its limited use by aminoacyl-tRNA synthetases:its contribution to selectivity can be seriously limited by thedissociation of aminoacyl-tRNA from the synthetase. Afterdissociation, the free aminoacyl-tRNA would be protectedby elongation factor Tu against any further deacylation (66,74, 99, 147) and will insert its amino acid, regardless ofwhether it is correct, into protein at the positions specifiedby the anticodon-codon interaction (18). For the k4 pathwayto contribute a factor of 10 to the selectivity would requirethat the deacylation rate constant (kh) be ninefold higherthan the dissociation rate constant (kd). However, availabledata indicate that kd and kh are of rather similar magnitude.For example, with yeast phenylalanyl-tRNA synthetase, kd= 40 s-5 (83) and kh = 62 s-' (89). The minimum values forkd estimated from the molecular activity of isoleucyl- andvalyl-tRNA synthetases in vivo are 12.4 and 26.6 s-1,respectively (78), not much different from the respective khvalues of 10 s- (31) and 40 s-1 (37, 41). From competitiveinhibition of enzymatic deacylation of Thr-tRNAVal bytRNAVal and from the fact that the kinetics of hydrolysisextrapolate back to 5.7% (no excess tRNAVal) and 3.6%(excess tRNAVal) misacylated Thr-tRNAVal at zero time(41), one can calculate that 63% of Thr-tRNAVal dissociatesfrom the enzyme before hydrolysis. Thus, the deacylation ofaminoacyl-tRNA contributes only a factor of about 2 toselectivity.

EDMTNG IN LIVING CELLS

There is no obvious straightforward way to study editingin vivo. It has even been stated that direct measurements ofexcess energy dissipation for proofreading in vivo is nottechnically feasible (69). The question of the in vivo signifi-cance of error-correcting processes would have remainedunanswered if nature had not provided a useful feature toapproach this question in living cells. In particular, severalaminoacyl-tRNA synthetases edit misactivated homocys-teine by the adenylate pathway with the formation of aunique compound, homocysteine thiolactone (equation 5).This feature of the homocysteine-editing reaction provided ameans to assay for editing in vivo by looking for a specialchemical product of editing, i.e., homocysteine thiolactone.Indeed, the thiolactone has been detected in both E. coli (75)and S. cerevisiae (76). Although several aminoacyl-tRNAsynthetases (MetRS, IleRS, ValRS, and LeuRS) edit homo-cysteine by converting it into the thiolactone in vitro, onlyone synthetase, i.e., methionyl-tRNA synthetase, is in-volved in the thiolactone synthesis in living cells. In prevent-ing errors, it is important that IleRS, ValRS, and LeuRS donot interact with homocysteine in vivo, since at least LeuRSand ValRS form homocysteinyl-tRNAIeu (27) and homocys-teinyl-tRNAVal (28), respectively, in vitro.The observations that establish the importance of error-

editing mechanisms in living cells are summarized below. (i)Homocysteine thiolactone is a major component of sulfuramino acid pools in E. coli and S. cerevisiae. Most probably,the thiolactone is also present in mammalian cells (134),although this should be independently confirmed. (ii) E. coliand S. cerevisiae mutants that are expected to accumulatehomocysteine synthesize massive amounts of the thiolac-

NH3

=0

S

+NH3

=0

0

NH3

H

Homocysteine Homoserine Ornithinethioloctone loctone 6-IactomFIG. 2. Cyclic forms of some amino acids arising through car-

boxyl group activation. Homoserine lactone is also referred to asa-aminobutyryl lactone. Ornithine B-lactam is also referred to as3-amino-2-piperidone.

tone. This establishes a substrate-product relationship be-tween homocysteine and the thiolactone in vivo. (iii) Me-thionyl-tRNA synthetase mutants defective in themethionine-binding site of the enzyme, in both E. coli and S.cerevisiae, are also defective in homocysteine thiolactonesynthesis. In addition, overproduction of methionyl-tRNAsynthetase in E. coli and S. cerevisiae leads to proportionaloverproduction of the thiolactone. This demonstrates thatmethionyl-tRNA synthetase is responsible for most if not allof the thiolactone synthesis in both E. coli and S. cerevisiae.In fact, there is no evidence for participation of any otheraminoacyl-tRNA synthetases or other enzymes in the thio-lactone synthesis in vivo. (iv) Synthesis of homocysteinethiolactone in vivo is inhibited by methionine and norleucinebut not by any other amino acid; this behavior was previ-ously described for the in vitro synthesis of the thiolactoneby bacterial methionyl-tRNA synthetases (77). This alsoexcludes the participation of isoleucyl-, valyl-, and leucyl-tRNA synthetases in the thiolactone synthesis in vivo. Thatisoleucyl-, valyl-, and leucyl-tRNA synthetases do not makethe thiolactone in vivo is further supported by the observa-tion that ilv mutants unable to synthesize isoleucine, valine,and leucine do not make more thiolactone (as one might haveexpected from the in vitro data) than wild-type cells. How-ever puzzling, these observations indicate that some specificediting reactions described in vitro may not, in fact, occur incells (see Relationship between Editing, Selectivity, andAmino Acid Biosynthetic Pathways, below, for further dis-cussion).The in vivo studies of editing in amino acid selection for

protein synthesis were possible because of the distinctnature of the by-product of editing by MetRS, homocysteinethiolactone, which can, by standard procedures, be easilyseparated from homocysteine and other sulfur compoundspresent in the cell. There are several other cases in whichmisactivation and editing can possibly lead to unique chem-ical products which could be exploited in vivo. For instance,homoserine, a precursor of methionine in microorganisms, ismisactivated by isoleucyl- and valyl-tRNA synthetases (Ta-ble 1) and subsequently edited by cyclization of homoseryladenylate to the lactone of homoserine, a-aminobutyryllactone (Fig. 2) (76a). Homoserine is a significant componentof amino acid pools, at least in S. cerevisiae, and homo-serine-overproducing yeast mutants (BOR1) exist (130),which makes S. cerevisiae a plausible biological system forin vivo studies of editing by other aminoacyl-tRNA syn-thetases. Cyclization is expected to also occur with theadenylates of ornithine, aspartate, glutamate, and lysine. Itis not known whether these amino acids are misactivatedand edited, but assaying for cyclic forms of these amino acidin vivo may provide a means of establishing this point and offurther extending in vivo studies of editing. The cyclic

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B-lactam of omithine, 3-amino-2-piperidone (Fig. 2) (30,106), is found in urine and, at lower concentrations, inplasma from patients with gyrate atrophy and other condi-tions associated with hyperomithinemia (reviewed in refer-ence 144). However, the mechanism of its formation fromornithine is not known.

COST OF EDITING IN VIVO

Some proofreading models postulate that more than onemolecule of ATP must be hydrolyzed for each molecule ofaminoacyl-tRNA formed in order to maintain the high selec-tivity of aminoacyl-tRNA synthetases (65). Although this istrue for charging of noncognate amino acids to tRNA (66,147), direct measurements with microbial ArgRS, IleRS,MetRS, PheRS, TyrRS, and ValRS indicate that in thecharging of a cognate amino acid to tRNA, the ATP/aminoacyl-tRNA stoichiometry is one with an upper limit ofexperimental error of 0.05 (99). Similar ATP/aminoacyl-tRNA stoichiometries were also obtained in reactions cata-lyzed by plant SerRS (73) and avian PheRS (54).However, as an exception to this stoichiometry rule,

Freist et al. (50) reported an astonishingly high ATP/ami-noacyl-tRNA stoichiometry of 5.5 for Ile-tRNA le formationcatalyzed by yeast isoleucyl-tRNA synthetase. At leastsome of the ATP excess is most probably a consequence ofthe double-labeling method used, since the same group alsoreported a high ATP/aminoacyl-tRNA stoichiometry of 1.7for yeast Arg-tRNA'g (53), in contrast to a stoichiometry of0.99 + 0.02 reported for the same system by Mulvey andFersht (99), who used a more accurate kinetic method. It istherefore expected that the extra energy expenditure neededto maintain high selectivity of an aminoacyl-tRNA syn-thetase will come from editing of a noncognate amino acid.Thus, we can estimate the energy cost of homocysteineediting by methionyl-tRNA synthetase in vivo by relating theamount of edited homocysteine (in the form of thiolactone)to the amount of methionine incorporated into protein.

In E. coli, one molecule of homocysteine is edited asthiolactone per 109 molecules of methionine incorporatedinto protein (75), a value that is in good agreement with thetheoretical prediction based on in vitro measurements of Kmand kcat values for homocysteine and methionine with me-thionyl-tRNA synthetase (77) and on in vivo concentrationsof the amino acids (75). This is equivalent to an extra energycost of 0.01 mol of ATP for editing per mol of Met-tRNAformed, a value about 10-fold smaller than previously as-sumed (69) from calculations based on in vitro data forediting of valine by isoleucyl-tRNA synthetase and of thre-onine by valyl-tRNA synthetase (121). The extra energy costof 0.01 mol of ATP per mol of cognate aminoacyl-tRNAformed may seem insignificant per se. However, assumingthat the extra energy cost for proofreading during synthesisof Met-tRNA is representative of all aminoacyl-tRNAs, andgiven the fact that protein synthesis requires 95% or more ofall energy used for polymerization reactions in E. coli (69),the energy cost of editing becomes significant in relation toother energy requirements, e.g., for DNA or RNA synthesis.The energy cost of editing in selection of amino acids forprotein synthesis can be calculated to be roughly the equiv-alent of 67 and 39% of the energy used by E. coli for DNAand RNA synthesis, respectively.

In S. cerevisiae, one molecule of homocysteine is editedas thiolactone per 500 molecules of methionine incorporatedinto protein (76), the equivalent of an extra 0.002 mol of ATPexpended for editing per mol of Met-tRNA formed. Al-

though at the wild-type level of editing the extra energyexpended is very small, increasing it can lead to growthinhibition. A homocysteine-overproducing cys2cys4 yeaststrain edits 1 molecule of homocysteine as thiolactone per 8molecules of methionine incorporated into protein (76), thusdissipating 0.13 mol of ATP for homocysteine editing; thisstrain grows 16% more slowly than the isogenic cys4 strain,which dissipates only 0.0095 mol ATP for editing. The levelsof S-adenosylhomocysteine and S-adenosylmethionine aresimilar in the two strains (76a), making it unlikely that theobserved growth inhibition is due to effects of excess homo-cysteine on intracellular methylations. This magnitude ofgrowth inhibition is astonishing and may indicate that S.cerevisiae is energy limited and cannot tolerate energywaste.The in vivo studies also indicated that cellular levels of

MetRS are not limiting for protein synthesis: overexpressionof MetRS did not affect the rate of protein synthesis in E. coli(75) or S. cerevisiae (76). At the same time, the rate ofhomocysteine editing by MetRS in vivo increased in propor-tion to MetRS overproduction (75, 76). Thus, excess MetRSin the cell becomes detrimental since it leads to wastefulhydrolysis of ATP inadvertently associated with homocys-teine editing. This provides at least one reason why cellularlevels of aminoacyl-tRNA synthetases have to be carefullyregulated (see reference 63 for a review of regulation ofsynthetase expression).

RELATIONSHIP BETWEEN EDITING, SELECTIVITY,AND AMINO ACID BIOSYNTHETIC PATHWAYS

Amino acid biosynthesis has to be regulated not only toallow economical use by microorganisms of frequently lim-ited resources but also to provide a proper balance ofmetabolites in the cell. A disturbance of this intricate bal-ance, for example by exposing susceptible bacterial cells toa great excess of certain individual amino acids, can result ingrowth inhibition (20). It is also becoming increasingly clearthat high-level expression of recombinant proteins in E. coliproduces imbalances in amino acid pools that lead to highlevels of errors in proteins (9). In humans, several geneticdisorders affect interconversion of amino acids and lead toamino acid pool imbalances in body fluids. This results insevere diseases with numerous clinical manifestations. Forinstance, cystathionine ,B-synthase deficiency leads to eleva-tion of concentrations of homocysteine in plasma and urine(reviewed in reference 97) (see below).

Methionine Pathway and HomocysteineSome amino acid biosynthetic pathways lead to interme-

diate metabolites which can create selectivity problems forthe protein biosynthetic apparatus of the cell, becomingparticularly severe under unbalanced or deregulated condi-tions. In the methionine biosynthetic pathway, the presenceof an obligatory intermediate homocysteine poses an acuteselectivity problem for methionyl-tRNA synthetase.The relationship between the methionine biosynthetic

pathway and the editing reaction in E. coli is depicted in Fig.3. In the final step of the biosynthetic pathway, homocys-teine (Hcy) is methylated to methionine by the product ofeither the metE or metH gene. The metH gene product is a

vitamin B12-dependent homocysteine methyltransferase(137). In the absence of vitamin B12, homocysteine is trans-methylated by the metE gene product, which makes up 5%of the total protein in E. coli (146). Any amount of homo-

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Sercys2 Hse

i SH-Cys-

t i 11 met6* Hcy -.- Metcys4

)mes1I

Hcy thioloctone Met-tRNAmet Hcy thiolactone Met-tRNAMet1 100 0.2 100

E. coli S. cerevisioe

FIG. 3. Schematic representation of the relationship betweenhomocysteine editing and methionine biosynthetic pathways in E.coli and S. cerevisiae. Each arrow represents a step catalyzed by aseparate enzyme. Mutations that helped to establish the relation-ships are indicated. Relative numbers of molecules of homocysteinethiolactone and Met-tRNAMet formed in vivo are given. Abbrevia-tions: Hse, homoserine; Hcy, homocysteine.

cysteine that cannot be processed to methionine is edited as

homocysteine thiolactone by the product of the metG gene(MetRS), whose major function, of course, is to provideMet-tRNAMet for protein synthesis. Quantitation of themetabolite flow through methionyl-tRNA synthetase indi-cates that, in the absence of vitamin B12, 1 mol of homocys-teine is edited as thiolactone per 109 mol of methionineincorporated into protein (75). In the presence of vitaminB12, E. coli cells produce three times less thiolactone,therefore dissipating less energy on homocysteine editing(76a). This may explain why E. coli evolved to use exclu-sively the vitamin B12-dependent homocysteine transmeth-ylase in its natural habitat in the gut (16, 137, 143).Two features of homocysteine editing in E. coli are

unexpected from in vitro studies. First, as described above,in addition to methionyl-tRNA synthetase, several otheraminoacyl-tRNA synthetases edit misactivated homocys-teine by transforming it into thiolactone in vitro (27, 77).However, only methionyl-tRNA synthetase is involved inhomocysteine editing in vivo (75). The contribution of valyl-and leucyl-tRNA synthetases to homocysteine editing invivo can be calculated to be negligible (less than 1%). Theisoleucyl-tRNA synthetase, on the other hand, is only two-fold less efficient in the editing than methionyl-tRNA syn-

thetase in vitro and should therefore measurably contributeto the thiolactone formation at least in appropriate mutantsand even in wild-type cells, in which the isoleucine pool isonly twice as large as the methionine pool (113). Second, a

methionine-starved metE mutant, which is expected to ac-

cumulate homocysteine, does not do so. Instead, the metEmutant elitninates excess homocysteine as the thiolactone.These two observations can be rationalized by proposingthat an interenzyme metabolite transfer may be involved inthe metabolism of homocysteine in E. coli. This kind oftransfer in metabolic pathways has been termed channeling(136). The interenzyme metabolite transfer would lead tocompartmentalization of homocysteine reactions to withinthe methionine biosynthetic pathway. The compartmenta-tion would be important since otherwise homocysteine mightalso be misactivated by IleRS, ValRS, and LeuRS andpossibly charged to the respective tRNAs, as has beenobserved in vitro with LeuRS (27) and ValRS (28); thiswould lead to incorporation of homocysteine in protein. This

model proposes interactions between the metE or metH andmetG genes and should be readily testable.The strategy used by S. cerevisiae to minimize the need

for homocysteine editing is different. This minimization isachieved by the evolution of a capability to transformhomocysteine into cysteine (Fig. 3), a pathway that alsoexists in mammals. As a result, in contrast to E. coli,wild-type yeast cells have very small homocysteine pools.This is illustrated by a yeast cys4 mutant (a prototroph)which is an equivalent of wild-type E. coli in terms of theorganization of its methionine biosynthetic pathway. Themutant has relatively large homocysteine pools, as does E.coli, and it edits 1 homocysteine per 100 methionines incor-porated into protein, five times more than wild-type yeastbut almost exactly the same as E. coli (76). In contrast to E.coli, yeast cells apparently are not under great evolutionarypressure to cope with excess homocysteine, and, whenforced by an experimenter to do so, they are very sluggish inthis respect. For example, whereas a metE mutant of E. coliis able to eliminate all excess homocysteine as the thiolac-tone, a yeast met6 mutant, which is an equivalent of an E.coli metE mutant (Fig. 3), accumulates homocysteine andthe thiolactone to levels 90- and 500-fold higher, respec-tively, than the wild-type yeast does (76). It remains to bedetermined whether homocysteine is incorporated intotRNA and protein in the homocysteine-overproducing yeastmet6 mutant.

Expression of the metE gene in E. coli and Salmonellatyphimurium is regulated by the metR gene. MetR protein isa transactivator of metE expression. Homocysteine has beenimplicated as a cofactor (143) which enhances binding of theMetR protein to a regulatory region of the metE gene (17).The evidence that homocysteine participates in regulation ofmetE expression came from molecular genetic studies ofexpression of metE::lacZ fusions. The expression was en-hanced when exogenous homocysteine was included inculture media or when metE or metF strains, which wereexpected to accumulate homocysteine, were used (143). Inaddition, in vitro studies of expression of the metE gene,involving coupled transcription-translation with extractsprepared from a metE strain, have shown that exogenouslyadded homocysteine stimulated synthesis of the MetE trans-methylase enzyme (17). Homocysteine used in these studieswas prepared by base hydrolysis of commercial homocys-teine thiolactone, and its purity was not reported. However,since E. coli strains, including metE and metF mutants,efficiently transform any intracellular excess homocysteineinto homocysteine thiolactone (75), it is possible that theobserved stimulation of metE expression is due to accumu-lation of the thiolactone, even in the in vitro system.Moreover, although Urbanowski and Stauffer (143) state thatthey used homocysteine prepared from the thiolactone, theirconditions of hydrolysis would result in transformation ofless than 10% of the thiolactone to homocysteine. Therefore,more careful studies are needed to determine whether ho-mocysteine or the thiolactone, whose synthesis by MetRSdepends on relative concentrations of both homocysteineand methionine (75), participates in MetR-dependent regu-lation of metE.

Branched-Chain Amino Acid Pathway and Norleucine(Norvaline, at-Aminobutyrate)

The branched-chain amino acid biosynthetic pathwayshave certain degrees of flexibility which can be eitheradvantageous or harmful to the cell. This flexibility can also

Ser

Hse

SH-C s

Cys -

metEHcy --.- Met

metH

)metG(

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A. ISOLEUCINE-VALINE PATHWA'

pyruvate - -

oe-ketobutyrate - --+

B. LEUCINE PATHWAY

a-ketoisovalerate - -+

0

a-ketoisocaproate CH3- H-CH2-CH2-C-COOH - homoleucine

CH3E-ketoisohexanoate

0

a-keto-p-methylvalerate - CH3-CH-CH2-C-COOH- homoisoleucine

CH2CH3

a-keto-y-methylcaproate

19pyruvate CH3-CH2-C-COOH- a-aminobutyrate

a-ketobutyrate

19a-ketobutyrate _ CH3-CH2-CH2-C-COOH - norvaline

a-ketovalermte0

a-ketovalerate - - CH3-CH2-CH2-CH2-C-COOH - norleucine

a-ketocaproate

FIG. 4. Schematic representation of the branched-chain aminoacid biosynthetic pathways in bacteria. The individual steps in thepathways are indicated by arrows. Each parallel step in the isoleu-cine-valine pathways (A) is catalyzed by the same enzyme. The foursteps of the leucine pathway (B) are catalyzed by another set ofenzymes. The transamination reactions in the pathways are cata-lyzed by two transaminases: one specific for each pathway and one

nonspecific. The leucine-forming enzymes exhibit limited specificity(80). For this reason, under certain conditions the pathway willincrease the chain length of several ,-ketoacids and produce un-usual amino acids, as indicated previously (8, 80).

create problems, only recently recognized, with using E. colistrains for high-level expression of recombinant proteins.Fortunately for commercial applications of recombinantDNA technology, these problems can be easily avoided.

ao-Ketoacid intermediates, in addition to being trans-formed along the major pathways ultimately into valine,isoleucine, or leucine, give rise to several nonprotein aminoacids as depicted in Fig. 4. Some of these nonprotein aminoacids are important components of antibiotics and thereforeconfer selective advantage to organisms producing them.For instance, a-aminobutyrate is a component of cyclo-sporin A, a cyclic undecapeptide with anti-inflammatory,immunosuppressive, antifungal, and antiparasitic propertiesthat is produced by the fungus Beauveria nivea (85). Norva-line is present in an antifungal peptide produced by Bacillussubtilis (102). Homoleucine, a homolog of leucine, is a

component of an antibiotic produced by Streptomyces dia-staticus (131). However, some by-products of the branched-chain amino acid biosynthetic pathways, such as a-aminobu-tyrate (80, 86), norleucine, and norvaline (8, 80, 141), can

create serious selectivity problems for the protein biosyn-thetic apparatus of the cell. a-Aminobutyrate is easily mis-activated by valyl-tRNA synthetase and then edited (37).

Norvaline is efficiently misactivated by isoleucyl-tRNA syn-thetase (64, 91) and most probably edited, but this has neverbeen tested. ex-Aminobutyrate accumulates in E. coli cul-tures supplemented with valine (86). Wild-type strains ofSerratia marcescens contain small pools of ao-aminobutyratewhich increase in certain regulatory mutants (80). The Ser-ratia marcescens mutants also accumulate norvaline (80).Norleucine is not only misactivated but also charged tomethionine tRNAs by methionyl-tRNA synthetase (140).Since the methionyl-tRNA synthetase does not edit norleu-cine efficiently (39) (Table 1), this amino acid is subsequentlyincorporated into protein in place of methionine (6, 12, 79).Norleucine is bacteriostatic to many microorganisms (114).In E. coli and Serratia marcescens, mutations resulting inresistance to norleucine occur in metK (62) and metA (81)loci, respectively. However, E. coli is also surprisinglytolerant to norleucine and can exhibit limited growth in itspresence. Substitution of one-half of the normal methionineresidues with norleucine in E. coli proteins does not lead toimmediate loss of cell viability (6).

Proteins containing norleucine at all of their methioninesites have been made. In a few cases studied so far, thebiological activities of the norleucine-containing proteinswere identical to those of unsubstituted proteins. In addi-tion, two E. coli norleucine-substituted proteins, P-galactosi-dase (101) and adenylate kinase (59), were more resistant toalkylation and oxidation, respectively, than their normalmethionine-containing counterparts. Three norleucine-con-taining proteins from other species, Staphylococcus aureusnuclease (4), recombinant human epidermal growth factor(82), and interleukin-2 (141), produced in E. coli, all exhib-ited full biological activity. Biological activity of anothernorleucine-containing recombinant protein produced in E.coli, bovine somatotropin, was not reported (8).

Norleucine, first detected in vivo in an isoleucine-valineauxotroph of Serratia marcescens when threonine was in-cluded in the frementation medium (80), has been proposedto be synthesized from a-ketobutyrate through two cycles ofthe leucine biosynthetic pathway (Fig. 4B). Norvaline,which is also present in these cells, arises from a-ketobu-tyrate in one cycle of the leucine pathway (80) (Fig. 4B).Unexpectedly, norleucine has been detected in recombinantinterleukin-2 and bovine somatotropin produced in E. coligrown in the absence of exogenous norleucine (8, 93, 141).Free norleucine and norvaline, which appear during high-level expression of these leucine-rich proteins, have beenshown to be products of the derepressed leucine biosyn-thetic pathway. Norleucine, norvaline, and several a-ke-toacids were detected in recombinant E. coli cultures butwere absent when an E. coli strain with a deletion of theleucine operon was used. The derepression of the leucinepathway was brought about by leucine limitation caused byhigh-level synthesis of leucine-rich recombinant proteins (8).Incorporation of norleucine into protein is prevented byincluding leucine and/or methionine in the fermentationmedia (141) or by using a leucine operon deletion E. colistrain (8).

Incorporation of norleucine into proteins is due to mis-charging of tRNAMet with norleucine by methionyl-tRNAsynthetase. This is the only mischarging error known inwhich a naturally produced noncognate amino acid is mis-takenly used by a synthetase to charge its cognate tRNA invivo. Methionyl-tRNA synthetase has an efficient proofread-ing mechanism which prevents incorporation of homocys-teine into tRNAMet and protein in vivo (75, 76) but thismechanism is not used for editing of norleucine (39). Appar-

0CH3-5H-C-COOH-- valine

CH3a-ketoisovalerate

0CH3-CH-C-COOH - isoleucine

CH2CH3

a-keto- -methylvalerate

0CH3-jH-CH2-C-COOH - leucine

CH3a-ketoisocaproate

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ently, E. coli has not been under evolutionary pressure toevolve such a mechanism to prevent incorporation of nor-leucine into tRNA and protein. Instead, E. coli developedtight controls over the branched-chain amino acid biosyn-thetic pathways which prevent norleucine accumulationunder most growth conditions in the first place.

Other Possible tRNA Misacylation Errors Due to AminoAcid Pool Imbalances

High-level expression of recombinant proteins in E. colimay lead to other types of missense errors. As much as 11%of the total recombinant atrial peptide III molecules containlysine at sites coded for by AGA, but not by CGU, argininecodons. Similar levels of lysine misincorporation at AGAarginine codons have been observed in insulinlike growthfactor type 1 obtained from the overproducing strain. WhenAGA codons were replaced with CGU codons, lysine mis-incorporation was eliminated from both peptides (9, 129). Itis not known whether lysine misincorporation is due to thelysine AAA tRNA misreading the rare arginine codon AGAor the arginyl-tRNA synthetase mischarging the rare argin-ine AGA tRNA with lysine. However, amino acid poolimbalances seem to play a role: supplementation of theculture medium with arginine reduced lysine misincorpora-tion to 1% (9).Measurements of phenylalanine misincorporation into

mouse epidermal growth factor overproduced in E. coliindicated an error frequency of about 1 in 40 for codonsdiffering by a single base from phenylalanine codons (127).In these experiments, a synthetic mouse epidermal growthfactor gene with a codon bias optimized for high-levelexpression in E. coli was used. Despite this, the genegenerated a product with an error frequency at least 10-foldhigher than the error rate found for normal E. coli proteins.Phenylalanine misincorporation into mouse epidermalgrowth factor was not significantly affected by using a strain(rpsL282) with hyperaccurate ribosomes (127), which mayindicate tRNA mischarging rather than codon misreading(61) as the basis for the errors.The unexpected proliferation of missense errors during

high-level expression of recombinant proteins in E. coli mayresemble problems presumably encountered by specialized(differentiated) eucaryotic cells early in evolution. For thesake of argument, we can assume that an E. coli cellproducing high levels of a recombinant protein product is anequivalent of a differentiated eucaryotic cell, for instance anerythrocyte which is a high-level globin producer. There isone important difference, however. A recombinant E. colicell produces a significant proportion of faulty proteinsbecause it cannot appropriately regulate its amino acidbiosynthetic pathways during high-level recombinant geneexpression. An erythrocyte produces essentially perfectglobin molecules, since it has, as all mammalian cells havedone a long time ago, disposed of most of its amino acidbiosynthetic genes. However, by deleting an amino acidbiosynthetic operon, e.g., the leucine operon, from ourrecombinant E. coli, we avoid deregulation of the pathwaywhich would lead to imbalances in amino acid pools andeven to production of novel amino acids, normally not madeby a nonrecombinant bacterial cell. Such a deletion strainproduces essentially error-free proteins, as the erythrocytedoes.

Implications for Disorders of Amino Acid Metabolismin Humans

The evidence discussed above indicates that imbalances inamino acid pools in microorganisms lead to errors in tRNAcharging. Although studies of errors in amino acid selectionin higher eucaryotic systems are virtually nonexistent, it islikely that amino acid pool imbalances would also lead tosimilar tRNA mischarging errors in mammalian cells. Thismight happen in a variety of human genetic disorders whosemost conspicuous biochemical manifestations include aminoacid pool imbalances. As an example, the possible involve-ment of mischarged tRNA in cystathionine j-synthase defi-ciency is considered below.

In humans, as in all mammals, the transsulfuration path-way converts methionine, an essential amino acid, intocysteine (reviewed in reference 97). The pathway starts withformation of S-adenosylmethionine, which yields S-adeno-sylhomocysteine in subsequent transmethylation reactions.S-Adenosylhomocysteine is further metabolized to homo-cysteine and adenosine. In addition to being remethylatedback to methionine, homocysteine is condensed with serineto form cystathionine in a reaction catalyzed by cystathio-nine ,B-synthase. Cystathionine is finally cleaved by theenzyme y-cystathionase to cysteine and a-ketobutyrate.

Cystathionine (-synthase deficiency (reviewed in refer-ence 97) is a genetic disorder of transsulfuration whichoccurs with a frequency of 1 in 200,000 in the generalpopulation. The disorder is inherited as an autosomal reces-sive trait. The most characteristic feature of cystathionineP-synthase deficiency is the presence of elevated levels ofboth homocysteine and methionine in plasma. However, asmany as 6% of older cystathionine ,B-synthase-deficientpatients who are homocysteinemic and homocysteinuric donot have methionine levels in plasma above the normalrange. Excess homocysteine is excreted in urine. Severalorgan systems or organs are affected: the eye; the skeletal,central nervous, and vascular systems; the liver; the hair;and the skin. Mental retardation or illness is frequent amongindividuals with cystathionine ,-synthase deficiency. A ma-jor cause of premature death of these individuals is throm-boembolism.No aspect of cystathionine ,B-synthase deficiency has

remained as obscure as the intermediate steps by which theenzyme deficiency leads to the specific clinical manifesta-tions associated with it.Although cystathionine ,B-synthase deficiency always

leads to elevation of homocysteine levels, homocysteinethiolactone has not been detected in the serum or plasma ofhomocysteinuric patients (22) or normal subjects (94, 98).However, the sensitivity of the methods used in thesestudies was relatively poor and would allow detection of thethiolactone only at concentrations greater than 32 ,M (98) or50 ,uM (22); this sensitivity may not be sufficient since totalconcentrations of homocysteine in plasma are only up to 10,M in normal subjects and up to 200 ,M in homocysteinuricpatients (97). Elevation of homocysteine levels in S. cerevi-siae, including cystathionine 13-synthase-deficient (cys4) mu-tants, leads to elevation of the thiolactone levels (76).Homocysteine is detrimental to growth both in S. cerevisiae(76) and in rats (97). In E. coli, all excess homocysteine thatis not used for transmethylation to methionine is trans-formed into the thiolactone. Homocysteine never accumu-lates in metE and metF mutants of E. coli; homocysteinethiolactone accumulates instead (75). It would be interestingto use more sensitive methods to determine whether the

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thiolactone is present in humans. The presence of thiolac-tone, at least in homocysteinuric patients, and participationof MetRS in its formation would indicate preservation of theediting mechanism of MetRS in humans. On the other hand,since homocysteine is synthesized by entirely different path-ways in humans and microorganisms, human MetRS maynot have the need and therefore may have lost the ability toedit homocysteine by transforming it into the thiolactone.Amino acid pool imbalances in microorganisms can lead to

high levels of errors in proteins, often as a result of tRNAmischarging (see previous sections). Since thioamino acidpool imbalances are always associated with cystathionine,B-synthase deficiency, it is tempting to suggest that thepleiotropic manifestations of a single genetic defect in thisdisorder are also due to errors in proteins. These errors canbe due to mischarging of tRNA with homocysteine, whichcan be facilitated by high levels of homocysteine in cys-tathionine ,-synthase-deficient patients. Of four microbialaminoacyl-tRNA synthetases that misactivate and edit ho-mocysteine in vitro, LeuRS and ValRS are able to formhomocysteinyl-tRNA in vitro (27, 28). Corresponding mam-malian synthetases may have this ability as well. IftRNAL'u, tRNAVal, and possibly tRNAIle are mischargedwith homocysteine in patients with cystathionine ,-synthasedeficiency, simple supplementation of diets with leucine,valine, and isoleucine may ameliorate harmful effects of thedeficiency. Whether this hypothesis is correct can be deter-mined by studying the amino acid selectivity of mammalianMetRS, IleRS, ValRS, and LeuRS and of errors in proteinsynthesis in homocysteinuric patients.Amino acid pool imbalances leading to errors in tRNA

charging may also play a role in several other humandisorders. Possible examples include gyrate atrophy andother conditions associated with hyperornithinemia (see thelast paragraph of the section on editing in living cells) andphenylketonuria (characterized by excess phenylalanine).

MOLECULAR BASIS FOR SELECTIVITY OFAMINOACYL-tRNA SYNTHETASES

Aminoacyl-tRNA synthetases differ considerably in sizeand quatemary structure and have limited sequence homol-ogy. However, a common pattern can be found in theorganization of the structures of these enzymes (15). Thefunctional units of aminoacyl-tRNA synthetases are ar-ranged along the amino acid chains in such a way that theaminoacyl adenylate domain is located in the N-terminal halfof the protein. The tRNA recognition domain partiallyoverlaps the adenylate domain and extends far to the C-ter-minal portion of the protein (122, 133). X-ray crystallographyand computer analysis of amino acid sequences have re-vealed structural similarities between aminoacyl-tRNA syn-thetases and have led to the classification of this family ofenzymes into two groups (15, 19, 29, 100, 123). Class Isynthetases (ArgRS, CysRS, GlnRS, GluRS, IleRS, LeuRS,MetRS, TrpRS, TyrRS, and ValRS) have two short consen-sus sequences (HXGH, where X is a hydrophobic aminoacid that is frequently isoleucine, and KMSKS) which formpart of the structural domain (the Rossman fold) that bindsATP, as observed in three crystal structures (GlnRS,MetRS, and TyrRS). Class II synthetases do not have theRossman fold (e.g., SerRS [19]) but share three new se-

quence motifs (29).Since aminoacyl-tRNA synthetases share the common

substrate ATP and recognize a-amino and carboxyl groupsof their cognate amino acid substrates, it may not be

surprising that homologous amino acid residues that makeup the aminoacyl adenylate domain of class I synthetases areengaged in common functions, i.e., interactions with ATPand a-amino and carboxyl groups of amino acid substrates.The first histidine residue of the HIGH sequence interactswith ATP in the crystal structures of MetRS and GlnRScomplexed with ATP (13, 118). Site-directed mutagenesisexperiments indicate that both histidine residues participatein the activation of tyrosine by TyrRS (36). The secondlysine of the KMSKS sequence lies near the y-phosphate ofATP in the crystal structure of the MetRS ATP complexand is seen to interact with the phosphates of ATP in thestructure of the GlnRS ATP tRNA In complex (118). InTyrRS the two corresponding lysine residues stabilize thetransition state for Tyr-AMP formation by strongly interact-ing with the PP1 moiety of ATP, as deduced from site-directed mutagenesis experiments (42).During interaction of a synthetase with its cognate amino

acid substrate, the enzyme recognizes nonspecific featurescommon to all amino acids, i.e., c-amino and carboxylgroups in an appropriate configuration around the a-carbon,as well as specific features unique to each amino acid, i.e.,the side chain. One may therefore expect to find in thestructures of aminoacyl-tRNA synthetases homologousamino acid residues which interact with a-amino and car-boxyl groups of the substrate as well as unique residueswhich provide specificity. What little is known about aminoacid-binding sites of the synthetases supports this notion.The most detailed picture of the amino acid-binding site,

including specificity determinants, comes from X-ray crys-tallographic and site-directed mutagenesis studies of tyrosyl-tRNA synthetase (36). Residues Asp-78, Tyr-169, and Gln-173 form a binding site for the a-amino group of tyrosine(Fig. SA). Glu-195 has been postulated to interact with thecarboxyl group of tyrosine during the activation reaction (11,43). The specificity site for tyrosine against phenylalanine iscomposed of Asp-176 and Tyr-34 (43). Asp-176 functions asa hydrogen bond acceptor of the substrate hydroxyl; Tyr-34functions as a hydrogen bond donor. Replacement of Tyr-34by Phe does not significantly alter kca, and increases Km fortyrosine only twofold. However, the kcatlKm value for acti-vation of phenylalanine increases sixfold. Altogether, themutation Tyr-34 -- Phe decreases discrimination againstphenylalanine 15-fold. Unfortunately, mutation of Asp-176has not yielded an active enzyme (36).

Studies of tyrosyl-tRNA synthetase have led to an under-standing of the molecular mechanism of amino acid activa-tion and specificity in unprecedented detail and also consid-erably expanded at the molecular level our ideas of generalconcepts in enzymatic catalysis and biological specificitysuch hydrogen bond and induced fit (42, 43). Because thissynthetase is essentially absolutely specific for tyrosine, theenzyme does not possess or need editing ability. Phenylala-nine is activated 1.5 x 105 times less efficiently than tyrosineby tyrosyl-tRNA synthetase (44). This discrimination isachieved exclusively through differences in initial bindingenergies of cognate (Tyr) and noncognate (Phe) substrates.Therefore we must look for another system to study themolecular basis of the other component of selectivity,namely, editing.

Fortunately, methionyl-tRNA synthetase provides such a

system. The methionyl-tRNA synthetase does have a prob-lem discriminating against homocysteine, the immediateprecursor of methionine in the cell. Homocysteine is acti-vated by the methionyl-tRNA synthetase only 100 times lessfrequently than methionine is in vivo. Incorporation of

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A

Arg86

NH

\C+=NH2

Thr4O

UH .

\N. O U

Lys82 NH2*. p+ ~~0 0NH- .IH.3oNH

73-C=O* H HH

.H-N -C- CO-P-0O

-COz A NH.2 *CH 00- *

Tyri1 69-6H iI2:i CH2K76C2. 0kpz..V¶o

Tyr34-OH

His45

+ Lys230. H3 N /*+ Lys2333

:NThr5l

I...HO

*His48

HS- Cys35

B

Arg233Ns- ~t-NH

`C+-=NH2

NH2.

H

.H -N+/

Asp52 - CO2- * H

Trp3O5

Tyr358 + Lys335

I~ ~ * H3 NOH 0 3

*p

0 0

H . I /-C-CO2P-p- 0

IH / \ NH2CH2 0 0

N

CH CH2 I'll

0 ~~~~+

/0 0\ 11e231

H H.. . . . . 0

FIG. 5. Schematic drawing of the interactions between TyrRS and the transition state in the formation of tyrosyl adenylate (A) and MetRSand the transition state in the formation of methionyl adenylate (B). Respective amino acid substrates and specificity residues of the enzymesare in boldface. Panel A adapted with permission from reference 36; copyright 1987, American Chemical Society. Panel B adapted withpermission from reference 58; copyright 1991, American Chemical Society.

Glnl,

Asp7l

Aspl

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homocysteine into tRNA and protein is prevented by anefficient editing function of the methionyl-tRNA synthetase(75, 76). Crystal structures of free methionyl-tRNA syn-thetase and of its complex with ATP have been solved (13),and site-directed mutagenesis studies of the methionyl ade-nylate domain of the enzyme have begun (57, 58).A possible binding site for methionine has been revealed

by studies of the crystal structure of MetRS (13). Subsequentsite-directed mutagenesis of some of the putative methion-ine-binding-site residues led to identification of Asp-52 andArg-233 as playing an important role in stabilization of thetransition state for Met-AMP formation, possibly by inter-acting with the a-amino and carboxyl groups, respectively,of the methionine substrate (Fig. SB). The Asp-52 residue ofMetRS corresponds to Asp-78 of TyrRS, and all but one ofclass I aminoacyl-tRNA synthetases have an aspartic acidresidue at a site corresponding to Asp-52 in MetRS (58). Thesynthetases most closely related to MetRS (such as IleRS,LeuRS, and ValRS) also have a conserved arginine at theposition corresponding to Arg-233. The specificity site formethionine contains Trp-305. Substitution of Ala for Trp-305leads to a 200-fold decrease in efficiency of methionineactivation (58). The Trp-305 -- Ala substitution does notaffect interaction of the synthetase with the noncognatesubstrate homocysteine, since mutant methionyl-tRNA syn-thetase with an alanine residue at position 305 misactivatesand edits homocysteine as efficiently as the wild-type en-zyme does (76a).

CONCLUSIONS

In this review, we have described and compared thepathways and consequences of editing of errors in selectionof amino acids by aminoacyl-tRNA synthetases, which is acrucial quality control point in maintaining the accuracy ofreading the genetic code. Several synthetases have to distin-guish between the correct substrate and a homolog differingby just one methyl group; this binding energy has beenestimated to contribute only a factor of 100 to selectivity,whereas synthetases distinguish such closely related sub-strates by a factor of 104 to 105, by editing. Although theearly information about editing by aminoacyl-tRNA syn-thetases was developed through in vitro studies, new ap-proaches taking advantage of the unique chemical productsof some editing reactions have enabled editing to be studiedin vivo as well. The in vivo studies have established theimportance of editing in living cells, assessed the energy costof editing (which can be reflected in the growth rate), andadded new perspectives on the necessity of cells to carefullyregulate both synthetase levels (to limit energy waste fromexcessive editing) as well as amino acid biosynthetic path-ways, which might otherwise create selectivity problems foraminoacyl-tRNA synthetases and the protein-synthesizingapparatus of the cell in general. In this regard, high-levelexpression of recombinant proteins stresses the proteinsynthesis machinery, exacerbating misincorporation, whichis in part a consequence of misactivation by aminoacyl-tRNA synthetases. Unbalanced amino acid pools associatedwith some genetic disorders in humans may also lead toerrors in tRNA charging, adding a new perspective to futurestudies of such disorders. The molecular basis for selectiv-ity, including editing function, of aminoacyl-tRNA syn-thetases at the physical-chemical level is also beginning to beelucidated in some systems, by combining X-ray crystallo-graphic structures and site-directed mutagenesis with func-tional assays or phenotypes. A considerable body of knowl-

edge and understanding has now evolved about thisimportant biological phenomenon of editing of errors byaminoacyl-tRNA synthetases, which is a universal propertyof all living cells.

ACKNOWLEDGMENTWe gratefully acknowledge support by a research grant

(GM27711) from the National Institutes of Health.

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Kurland. 1982. Translation rates and misreading characteris-tics of rpsD mutants in Eschenchia coli. Mol. Gen. Genet.187:467-472.

2. Anderson, D. I., and C. G. Kurland. 1983. Ram ribosomes aredefective proofreaders. Mol. Gen. Genet. 191:378-381.

3. Anderson, R. P., and J. R. Menninger. 1986. The accuracy ofRNA synthesis, p. 159-189. In T. B. L. Kirkwood, R. F.Rosenberger, and D. J. Galas (ed.), Accuracy of molecularprocesses. Chapman & Hall, New York.

4. Anfinsen, C. B., and L. G. Corley. 1969. An active variant ofstaphylococcal nuclease containing norleucine in place ofmethionine. J. Biol. Chem. 244:5941-5953.

5. Baldwin, A. N., and P. Berg. 1966. Transfer ribonucleic acid-induced hydrolysis of valyl adenylate bound to isoleucyl ribo-nucleic acid synthetase. J. Biol. Chem. 241:839-845.

6. Barker, D. G., and C. J. Bruton. 1979. The fate of norleucineas a replacement for methionine in protein synthesis. J. Mol.Biol. 133:217-231.

7. Bergman, F. H., P. Berg, and M. Dieckman. 1961. The enzy-matic synthesis of aminoacyl derivatives of ribonucleic acid.II. The preparation of leucyl-, valyl-, isoleucyl-, and methio-nyl-ribonucleic acid synthetases from Escherichia coli. J. Biol.Chem. 236:1735-1740.

8. Bogosian, G., B. N. Violand, E. J. Dorward-King, W. E.Workman, P. E. Jung, and J. F. Kane. 1989. Biosynthesis andincorporation into protein of norleucine by Escherichia coli. J.Biol. Chem. 264:531-539.

9. Bogosian, G., B. N. Violand, P. E. Jung, and J. F. Kane. 1990.Effect of protein overexpression on mistranslation in Esche-richia coli, p. 546-558. In W. E. Hill, A. Dahlberg, R. A.Garrett, P. B. Moore, D. Schlessinger, and J. R. Warner (ed.),The ribosome: structure, function, and evolution. AmericanSociety for Microbiology, Washington, D.C.

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