for Protein Synthesis - Europe PMC

Vol. 56, No. 3

MICROBIOLOGICAL REVIEWS, Sept. 1992, p. 412-429 0146-0749/92/030412-18$02.00/0 Copyright X 1992, American Society for Microbiology

Editing of Errors in Selection of Amino Acids for Protein Synthesis HIERONIM JAKUBOWSKI* AND EMANUEL GOLDMAN Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, 185 South Orange Avenue, Newark New Jersey 07103 INTRODUCTION .............................................................................. 412 THE PROBLEM .............................................................................. 413 DISCOVERY OF EDITING .............................................................................. 413 CONTRIBUTION OF EDITING TO SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES ...........413 EDITING PATHWAYS .............................................................................. 415 416 Misacylation-Deacylation Pathway .............................................................................. Deacylase Activity of Aminoacyl-tRNA Synthetases ...................................................................416 417 Adenylate Pathway .............................................................................. Relative Contribution of Editing Pathways to Selectivity .............................................................417 EDITING IN LIVING CELLS .............................................................................. 418 COST OF EDITING IN VIVO .............................................................................. 419 RELATIONSHIP BETWEEN EDITING, SELECTIVITY, AND AMINO ACID BIOSYNTHETIC PATHWAYS .............................................................................. 419 Methionine Pathway and Homocysteine .............................................................................. 419 Branched-Chain Amino Acid Pathway and Norleucine (Norvaline, a-Aminobutyrate)........ 420 422 Other Possible tRNA Misacylation Errors Due to Amino Acid Pool Imbalances ....................... 422 Implications for Disorders of Amino Acid Metabolism in Humans ...................................... MOLECULAR BASIS FOR SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES ......................423 CONCLUSIONS .............................................................................. 425 ACKNOWLEDGMENT .............................................................................. 425 REFERENCES ...............................................................................425 ................ ....

* .........

by two crucial steps of protein synthesis which are also important for accurate translation of genetic information. The first step involves the selection of a cognate amino acid and tRNA by an aminoacyl-tRNA synthetase to provide correctly aminoacylated (also referred to as charged) tRNA. In the second step, a correct aminoacyl-tRNA is selected in the codon-programmed ribosomal A site. The in vivo error frequencies in protein synthesis (substitution of one amino acid for another) are in the range of 10-3 to 10' (see references 24, 90, 92, and 110 and references therein). Most translational errors measured in vivo are due to mistakes in the selection of correct aminoacyl-tRNAs by the ribosome (149). The in vitro measurements of the selectivity of aminoacyl-tRNA synthetases indicate that the upper limit for errors in the selection of correct amino acids for protein synthesis is in the range of 10' to 10-5 (66, 89, 147). The selectivity of aminoacyl-tRNA synthetases toward tRNA is even greater. The frequency of errors involving noncognate tRNA 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 the codon-programmed ribosomal A site, which are in many instances limited, but also on subsequent editing of errors in the initial selection. The editing in amino acid selection for protein synthesis by an aminoacyl-tRNA synthetase was the first proofreading process to be discovered in the flow of genetic information (5, 105). The proofreading of an amino acid has recently been shown to be an important in vivo process which prevents incorporation of a wrong amino acid into tRNA and protein in both Escherichia coli (75) and Saccharomyces cerevisiae (76). Editing was also postulated

INTRODUCTION The accurate processing of genetic information is fundamental to the growth, development, and function of living cells. As with any genetic trait, the accuracy of macromolecular synthesis is under evolutionary pressure. The levels of accuracy observed in DNA replication, transcription, and translation are the result of a balance between the need, on the one hand, to preserve the gene and its function and, on the other hand, to be flexible enough to allow adaptation to a changing environment. Maximum accuracy, owing to its high energy cost, is never achieved by a living cell (34, 107, 108, 120, 121). The existence of proofreading mutants with enhanced (10, 55, 61) or lowered (1, 2, 61) accuracy indicates that present-day cells operate at an optimal, i.e., intermediate, level of accuracy (25) and have a reserve of proofreading capacity 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 genetic information, with error rates in the range of 10-6 to 10-10 (21, 60). These low error rates in DNA replication are possible due to the existence of proofreading (14) and repair processes (115, 128). The accuracy of subsequent steps of expression of genetic information is several orders of magnitude lower than the accuracy of DNA replication. The error rate for transcription is about 10-4 (3, 116, 117, 135). An editing mechanism has been implicated in maintaining the accuracy of RNA synthesis (87). The shape of the present-day genetic code is determined *

Corresponding author. 412

VOL. 56, 1992

(65, 103) and eventually found (119, 138, 139) to occur during ribosomal selection of aminoacyl-tRNA. This selection is also influenced by the relA locus in bacteria (reviewed in references 56 and 110; see also reference 74). There is also evidence for editing of an aberrant polypeptide on the ribosome immediately after peptide bond formation, when an incorrect aminoacyl-tRNA has donated the wrong amino acid to the growing peptide chain (95). Since at least 95% of metabolic energy is consumed for protein synthesis in E. coli (69), and perhaps not much less in S. cerevisiae, the editing of errors in translation can be quite costly to the cell (75, 76). This review will focus on the nature and magnitude of errors made by aminoacyl-tRNA synthetases during amino acid selection, alternative pathways for editing of these errors, the magnitude of the contribution of editing to the selectivity of aminoacyl-tRNA synthetases, the energy cost of editing, and the in vivo significance of editing. We will also examine the relationship between editing, selectivity, and amino acid biosynthetic pathways in living cells. Implications of limited selectivity of synthetases for disorders of amino acid metabolism in humans will be discussed. Selectivity of aminoacyl-tRNA synthetases will be examined at the molecular level. For the purposes of this review, the terms "editing" and "proofreading" refer to the ability of aminoacyl-tRNA synthetases to correct or prevent incorrect aminoacylation of tRNA. THE PROBLEM Aminoacylation of tRNA is a two-step reaction. In the first step (equation 1), which is generally referred to as amino acid activation, an amino acid (AA) is activated to form enzyme (E)-bound aminoacyl adenylate. E + AA + ATP ; E AA-AMP + PPi (1) In the second step (equation 2), the amino acid is transferred from 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 protein amino acids (and a few nonprotein ones such as homocysteine, homoserine, or ornithine, which are intermediates in amino acid biosynthetic pathways) and 1 cognate tRNA family out of 20 tRNA families. Selection of tRNAs is not a major problem since these are relatively large molecules with adequate scope for distinctive structural variation. For example, the selectivity of E. coli isoleucyl-tRNA synthetase for tRNAI'le is 4 x 107 against tRNAP ie and 2 x 107 against tRNAf"et (96, 148, 149), and many other synthetases exhibit similar selectivities (23, 112, 126). The molecular basis for such discrimination, aptly called tRNA identity, is now known in considerable detail for several tRNAs (reviewed in references 104, 122, 125, 133, and 150). In contrast, amino acids are small molecules; some of them are so similar in structure that aminoacyl-tRNA synthetases cannot initially distinguish between them with adequate selectivity and often mistakenly activate them to form enzyme-bound noncognate aminoacyl adenylates. This is generally referred to as misactivation. The problem was first recognized by Pauling (111), well before the basic framework for protein synthesis had been established. He calculated an error rate of about 1 in 5 for glycine replacing alanine, valine replacing isoleucine, and so on. Pauling's calculation was based on a value of about 1 kcal/mol (4.2 kJ/mol) for the hydrophobic binding energy of a methylene group, the figure

EDITING OF ERRORS IN AMINO ACID SELECTION

413

usually found by physical-chemical methods from partitioning between hydrophilic and hydrophobic solvents. Although subsequent enzymatic measurements indicated tighter binding to proteins than to hydrophobic solvents (3.4 kcal/mol [14.2 kJ/mol] per methylene group [44]), the initial discrimination between amino acids differing just by one methylene group in their structures cannot be better than a factor of -200, as found for many synthetases in equation 1. This is still not adequate to account for the ability of aminoacyl-tRNA synthetases to distinguish such closely related amino acids with an overall discrimination factor of 104 to 105 (66, 147). In these cases, paradoxically, the overall tRNA aminoacylation reaction is more accurate than the partial activation reaction. DISCOVERY OF EDITING The solution to the problem of how the overall reaction can be more accurate than the partial reaction came from studies of enzyme-bound aminoacyl adenylates formed by isoleucyl-tRNA synthetase (IleRS) (5, 105). The enzyme forms relatively stable cognate E . Ile-AMP and noncognate EIe Val-AMP complexes in the presence of ATP and either isoleucine or valine. Whereas the cognate Eile. Ile-AMP complex reacts with tRNAile to form IletRNAile, the noncognate EIle Val-AMP complex is quantitatively hydrolyzed in the presence of tRNAile and no Val-tRNAile is formed. Thus, in the presence of tRNAI'e, ATP, and valine, the isoleucyl-tRNA synthetase acts as an ATP pyrophosphatase, hydrolyzing ATP to AMP (5). Theoretical analysis of the specificity problem in macromolecular biosynthesis led to Hopfield's proposal of a kinetic proofreading scheme in which intermediate complexes have access to a rejection path in addition to the main path leading to the final product (65, 147). A similar proofreading scheme was independently proposed by Ninio (103). In the kinetic proofreading scheme, discrimination occurs at two steps: first during initial binding and then during editing of the intermediate. A cognate amino acid will flow through the main path, and a noncognate amino acid will be discarded through an irreversible (driven by hydrolysis of ATP) editing path. A diagnostic feature of this editing is ATP hydrolysis in the presence of a noncognate amino acid. The experimental discovery of editing did not demonstrate a specific mechanism. Also, the kinetic proofreading scheme did not propose any specific mechanism of editing (65), as emphasized by Yamane and Hopfield (147). Experimental proof of specific pathways of editing came later (37, 41, 70, 72, 77, 88, 89).

CONTRIBUTION OF EDITING TO SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES The initial recognition of amino acids by an aminoacyltRNA synthetase can be conveniently studied by measuring ATP-PPi exchange in equation 1. Although the measurements are straightforward, they may lead to artifactual results if care is not taken to quantitatively control the purity of amino acid and aminoacyl-tRNA synthetase preparations. In studying errors in amino acid activation, it is clearly important to use amino acid preparations of extreme purity so that error rates as low as 10-5 can be unambiguously detected. Commercial samples of amino acids often contain significant trace quantities of contaminants, which in some cases is as high as 0.5% (for tyrosine contamination in phenylalanine preparations) (88). Extremely poor discrimi-

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JAKUBOWSKI AND GOLDMAN

414

nation of valyl-tRNA synthetase against isoleucine and of phenylalanyl-tRNA synthetase against tyrosine, which was thought to implicate the need for an editing mechanism (68), turned out to be due to the presence of cognate amino acid as a contaminant in preparations of noncognate amino acids (40, 88). Apparent misactivation of isoleucine and valine by methionyl-tRNA synthetase was in fact due to the presence of 0.2% methionine in commercial isoleucine and valine preparations (39). Fortunately, when recognized, these problems can be adequately controlled and will not obscure genuine misactivations and requirements for editing mechanisms.

acids can be inferred from apparent ATP pyrophosphatase activity

Editing of misactivated measurements of an

of

an

amino

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 the editing reaction, in many cases efficient editing occurs in the absence of tRNA (77). This point will be developed further in the section on editing pathways (below). The notion of limited specificity of aminoacyl-tRNA synthetases in initial selection of amino acids and of essentially absolute specificity in tRNA aminoacylation originated in 1961 when Bergman et al. described misactivation of valine by E. coli isoleucyl-tRNA synthetase and of threonine by E. coli valyl-tRNA synthetase (7). The spectra of misactivations by these two synthetases were considerably expanded in subsequent years (68, 72, 77), and new misactivations were discovered for alanyl- (142), leucyl- (27), methionyl(39, 109), and phenylalanyl- (68, 88) tRNA synthetases; valyl- and isoleucyl-tRNA synthetases hold the distinction of being the most promiscuous among the family of synthetases. In all cases, the original notion of an extremely high specificity of an aminoacyl-tRNA synthetase in the tRNA aminoacylation reaction has been confirmed. In general, most misactivations occur at a relatively high frequency and require subsequent correction by editing. Some misactivations, although measurable, are so inefficient that no editing 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 the initial activation reaction that there is no need, and in fact no evidence, for an editing mechanism. The data on misactivation and editing of noncognate amino acids by E. coli aminoacyl-tRNA synthetases, compiled in Table 1, include only studies in which amino acid purity has been documented and both misactivation and editing rates have been measured. Overall selectivities against noncognate amino acids are also given. The data are summarized below. Isoleucyl-tRNA synthetase activates, in addition to its cognate substrate isoleucine, seven other naturally occurring amino acids: isoleucine >> valine > homocysteine > cysteine threonine > homoserine >> ca-aminobutyrate 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 isoleucyltRNA synthetase against noncognate amino acids. Given that valine is just one methylene group smaller than isoleucine, it is not surprising that isoleucyl- and valyl-tRNA synthetases share the same amino acid substrates. Thus, valyl-tRNA synthetase will activate, in addition to valine, -

TABLE 1. Misactivation, editing, and selectivity of E. coli aminoacyl-tRNA synthetases against noncognate amino acids Synthetase and amino acid

Isoleucyl-tRNA synthetased Isoleucine Valine

Threoninef ca-Aminobutyratef Cysteine Homocysteine

Homoserinef Alaninef Valyl-tRNA synthetased Valine Threonine

ca-Aminobutyrate Cysteine Serine Alanine Homocysteine

Homoserinef Isoleucineg

Leucyl-tRNA synthetaseh Leucine Homocysteine

Relative rate of

Relative rate of

1 0.007 0.0002 0.0003 0.0003 0.0025 0.00004 1 x 10-6

1 (0.014 s-1) 43 23 24 100 76 5

1 0.004 0.005 0.001 0.00016 0.000i 0.0002 0.00014 0.000017

1 (0.02 s-') 180 58 90 10 48

editin edim

activation0

1

6,000w 115,000 80,000 330,000 30,000 125,000 8.5 x 106

8.5

1

45,000w 12,000 90,000

62,000 480,000 11,000 60,000

1.6 1

1 (0.08 s-1)i

1 0.0083

Select Slctviy

1

3,000

25

Methionyl-tRNA

synthetased Methionine Homocysteine Norleucinef Ethioninef

Alanyl-tRNA synthetase' Alanine Glycine Serine Cysteine

0.0054 0.005 0.035

1 (0.04 s-1)f 60 5 7

11,000

1 0.004 0.002 0.00008

1 (0.04 s-1) 11.5 23

1 2,900 11,500 > 12,500

1

1

1,000 200

ATP-PP1

a Relative values of kcat/Km in the exchange reaction. The reciprocal of these relative values is defined as the initial selectivity. 6 Relative values of kcat in the ATP pyrophosphatase reaction. Absolute kC,t values 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 elongation factor 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 selectivity against noncognate amino acids which are the most efficiently activated (oa-aminobutyrate, threonine, cysteine) by two orders of magnitude. The initial selectivity of valyltRNA synthetase against isoleucine, homoserine, and homocysteine is adequate (-104), and there is no evidence for their editing; despite similar initial selectivities against ala-

-

-

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nine and serine, these two amino acids are nevertheless edited. Homocysteine, an immediate precursor of methionine in all organisms from bacteria to humans, is the most frequently misactivated amino acid. Four aminoacyl-tRNA synthetases misactivate homocysteine. These include, not surprisingly, methionyl-tRNA synthetase (109), isoleucyland valyl-tRNA synthetases (77) (mentioned above), and leucyl-tRNA synthetase (27). Misactivated homocysteine is transformed into homocysteine thiolactone during editing by the four synthetases in vitro (27, 77). However, homocysteine has also been reported to be stably aminoacylated to tRNAIeU and tRNAVal by leucyl- (27) and valyl- (28) tRNA synthetases, respectively, although this has not been quantitated. There is no mischarging of tRNAMet during editing of homocysteine by methionyl-tRNA synthetase (39). Misactivation and editing of homocysteine, as a result of its unique chemistry (77), have also been studied in vivo (see Editing in Living 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 against glycine and serine is further improved by a factor of 10 to 20 by subsequent editing. The initial selectivity of yeast phenylalanyl-tRNA synthetase against tyrosine is about 2,000 (88). Editing improves the 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 better than the error rate in protein synthesis. (ii) The initial selectivity against noncognate naturally occurring amino acids may be anywhere from 120- to 10,000-fold or better and sets an upper limit for the overall selectivity. (iii) Editing contributes at least a factor of 10 to 100 to the selectivity of an aminoacyl-tRNA synthetase and sets a lower limit for the selectivity. The most efficient misactivations are also the most efficiently edited, and the least efficient misactivations are the least efficiently edited. (iv) Finally, the figures given in the last column of Table 1 show that selectivity, albeit very high, is limited: the initial selectivity component of overall selectivity is variable and depends on concentrations of cognate and noncognate amino acids. This may lead to lower selectivity during unbalanced conditions when one amino acid is in great excess over another, which is observed in vivo (see Relationship between Editing, Selectivity, and Amino Acid Biosynthetic Pathways, below). There have been indirect investigations of the discriminaE + AA + ATP + tRNA

would be difficult to achieve even in the presence of elongation factor Tu to protect putative mischarged tRNA against deacylation (66, 147). However, the authors do not present evidence confirming that the radioactive material incorporated into tRNA is in fact noncognate amino acid. Further, the description in their original Materials and Methods is confusing because they state that no incorporation of noncognate amino acids above background was observed (51). In many cases the reported Km values for noncognate amino acids were equal to or lower than that for the cognate amino acid. This implies that the synthetases would be severely inhibited by noncognate amino acids, which is apparently not the case. Moreover, the authors appear to have measured, under one set of experimental conditions, aminoacylation rates differing as much as 4 x 104-fold (for isoleucyl-tRNA synthetase) or 5 x 105-fold (for tyrosyl-tRNA synthetase), which does not seem to be feasible. In the absence of any supporting evidence, it is difficult to justify quantitative conclusions made from the data obtained with modified tRNAs. Since chemical modifications of the 3' terminus of tRNA influence the mechanism of tRNA aminoacylation (46), the kinetic data obtained with modified tRNAs have different meaning from data obtained with intact tRNAs. This could account for conflicting conclusions reached by the same group with differently 3'-modified tRNAs (50, 51). Moreover, the discrimination factors calculated from measurements with various 3'-modified tRNAs appear to be artifactual at least for E. coli isoleucyl-tRNA synthetase. For example, the initial selectivities calculated by Freist et al. (52) for valine, alanine, cysteine, and threonine are 53, 430, 18, and 85, respectively; however, the initial selectivities obtained from direct measurements in well-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-tRNA synthetase against leucine and glycine, calculated to be 20 and 1,970, respectively, by Freist et al. (52), differ by at least an order of magnitude from the values of 640 and >20,000, which can be calculated from classical direct physicalchemical measurements with the same enzyme (45). EDITING PATHWAYS There are two intermediates on the pathway to aminoacyltRNA. The first is an enzyme-bound aminoacyl adenylate, and the second is an enzyme-bound aminoacyl-tRNA (equation 4).

E AA-AMP. tRNA a E- tRNA + AA + AMP ;

tion against all 19 noncognate amino acids by yeast isoleucyl- (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 from measurements of aminoacylation of native and 3'-modified tRNA with 20 protein amino acids and from accompanying AMP production. Remarkably, the authors appear to have measured, under standard conditions, the formation of misacylated tRNAs with all protein amino acids, a feat that

415

;

E. AA-tRNA

z

E + AA-tRNA

(4)

lb E. tRNA + AA

Both of these intermediates can be proofread, indicated by side reactions a and b in equation 4. Although it was recognized by Baldwin and Berg (5) that editing during rejection of valine by the isoleucyl-tRNA synthetase could occur by the hydrolysis of EI' Val-AMP or could involve transient formation of incorrect Val-tRNAIle followed by its hydrolysis by the enzyme, the mechanism proved to be difficult to identify unequivocally (31). However, there are several clear-cut cases in which either the adenylate (side

416


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

Misacylation-Deacylation Pathway

Following the discovery of editing in tRNA aminoacylation, a substantial amount of circumstantial evidence supporting the misacylation-deacylation pathway for the rejection of valine by isoleucyl-tRNA synthetase accumulated. For example, it was found that synthetases are weak deacylases toward cognate aminoacyl-tRNA but the deacylase activity is higher with some mischarged tRNAs (26, 124, 148). Also, 3'-modified tRNAIl' can be aminoacylated with valine by isoleucyl-tRNA synthetase (145). However, definitive proof was elusive and has never been obtained with this system. In fact, even fast kinetic approaches failed to detect Val-tRNAI'e in reaction mixtures containing substrate amounts of isoleucyl-tRNA synthetase, ATP, valine, and tRNAI'le (31). In addition, the kinetics of deacylation of Val-tRNAI'le (which can be made under artificial conditions) by the enzyme were not consistent with the proposed

misacylation-deacylation pathway (31). The proof of the misacylation-deacylation pathway came

from studies of misactivation of threonine and a-aminobutyrate by valyl-tRNA synthetases from Bacillus stearothermophilus, E. coli, S. cerevisiae, and lupin seeds by using a rapid quenching apparatus to trap and observe the mischarged tRNAVal (33, 37, 41, 77). Threonine is isosteric with valine and is readily activated by valyl-tRNA synthetases to form the enzyme-bound threonyl adenylate. On mixing EVal Thr-AMP with tRNAVal in a rapid-quenching apparatus, Thr-tRNAVal is detected transiently at a maximum of 22% at 25 ms and then disappears after 150 ms (41). Using a similar fast kinetic approach with yeast phenylalanyl-tRNA synthetase, Lin et al. (89) have shown that a minor fraction (4 to 5%) of misactivated tyrosine is transferred to tRNAPhe but the resulting Tyr-tRNAPh, is very rapidly deacylated. By monitoring the fate of Tyr-AMP, these authors also demonstrated that the major part of the misactivated tyrosine is edited by fast hydrolysis of enzymebound tyrosyl adenylate. In an attempt to rationalize observed misactivations in one simple framework, a "double-sieve" model was proposed (32, 33, 40). The experimental basis of the model came from measurements of editing of noncognate amino acids by valyl-tRNA synthetase. The essence of the double-sieve model is the proposal of the existence of two sites: one synthetic site and one hydrolytic site. The synthetic site excludes amino acids larger than the cognate one by steric repulsion. The smaller (and isosteric) amino acids are activated by the enzyme at progressively lower rates as their structures differ more and more from that of the cognate substrate. The hydrolytic site edits the products of misactivation of smaller (and naturally occurring isosteric) substrates. The model was formulated at a time when a limited number of misactivations and only one editing pathway were known (32) and was later modified (33, 35, 40, 142). However, the number of exceptions outweigh the number of cases fitting the model. The double-sieve model breaks down for MetRS, which misactivates and edits noncognate amino acids that are either larger (e.g., ethionine) or smaller (e.g., homocysteine) than the cognate substrate, methionine (Table 1). In addition, efficient editing of homocysteine by MetRS is achieved by just one active site and by a different mechanism from the proposed double sieve (77, 142). Simi-

MICROBIOL. REV.

larly, one active site can account for editing of homocysteine, cysteine (77), and possibly valine (31) by IleRS. Although editing of threonine and a-aminobutyrate by ValRS fit the model by definition, editing of alanine by the enzyme may involve only one active site (31, 77). Editing of tyrosine (89) and serine (142) by PheRS and AlaRS, respectively, also does not obey the double-sieve mechanism since the noncognate amino acids are larger (by an -OH group) than the cognate substrate, yet they both are misactivated and then efficiently edited. In addition, a multistep mechanism of editing of tyrosine by PheRS (88, 89) does not fit the simple idea of a double sieve.

Deacylase Activity of Aminoacyl-tRNA Synthetases The two aminoacyl-tRNA synthetases which edit noncognate amino acids through the misacylation-deacylation pathway also exhibit a weak deacylase activity toward their respective cognate aminoacyl-tRNAs. Moreover, tyrosyltRNA synthetase, which is extremely selective in the initial recognition of its cognate amino acid substrate, tyrosine (68), and does not possess any editing mechanism (44), also does not exhibit any deacylase activity toward Tyr-tRNATYr (99, 145). This may suggest that weak deacylase activity is indicative of the existence of a misacylation-deacylation pathway in amino acid selection by a synthetase. However, several other aminoacyl-tRNA synthetases which either are unlikely to require an editing function, such as seryl-tRNA synthetase (145), or are known not to edit through the misacylation-deacylation pathway, such as methionyl-tRNA synthetase (77), or not to edit at all, such as cysteinyl-tRNA synthetase (38), still possess a weak deacylase activity toward 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 toward Val-tRNAI'le (k = 10 s-1 [26, 31]), but Val-tRNAIle does not

form during editing of valine by isoleucyl-tRNA synthetase (31). Valyl-tRNA synthetase possesses a weak deacylase activity toward Val-tRNAVal (k = 0.02 s-1 [Table 1]) and edits misactivated threonine via deacylation of Thr-tRNAVal (k = 40 s-1 [37]), yet it has been reported to form stable homocysteinyl-tRNAVal (28). Leucyl-tRNA synthetase is known to possess an efficient deacylase activity toward Leu-tRNAJU (k = 0.08 s-1 [71]), but nevertheless it forms mischarged homocysteinyl-tRNAIeu which does not seem to be deacylated by the enzyme (27). A half-life of 15 min given for deacylation of homocysteinyl-tRNAIeu in the presence of leucyl-tRNA synthetase (27) is approximately what can be expected for nonenzymatic deacylation of the mischarged tRNA. Thus, although in some cases a weak deacylase activity is associated with a specific editing function, the general significance of this activity of aminoacyltRNA synthetases toward cognate aminoacyl-tRNA is not clear. A neighboring hydroxyl group of the terminal adenosine is required for enzymatic deacylation of aminoacyl-tRNA. Chemically modified tRNAI'le whose terminal adenosine has been replaced with 3'-deoxyadenosine still accepts amino acids, but the aminoacyl-tRNAIle_C_C-3'dA is not deacylated by isoleucyl-tRNA synthetase (145). This suggested that a 2'-OH is an acceptor site for amino acids and that a 3'-OH is required for deacylation. The requirement for the 3'-OH was explained by proposing that it activates a water molecule that participates in hydrolysis of the adjacent bond of aminoacyl-tRNA (chemical proofreading [28, 67, 68, 145]). Alternatively, the 3'-OH may be required so that the

VOL. 56, 1992


amino acid can migrate onto it and become accessible to the hydrolytic site (hydrolytic editing [41]). This notion of two sites was subsequently elaborated into the double-sieve model (32, 40) (see preceding section). Although tRNA-CC-2'dA was available to test those proposals, the control experiments were not done until 13 years later, when Freist and Sternbach showed that aminoacyl esters of tRNA"'e-CC-2'dA are as resistant to enzymatic deacylation, as are the aminoacyl esters of tRNA'le-C-C-3'dA, implying that the only requirement for deacylation is a free cis-OH, regardless of the position of the aminoacylated hydroxyl group (49). Adenylate Pathway Initial studies of editing in tRNA aminoacylation were greatly influenced by the notion that tRNA directly contributes to the specificity of an aminoacyl-tRNA synthetase by being first misacylated and then deacylated to remove an error. This effectively delayed for several years the discovery of a much more effective and widespread editing pathway in which an enzyme-bound noncognate aminoacyl adenylate is rejected before it can misacylate tRNA; this is known as the adenylate pathway. The evidence for the adenylate editing pathway came from studies with valyl-tRNA synthetase. Valyl-tRNA synthetase forms enzyme-bound adenylates with several noncognate amino acids. Enzyme-bound noncognate aminoacyl adenylates can be isolated by nitrocellulose disk filtration (70, 72). In contrast to enzyme-bound valyl adenylate, which is very stable, enzyme-bound noncognate aminoacyl adenylates are rapidly hydrolyzed and disappear within less than 1 min (70). In effect, valyl-tRNA synthetase acts as an ATP pyrophosphatase in the presence of threonine, ot-aminobutyrate, cysteine, alanine, and serine but in the absence of tRNA (72). Subsequently, very efficient tRNA-independent editing was discovered with isoleucyl-tRNA synthetase (rejecting cysteine and homocysteine) and methionyl-tRNA synthetase (rejecting homocysteine) (77, 132). The unique feature of the editing of homocysteine is the formation of homocysteine thiolactone (equation 5) (77): .+

NH3

NH3

=0

'AMP SH

+ AMP

S

There are three distinct mechanisms by which noncognate amino acids are edited through the adenylate pathway. Examples of each, as well as the relative importance of misacylation-deacylation and adenylate pathways, are discussed in the following section. Relative Contribution of Editing Pathways to Selectivity Editing of errors in amino acid selection by an aminoacyltRNA synthetase can take place by the four routes shown in Fig. 1. (i) The first route is via k1, the dissociation of an enzymebound aminoacyl adenylate to give free aminoacyl adenylate which hydrolyzes in solution (70, 72, 77). This route may contribute to specificity in cases in which the edited noncognate adenylate can be scavenged by its corresponding aminoacyl-tRNA synthetase, which might happen in vivo. The

417

E *AA-AMP * tRNA

-tRNA E + AA + ATP

- PP, + E * AA-AMP

1k,

AMP + E *

jk2

k3

E + AA-AMP - E +

AA*

AA-tRNA --protein

l k4 + AMP

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

best examples of the k1 route are isoleucyl-tRNA synthetase editing cysteine (77) and phenylalanyl-tRNA synthetase editing tyrosine, which is also edited by the k3 and k4 routes (89). (ii) The second is via k2, the tRNA-independent deacylation of an enzyme-bound aminoacyl adenylate (27, 70, 72, 77). Homocysteine misactivated by isoleucyl- and methionyl-tRNA synthetases is efficiently edited by this route, with the formation of homocysteine thiolactone (equation 5). tRNA does not affect the formation of the thiolactone during editing (77), and there is also no evidence for transient mischarging of tRNAMet with homocysteine (39). The k2 route has been shown to exist in vivo (see the following section). (iii) The third is via k3, the tRNA-dependent hydrolysis of an enzyme-bound aminoacyl adenylate without transient mischarging of tRNA (31, 72). The most publicized (although not always correctly) textbook case of editing of misactivated valine by isoleucyl-tRNA synthetase is exclusively by the k3 route. Incorrectly formed valyl adenylate is hydrolyzed by isoleucyl-tRNA synthetase in the presence of tRNAI'le (5), but there is no transient formation of mischarged tRNAI'e (31). There are two other well-documented cases in which efficient editing requires tRNA but the major fraction of the misactivated amino acid is edited by hydrolysis of noncognate aminoacyl adenylate with only a minor fraction (4%) of misacylated tRNA formed transiently: editing of threonine by plant valyl-tRNA synthetase (72, 77) and editing of tyrosine by yeast phenylalanyl-tRNA synthetase (89). In general, tRNA dependence does not necessarily mean that editing involves transient misacylation of tRNA as has frequently been assumed (35); an allosteric change in the synthetase upon binding of tRNA could account for stimulation of the editing. In fact, in some cases enzymatic hydrolysis of noncognate aminoacyl adenylates duirng editing is weakly stimulated by tRNA which has been deprived of its ability to accept amino acids by periodate oxidation

(72). (iv) The fourth is via k4, the deacylation of an enzymebound misacylated tRNA (26, 37, 41, 77, 89, 145, 148). There are only three examples thus far of editing through the k4 route: editing of threonine and a-aminobutyrate by valyltRNA synthetases (reviewed in reference 35) and of tyrosine by 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 misacylationdeacylation pathway. Although it was originally described as being edited exclusively through the k4 pathway, a signifi-

418


cant fraction of threonine misactivated by valyl-tRNA synthetase may also be edited by the k3 pathway. In fact, Lin et

al. (89) have shown that the data of Fersht and Kaethner (41) better fit a model in which a major fraction of Thr-AMP is hydrolyzed before transfer to tRNAVal. There is one problem with the k4 pathway which may account for its limited use by aminoacyl-tRNA synthetases: its contribution to selectivity can be seriously limited by the dissociation of aminoacyl-tRNA from the synthetase. After dissociation, the free aminoacyl-tRNA would be protected by elongation factor Tu against any further deacylation (66, 74, 99, 147) and will insert its amino acid, regardless of whether it is correct, into protein at the positions specified by the anticodon-codon interaction (18). For the k4 pathway to contribute a factor of 10 to the selectivity would require that the deacylation rate constant (kh) be ninefold higher than the dissociation rate constant (kd). However, available data 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 for kd estimated from the molecular activity of isoleucyl- and valyl-tRNA synthetases in vivo are 12.4 and 26.6 s-1, respectively (78), not much different from the respective kh values of 10 s- (31) and 40 s-1 (37, 41). From competitive inhibition of enzymatic deacylation of Thr-tRNAVal by tRNAVal and from the fact that the kinetics of hydrolysis extrapolate 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 dissociates from the enzyme before hydrolysis. Thus, the deacylation of aminoacyl-tRNA contributes only a factor of about 2 to

selectivity. EDMTNG IN LIVING CELLS There is no obvious straightforward way to study editing in vivo. It has even been stated that direct measurements of excess energy dissipation for proofreading in vivo is not technically feasible (69). The question of the in vivo significance of error-correcting processes would have remained unanswered if nature had not provided a useful feature to approach this question in living cells. In particular, several aminoacyl-tRNA synthetases edit misactivated homocysteine by the adenylate pathway with the formation of a unique compound, homocysteine thiolactone (equation 5). This feature of the homocysteine-editing reaction provided a means to assay for editing in vivo by looking for a special chemical 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-tRNA synthetases (MetRS, IleRS, ValRS, and LeuRS) edit homocysteine by converting it into the thiolactone in vitro, only one synthetase, i.e., methionyl-tRNA synthetase, is involved in the thiolactone synthesis in living cells. In preventing errors, it is important that IleRS, ValRS, and LeuRS do not interact with homocysteine in vivo, since at least LeuRS and ValRS form homocysteinyl-tRNAIeu (27) and homocysteinyl-tRNAVal (28), respectively, in vitro. The observations that establish the importance of errorediting mechanisms in living cells are summarized below. (i) Homocysteine thiolactone is a major component of sulfur amino 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. coli and S. cerevisiae mutants that are expected to accumulate homocysteine synthesize massive amounts of the thiolac-

MICROBIOL. REV. +

NH3

NH3

NH3 =0

=0 S

0

Homocysteine thioloctone

Homoserine loctone

H

Ornithine 6-Iactom

FIG. 2. Cyclic forms of some amino acids arising through carboxyl group activation. Homoserine lactone is also referred to as a-aminobutyryl lactone. Ornithine B-lactam is also referred to as 3-amino-2-piperidone.

tone. This establishes a substrate-product relationship between homocysteine and the thiolactone in vivo. (iii) Methionyl-tRNA synthetase mutants defective in the methionine-binding site of the enzyme, in both E. coli and S. cerevisiae, are also defective in homocysteine thiolactone synthesis. In addition, overproduction of methionyl-tRNA synthetase in E. coli and S. cerevisiae leads to proportional overproduction of the thiolactone. This demonstrates that methionyl-tRNA synthetase is responsible for most if not all of the thiolactone synthesis in both E. coli and S. cerevisiae. In fact, there is no evidence for participation of any other aminoacyl-tRNA synthetases or other enzymes in the thiolactone synthesis in vivo. (iv) Synthesis of homocysteine thiolactone in vivo is inhibited by methionine and norleucine but not by any other amino acid; this behavior was previously described for the in vitro synthesis of the thiolactone by bacterial methionyl-tRNA synthetases (77). This also excludes the participation of isoleucyl-, valyl-, and leucyltRNA synthetases in the thiolactone synthesis in vivo. That isoleucyl-, valyl-, and leucyl-tRNA synthetases do not make the thiolactone in vivo is further supported by the observation that ilv mutants unable to synthesize isoleucine, valine, and leucine do not make more thiolactone (as one might have expected from the in vitro data) than wild-type cells. However puzzling, these observations indicate that some specific editing reactions described in vitro may not, in fact, occur in cells (see Relationship between Editing, Selectivity, and Amino 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 distinct nature of the by-product of editing by MetRS, homocysteine thiolactone, which can, by standard procedures, be easily separated from homocysteine and other sulfur compounds present in the cell. There are several other cases in which misactivation and editing can possibly lead to unique chemical products which could be exploited in vivo. For instance, homoserine, a precursor of methionine in microorganisms, is misactivated by isoleucyl- and valyl-tRNA synthetases (Table 1) and subsequently edited by cyclization of homoseryl adenylate to the lactone of homoserine, a-aminobutyryl lactone (Fig. 2) (76a). Homoserine is a significant component of amino acid pools, at least in S. cerevisiae, and homoserine-overproducing yeast mutants (BOR1) exist (130), which makes S. cerevisiae a plausible biological system for in vivo studies of editing by other aminoacyl-tRNA synthetases. Cyclization is expected to also occur with the adenylates of ornithine, aspartate, glutamate, and lysine. It is not known whether these amino acids are misactivated and edited, but assaying for cyclic forms of these amino acid in vivo may provide a means of establishing this point and of further extending in vivo studies of editing. The cyclic

VOL. 56, 1992

B-lactam of omithine, 3-amino-2-piperidone (Fig. 2) (30, 106), is found in urine and, at lower concentrations, in plasma from patients with gyrate atrophy and other conditions associated with hyperomithinemia (reviewed in reference 144). However, the mechanism of its formation from ornithine is not known. COST OF EDITING IN VIVO Some proofreading models postulate that more than one molecule of ATP must be hydrolyzed for each molecule of aminoacyl-tRNA formed in order to maintain the high selectivity of aminoacyl-tRNA synthetases (65). Although this is true for charging of noncognate amino acids to tRNA (66, 147), direct measurements with microbial ArgRS, IleRS, MetRS, PheRS, TyrRS, and ValRS indicate that in the charging of a cognate amino acid to tRNA, the ATP/ aminoacyl-tRNA stoichiometry is one with an upper limit of experimental error of 0.05 (99). Similar ATP/aminoacyltRNA stoichiometries were also obtained in reactions catalyzed 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/aminoacyl-tRNA stoichiometry of 5.5 for Ile-tRNA le formation catalyzed by yeast isoleucyl-tRNA synthetase. At least some of the ATP excess is most probably a consequence of the double-labeling method used, since the same group also reported a high ATP/aminoacyl-tRNA stoichiometry of 1.7 for yeast Arg-tRNA'g (53), in contrast to a stoichiometry of 0.99 + 0.02 reported for the same system by Mulvey and Fersht (99), who used a more accurate kinetic method. It is therefore expected that the extra energy expenditure needed to maintain high selectivity of an aminoacyl-tRNA synthetase will come from editing of a noncognate amino acid. Thus, we can estimate the energy cost of homocysteine editing by methionyl-tRNA synthetase in vivo by relating the amount 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 as thiolactone per 109 molecules of methionine incorporated into protein (75), a value that is in good agreement with the theoretical prediction based on in vitro measurements of Km and kcat values for homocysteine and methionine with methionyl-tRNA synthetase (77) and on in vivo concentrations of the amino acids (75). This is equivalent to an extra energy cost of 0.01 mol of ATP for editing per mol of Met-tRNA formed, a value about 10-fold smaller than previously assumed (69) from calculations based on in vitro data for editing of valine by isoleucyl-tRNA synthetase and of threonine by valyl-tRNA synthetase (121). The extra energy cost of 0.01 mol of ATP per mol of cognate aminoacyl-tRNA formed may seem insignificant per se. However, assuming that the extra energy cost for proofreading during synthesis of Met-tRNA is representative of all aminoacyl-tRNAs, and given the fact that protein synthesis requires 95% or more of all energy used for polymerization reactions in E. coli (69), the energy cost of editing becomes significant in relation to other energy requirements, e.g., for DNA or RNA synthesis. The energy cost of editing in selection of amino acids for protein synthesis can be calculated to be roughly the equivalent of 67 and 39% of the energy used by E. coli for DNA and RNA synthesis, respectively. In S. cerevisiae, one molecule of homocysteine is edited as thiolactone per 500 molecules of methionine incorporated into protein (76), the equivalent of an extra 0.002 mol of ATP expended for editing per mol of Met-tRNA formed. Al-


419

though at the wild-type level of editing the extra energy expended is very small, increasing it can lead to growth inhibition. A homocysteine-overproducing cys2cys4 yeast strain edits 1 molecule of homocysteine as thiolactone per 8 molecules of methionine incorporated into protein (76), thus dissipating 0.13 mol of ATP for homocysteine editing; this strain grows 16% more slowly than the isogenic cys4 strain, which dissipates only 0.0095 mol ATP for editing. The levels of S-adenosylhomocysteine and S-adenosylmethionine are similar in the two strains (76a), making it unlikely that the observed growth inhibition is due to effects of excess homocysteine on intracellular methylations. This magnitude of growth inhibition is astonishing and may indicate that S. cerevisiae is energy limited and cannot tolerate energy waste. The in vivo studies also indicated that cellular levels of MetRS are not limiting for protein synthesis: overexpression of MetRS did not affect the rate of protein synthesis in E. coli (75) or S. cerevisiae (76). At the same time, the rate of homocysteine editing by MetRS in vivo increased in proportion to MetRS overproduction (75, 76). Thus, excess MetRS in the cell becomes detrimental since it leads to wasteful hydrolysis of ATP inadvertently associated with homocysteine editing. This provides at least one reason why cellular levels of aminoacyl-tRNA synthetases have to be carefully regulated (see reference 63 for a review of regulation of

synthetase expression). RELATIONSHIP BETWEEN EDITING, SELECTIVITY, AND AMINO ACID BIOSYNTHETIC PATHWAYS Amino acid biosynthesis has to be regulated not only to allow economical use by microorganisms of frequently limited resources but also to provide a proper balance of metabolites in the cell. A disturbance of this intricate balance, for example by exposing susceptible bacterial cells to a great excess of certain individual amino acids, can result in growth inhibition (20). It is also becoming increasingly clear that high-level expression of recombinant proteins in E. coli produces imbalances in amino acid pools that lead to high levels of errors in proteins (9). In humans, several genetic disorders affect interconversion of amino acids and lead to amino acid pool imbalances in body fluids. This results in severe diseases with numerous clinical manifestations. For instance, cystathionine ,B-synthase deficiency leads to elevation of concentrations of homocysteine in plasma and urine (reviewed in reference 97) (see below). Methionine Pathway and Homocysteine Some amino acid biosynthetic pathways lead to intermediate metabolites which can create selectivity problems for the protein biosynthetic apparatus of the cell, becoming particularly severe under unbalanced or deregulated conditions. In the methionine biosynthetic pathway, the presence of an obligatory intermediate homocysteine poses an acute selectivity 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, homocysteine (Hcy) is methylated to methionine by the product of either 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 transmethylated by the metE gene product, which makes up 5% of the total protein in E. coli (146). Any amount of homo-

420


MICROBIOL. REV.

Ser

Ser

cys2

Hse

Hse

SH-C s

i Cys

SH-

Cys -

-

metE

Hcy --.- Met metH

Met-tRNAmet 100

1

E. coli

i 11 met6

*

cys4

Hcy

-.-

Met

)mes1I

)metG( Hcy thioloctone

t

Hcy thiolactone 0.2

Met-tRNAMet 100

S. cerevisioe

FIG. 3. Schematic representation of the relationship between homocysteine editing and methionine biosynthetic pathways in E. coli and S. cerevisiae. Each arrow represents a step catalyzed by a separate enzyme. Mutations that helped to establish the relationships are indicated. Relative numbers of molecules of homocysteine thiolactone and Met-tRNAMet formed in vivo are given. Abbreviations: 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 provide Met-tRNAMet for protein synthesis. Quantitation of the metabolite flow through methionyl-tRNA synthetase indicates that, in the absence of vitamin B12, 1 mol of homocysteine is edited as thiolactone per 109 mol of methionine incorporated into protein (75). In the presence of vitamin B12, 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 exclusively the vitamin B12-dependent homocysteine transmethylase 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 other aminoacyl-tRNA synthetases edit misactivated homocysteine by transforming it into thiolactone in vitro (27, 77). However, only methionyl-tRNA synthetase is involved in homocysteine editing in vivo (75). The contribution of valyland leucyl-tRNA synthetases to homocysteine editing in vivo can be calculated to be negligible (less than 1%). The isoleucyl-tRNA synthetase, on the other hand, is only twofold less efficient in the editing than methionyl-tRNA synthetase in vitro and should therefore measurably contribute to the thiolactone formation at least in appropriate mutants and even in wild-type cells, in which the isoleucine pool is only twice as large as the methionine pool (113). Second, a methionine-starved metE mutant, which is expected to accumulate homocysteine, does not do so. Instead, the metE mutant elitninates excess homocysteine as the thiolactone. These two observations can be rationalized by proposing that an interenzyme metabolite transfer may be involved in the metabolism of homocysteine in E. coli. This kind of transfer in metabolic pathways has been termed channeling (136). The interenzyme metabolite transfer would lead to compartmentalization of homocysteine reactions to within the methionine biosynthetic pathway. The compartmentation would be important since otherwise homocysteine might also be misactivated by IleRS, ValRS, and LeuRS and possibly charged to the respective tRNAs, as has been observed in vitro with LeuRS (27) and ValRS (28); this would lead to incorporation of homocysteine in protein. This

model proposes interactions between the metE or metH and metG genes and should be readily testable. The strategy used by S. cerevisiae to minimize the need for homocysteine editing is different. This minimization is achieved by the evolution of a capability to transform homocysteine into cysteine (Fig. 3), a pathway that also exists 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 the organization of its methionine biosynthetic pathway. The mutant has relatively large homocysteine pools, as does E. coli, and it edits 1 homocysteine per 100 methionines incorporated into protein, five times more than wild-type yeast but almost exactly the same as E. coli (76). In contrast to E. coli, yeast cells apparently are not under great evolutionary pressure to cope with excess homocysteine, and, when forced by an experimenter to do so, they are very sluggish in this respect. For example, whereas a metE mutant of E. coli is able to eliminate all excess homocysteine as the thiolactone, a yeast met6 mutant, which is an equivalent of an E. coli metE mutant (Fig. 3), accumulates homocysteine and the thiolactone to levels 90- and 500-fold higher, respectively, than the wild-type yeast does (76). It remains to be determined whether homocysteine is incorporated into tRNA and protein in the homocysteine-overproducing yeast met6 mutant. Expression of the metE gene in E. coli and Salmonella typhimurium is regulated by the metR gene. MetR protein is a transactivator of metE expression. Homocysteine has been implicated as a cofactor (143) which enhances binding of the MetR protein to a regulatory region of the metE gene (17). The evidence that homocysteine participates in regulation of metE expression came from molecular genetic studies of expression of metE::lacZ fusions. The expression was enhanced when exogenous homocysteine was included in culture media or when metE or metF strains, which were expected to accumulate homocysteine, were used (143). In addition, in vitro studies of expression of the metE gene, involving coupled transcription-translation with extracts prepared from a metE strain, have shown that exogenously added homocysteine stimulated synthesis of the MetE transmethylase enzyme (17). Homocysteine used in these studies was prepared by base hydrolysis of commercial homocysteine thiolactone, and its purity was not reported. However, since E. coli strains, including metE and metF mutants, efficiently transform any intracellular excess homocysteine into homocysteine thiolactone (75), it is possible that the observed stimulation of metE expression is due to accumulation of the thiolactone, even in the in vitro system. Moreover, although Urbanowski and Stauffer (143) state that they used homocysteine prepared from the thiolactone, their conditions of hydrolysis would result in transformation of less than 10% of the thiolactone to homocysteine. Therefore, more careful studies are needed to determine whether homocysteine or the thiolactone, whose synthesis by MetRS depends on relative concentrations of both homocysteine and methionine (75), participates in MetR-dependent regulation of metE.

Branched-Chain Amino Acid Pathway and Norleucine (Norvaline, at-Aminobutyrate) The branched-chain amino acid biosynthetic pathways have certain degrees of flexibility which can be either advantageous or harmful to the cell. This flexibility can also

VOL. 56, 1992


A. ISOLEUCINE-VALINE PATHWA'

0 pyruvate -

CH3-5H-C-COOH--

-

valine

CH3 a-ketoisovalerate 0

oe-ketobutyrate

-

CH3-CH-C-COOH -

--+

isoleucine

CH2 CH3 a-keto- -methylvalerate B. LEUCINE PATHWAY

0

a-ketoisovalerate

-

CH3-jH-CH2-C-COOH -

-+

leucine

CH3

a-ketoisocaproate 0

a-ketoisocaproate

CH3- H-CH2-CH2-C-COOH

-

homoleucine

CH3 E-ketoisohexanoate 0

a-keto-p-methylvalerate

-

CH3-CH-CH2-C-COOH-

homoisoleucine

CH2 CH3 a-keto-y-methylcaproate 19 CH3-CH2-C-COOH-

pyruvate

a-aminobutyrate

a-ketobutyrate a-ketobutyrate

_

19

CH3-CH2-CH2-C-COOH

norvaline

-

a-ketovalermte 0

a-ketovalerate

-

-

CH3-CH2-CH2-CH2-C-COOH

-

norleucine

a-ketocaproate

FIG. 4. Schematic representation of the branched-chain amino acid biosynthetic pathways in bacteria. The individual steps in the pathways are indicated by arrows. Each parallel step in the isoleucine-valine pathways (A) is catalyzed by the same enzyme. The four steps of the leucine pathway (B) are catalyzed by another set of enzymes. The transamination reactions in the pathways are catalyzed 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 will increase the chain length of several ,-ketoacids and produce unusual amino acids, as indicated previously (8, 80).

create problems, only recently recognized, with using E. coli

strains for high-level expression of recombinant proteins. Fortunately for commercial applications of recombinant DNA technology, these problems can be easily avoided. ao-Ketoacid intermediates, in addition to being transformed along the major pathways ultimately into valine, isoleucine, or leucine, give rise to several nonprotein amino acids as depicted in Fig. 4. Some of these nonprotein amino acids are important components of antibiotics and therefore confer selective advantage to organisms producing them. For instance, a-aminobutyrate is a component of cyclosporin A, a cyclic undecapeptide with anti-inflammatory, immunosuppressive, antifungal, and antiparasitic properties that is produced by the fungus Beauveria nivea (85). Norvaline is present in an antifungal peptide produced by Bacillus subtilis (102). Homoleucine, a homolog of leucine, is a component of an antibiotic produced by Streptomyces diastaticus (131). However, some by-products of the branchedchain amino acid biosynthetic pathways, such as a-aminobutyrate (80, 86), norleucine, and norvaline (8, 80, 141), can create serious selectivity problems for the protein biosynthetic apparatus of the cell. a-Aminobutyrate is easily misactivated by valyl-tRNA synthetase and then edited (37).

421

Norvaline is efficiently misactivated by isoleucyl-tRNA synthetase (64, 91) and most probably edited, but this has never been tested. ex-Aminobutyrate accumulates in E. coli cultures supplemented with valine (86). Wild-type strains of Serratia marcescens contain small pools of ao-aminobutyrate which increase in certain regulatory mutants (80). The Serratia marcescens mutants also accumulate norvaline (80). Norleucine is not only misactivated but also charged to methionine tRNAs by methionyl-tRNA synthetase (140). Since the methionyl-tRNA synthetase does not edit norleucine efficiently (39) (Table 1), this amino acid is subsequently incorporated into protein in place of methionine (6, 12, 79). Norleucine is bacteriostatic to many microorganisms (114). In E. coli and Serratia marcescens, mutations resulting in resistance to norleucine occur in metK (62) and metA (81) loci, respectively. However, E. coli is also surprisingly tolerant to norleucine and can exhibit limited growth in its presence. Substitution of one-half of the normal methionine residues with norleucine in E. coli proteins does not lead to immediate loss of cell viability (6). Proteins containing norleucine at all of their methionine sites have been made. In a few cases studied so far, the biological activities of the norleucine-containing proteins were identical to those of unsubstituted proteins. In addition, two E. coli norleucine-substituted proteins, P-galactosidase (101) and adenylate kinase (59), were more resistant to alkylation and oxidation, respectively, than their normal methionine-containing counterparts. Three norleucine-containing proteins from other species, Staphylococcus aureus nuclease (4), recombinant human epidermal growth factor (82), and interleukin-2 (141), produced in E. coli, all exhibited full biological activity. Biological activity of another norleucine-containing recombinant protein produced in E. coli, bovine somatotropin, was not reported (8). Norleucine, first detected in vivo in an isoleucine-valine auxotroph of Serratia marcescens when threonine was included in the frementation medium (80), has been proposed to be synthesized from a-ketobutyrate through two cycles of the leucine biosynthetic pathway (Fig. 4B). Norvaline, which is also present in these cells, arises from a-ketobutyrate in one cycle of the leucine pathway (80) (Fig. 4B). Unexpectedly, norleucine has been detected in recombinant interleukin-2 and bovine somatotropin produced in E. coli grown in the absence of exogenous norleucine (8, 93, 141). Free norleucine and norvaline, which appear during highlevel expression of these leucine-rich proteins, have been shown to be products of the derepressed leucine biosynthetic pathway. Norleucine, norvaline, and several a-ketoacids were detected in recombinant E. coli cultures but were absent when an E. coli strain with a deletion of the leucine operon was used. The derepression of the leucine pathway was brought about by leucine limitation caused by high-level synthesis of leucine-rich recombinant proteins (8). Incorporation of norleucine into protein is prevented by including leucine and/or methionine in the fermentation media (141) or by using a leucine operon deletion E. coli strain (8). Incorporation of norleucine into proteins is due to mischarging of tRNAMet with norleucine by methionyl-tRNA synthetase. This is the only mischarging error known in which a naturally produced noncognate amino acid is mistakenly used by a synthetase to charge its cognate tRNA in vivo. Methionyl-tRNA synthetase has an efficient proofreading mechanism which prevents incorporation of homocysteine into tRNAMet and protein in vivo (75, 76) but this mechanism is not used for editing of norleucine (39). Appar-

422


ently, E. coli has not been under evolutionary pressure to evolve such a mechanism to prevent incorporation of norleucine into tRNA and protein. Instead, E. coli developed tight controls over the branched-chain amino acid biosynthetic pathways which prevent norleucine accumulation under most growth conditions in the first place. Other Possible tRNA Misacylation Errors Due to Amino Acid Pool Imbalances

High-level expression of recombinant proteins in E. coli may lead to other types of missense errors. As much as 11% of the total recombinant atrial peptide III molecules contain lysine at sites coded for by AGA, but not by CGU, arginine codons. Similar levels of lysine misincorporation at AGA arginine codons have been observed in insulinlike growth factor type 1 obtained from the overproducing strain. When AGA codons were replaced with CGU codons, lysine misincorporation was eliminated from both peptides (9, 129). It is not known whether lysine misincorporation is due to the lysine AAA tRNA misreading the rare arginine codon AGA or the arginyl-tRNA synthetase mischarging the rare arginine AGA tRNA with lysine. However, amino acid pool imbalances seem to play a role: supplementation of the culture medium with arginine reduced lysine misincorporation to 1% (9). Measurements of phenylalanine misincorporation into mouse epidermal growth factor overproduced in E. coli indicated an error frequency of about 1 in 40 for codons differing by a single base from phenylalanine codons (127). In these experiments, a synthetic mouse epidermal growth factor gene with a codon bias optimized for high-level expression in E. coli was used. Despite this, the gene generated a product with an error frequency at least 10-fold higher than the error rate found for normal E. coli proteins. Phenylalanine misincorporation into mouse epidermal growth factor was not significantly affected by using a strain (rpsL282) with hyperaccurate ribosomes (127), which may indicate 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 may resemble problems presumably encountered by specialized (differentiated) eucaryotic cells early in evolution. For the sake of argument, we can assume that an E. coli cell producing high levels of a recombinant protein product is an equivalent of a differentiated eucaryotic cell, for instance an erythrocyte which is a high-level globin producer. There is one important difference, however. A recombinant E. coli cell produces a significant proportion of faulty proteins because it cannot appropriately regulate its amino acid biosynthetic pathways during high-level recombinant gene expression. An erythrocyte produces essentially perfect globin molecules, since it has, as all mammalian cells have done a long time ago, disposed of most of its amino acid biosynthetic genes. However, by deleting an amino acid biosynthetic operon, e.g., the leucine operon, from our recombinant E. coli, we avoid deregulation of the pathway which would lead to imbalances in amino acid pools and even to production of novel amino acids, normally not made by a nonrecombinant bacterial cell. Such a deletion strain produces essentially error-free proteins, as the erythrocyte does.

MICROBIOL. REV.

Implications for Disorders of Amino Acid Metabolism in Humans

The evidence discussed above indicates that imbalances in amino acid pools in microorganisms lead to errors in tRNA charging. Although studies of errors in amino acid selection in higher eucaryotic systems are virtually nonexistent, it is likely that amino acid pool imbalances would also lead to similar tRNA mischarging errors in mammalian cells. This might happen in a variety of human genetic disorders whose most conspicuous biochemical manifestations include amino acid pool imbalances. As an example, the possible involvement of mischarged tRNA in cystathionine j-synthase deficiency is considered below. In humans, as in all mammals, the transsulfuration pathway converts methionine, an essential amino acid, into cysteine (reviewed in reference 97). The pathway starts with formation of S-adenosylmethionine, which yields S-adenosylhomocysteine in subsequent transmethylation reactions. S-Adenosylhomocysteine is further metabolized to homocysteine and adenosine. In addition to being remethylated back to methionine, homocysteine is condensed with serine to form cystathionine in a reaction catalyzed by cystathionine ,B-synthase. Cystathionine is finally cleaved by the enzyme y-cystathionase to cysteine and a-ketobutyrate. Cystathionine (-synthase deficiency (reviewed in reference 97) is a genetic disorder of transsulfuration which occurs with a frequency of 1 in 200,000 in the general population. The disorder is inherited as an autosomal recessive trait. The most characteristic feature of cystathionine P-synthase deficiency is the presence of elevated levels of both homocysteine and methionine in plasma. However, as many as 6% of older cystathionine ,B-synthase-deficient patients who are homocysteinemic and homocysteinuric do not have methionine levels in plasma above the normal range. Excess homocysteine is excreted in urine. Several organ 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 among individuals with cystathionine ,-synthase deficiency. A major cause of premature death of these individuals is thromboembolism. No aspect of cystathionine ,B-synthase deficiency has remained as obscure as the intermediate steps by which the enzyme deficiency leads to the specific clinical manifestations associated with it. Although cystathionine ,B-synthase deficiency always leads to elevation of homocysteine levels, homocysteine thiolactone has not been detected in the serum or plasma of homocysteinuric patients (22) or normal subjects (94, 98). However, the sensitivity of the methods used in these studies was relatively poor and would allow detection of the thiolactone only at concentrations greater than 32 ,M (98) or 50 ,uM (22); this sensitivity may not be sufficient since total concentrations of homocysteine in plasma are only up to 10 ,M in normal subjects and up to 200 ,M in homocysteinuric patients (97). Elevation of homocysteine levels in S. cerevisiae, including cystathionine 13-synthase-deficient (cys4) mutants, 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 that is not used for transmethylation to methionine is transformed into the thiolactone. Homocysteine never accumulates in metE and metF mutants of E. coli; homocysteine thiolactone accumulates instead (75). It would be interesting to use more sensitive methods to determine whether the

VOL. 56, 1992

thiolactone is present in humans. The presence of thiolactone, at least in homocysteinuric patients, and participation of MetRS in its formation would indicate preservation of the editing mechanism of MetRS in humans. On the other hand, since homocysteine is synthesized by entirely different pathways in humans and microorganisms, human MetRS may not have the need and therefore may have lost the ability to edit 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 tRNA mischarging (see previous sections). Since thioamino acid pool imbalances are always associated with cystathionine ,B-synthase deficiency, it is tempting to suggest that the pleiotropic manifestations of a single genetic defect in this disorder are also due to errors in proteins. These errors can be due to mischarging of tRNA with homocysteine, which can be facilitated by high levels of homocysteine in cystathionine ,-synthase-deficient patients. Of four microbial aminoacyl-tRNA synthetases that misactivate and edit homocysteine in vitro, LeuRS and ValRS are able to form homocysteinyl-tRNA in vitro (27, 28). Corresponding mammalian synthetases may have this ability as well. If tRNAL'u, tRNAVal, and possibly tRNAIle are mischarged with homocysteine in patients with cystathionine ,-synthase deficiency, simple supplementation of diets with leucine, valine, and isoleucine may ameliorate harmful effects of the deficiency. Whether this hypothesis is correct can be determined by studying the amino acid selectivity of mammalian MetRS, IleRS, ValRS, and LeuRS and of errors in protein synthesis in homocysteinuric patients. Amino acid pool imbalances leading to errors in tRNA charging may also play a role in several other human disorders. Possible examples include gyrate atrophy and other conditions associated with hyperornithinemia (see the last paragraph of the section on editing in living cells) and phenylketonuria (characterized by excess phenylalanine). MOLECULAR BASIS FOR SELECTIVITY OF AMINOACYL-tRNA SYNTHETASES Aminoacyl-tRNA synthetases differ considerably in size and quatemary structure and have limited sequence homology. However, a common pattern can be found in the organization of the structures of these enzymes (15). The functional units of aminoacyl-tRNA synthetases are arranged along the amino acid chains in such a way that the aminoacyl adenylate domain is located in the N-terminal half of the protein. The tRNA recognition domain partially overlaps the adenylate domain and extends far to the C-terminal portion of the protein (122, 133). X-ray crystallography and computer analysis of amino acid sequences have revealed structural similarities between aminoacyl-tRNA synthetases and have led to the classification of this family of enzymes into two groups (15, 19, 29, 100, 123). Class I

synthetases (ArgRS, CysRS, GlnRS, GluRS, IleRS, LeuRS, MetRS, TrpRS, TyrRS, and ValRS) have two short consensus sequences (HXGH, where X is a hydrophobic amino acid that is frequently isoleucine, and KMSKS) which form part of the structural domain (the Rossman fold) that binds ATP, as observed in three crystal structures (GlnRS, MetRS, and TyrRS). Class II synthetases do not have the Rossman fold (e.g., SerRS [19]) but share three new sequence motifs (29).

Since aminoacyl-tRNA synthetases share the common substrate ATP and recognize a-amino and carboxyl groups of their cognate amino acid substrates, it may not be


423

surprising that homologous amino acid residues that make up the aminoacyl adenylate domain of class I synthetases are engaged in common functions, i.e., interactions with ATP and a-amino and carboxyl groups of amino acid substrates. The first histidine residue of the HIGH sequence interacts with ATP in the crystal structures of MetRS and GlnRS complexed with ATP (13, 118). Site-directed mutagenesis experiments indicate that both histidine residues participate in the activation of tyrosine by TyrRS (36). The second lysine of the KMSKS sequence lies near the y-phosphate of ATP in the crystal structure of the MetRS ATP complex and is seen to interact with the phosphates of ATP in the structure of the GlnRS ATP tRNA In complex (118). In TyrRS the two corresponding lysine residues stabilize the transition state for Tyr-AMP formation by strongly interacting 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 features common to all amino acids, i.e., c-amino and carboxyl groups 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 the structures of aminoacyl-tRNA synthetases homologous amino acid residues which interact with a-amino and carboxyl groups of the substrate as well as unique residues which provide specificity. What little is known about amino acid-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 crystallographic and site-directed mutagenesis studies of tyrosyltRNA synthetase (36). Residues Asp-78, Tyr-169, and Gln173 form a binding site for the a-amino group of tyrosine (Fig. SA). Glu-195 has been postulated to interact with the carboxyl group of tyrosine during the activation reaction (11, 43). The specificity site for tyrosine against phenylalanine is composed of Asp-176 and Tyr-34 (43). Asp-176 functions as a hydrogen bond acceptor of the substrate hydroxyl; Tyr-34 functions as a hydrogen bond donor. Replacement of Tyr-34 by Phe does not significantly alter kca, and increases Km for tyrosine only twofold. However, the kcatlKm value for activation of phenylalanine increases sixfold. Altogether, the mutation Tyr-34 -- Phe decreases discrimination against phenylalanine 15-fold. Unfortunately, mutation of Asp-176 has not yielded an active enzyme (36). Studies of tyrosyl-tRNA synthetase have led to an understanding of the molecular mechanism of amino acid activation and specificity in unprecedented detail and also considerably expanded at the molecular level our ideas of general concepts in enzymatic catalysis and biological specificity such hydrogen bond and induced fit (42, 43). Because this synthetase is essentially absolutely specific for tyrosine, the enzyme does not possess or need editing ability. Phenylalanine is activated 1.5 x 105 times less efficiently than tyrosine by tyrosyl-tRNA synthetase (44). This discrimination is achieved exclusively through differences in initial binding energies of cognate (Tyr) and noncognate (Phe) substrates. Therefore we must look for another system to study the molecular basis of the other component of selectivity,

namely, editing. Fortunately, methionyl-tRNA synthetase provides such a

system. The methionyl-tRNA synthetase does have a problem discriminating against homocysteine, the immediate precursor of methionine in the cell. Homocysteine is activated by the methionyl-tRNA synthetase only 100 times less frequently than methionine is in vivo. Incorporation of

424

MICROBIOL. REV.


A Arg86 NH

\C+=NH2

Thr4O I UH .

His45

\N.

NH2*.

Lys82 NH

Asp7l

-COz2

H

*+

Lys230 /

Lys233

3

H

H

O .H-N -C- CO -P-0 A 00 - * *CH

iI2:i

CH

NH. :N

2K

.V¶o kpz. 0

76C2.

H3 N

.

0 .3o IH . ~~0

Tyri1 69-6H Aspl

+

p

+

NH-

Glnl,73-C=O*

U

,\

O

Tyr34 -OH

Thr5l I ... HO *His48

HS- Cys35

B Tyr358 Arg233

+

I~

OH

Ns-

t-NH ~

~

`C+-=NH2

H3 N

*

0

Lys335

3

*p 0

0

NH2.

H

H

/

I

.

0

.H -N+ -C-CO2P-p/

Asp52

-

CO2- *

H

IH

CH2 CH

NH2

/ \ 0

0

N

CH2

I'll

~~~~+

0

11e231

/0 0\ Trp3O5

H

H

. . . .

.

.

0

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

VOL. 56, 1992

homocysteine into tRNA and protein is prevented by an efficient editing function of the methionyl-tRNA synthetase (75, 76). Crystal structures of free methionyl-tRNA synthetase and of its complex with ATP have been solved (13), and site-directed mutagenesis studies of the methionyl adenylate 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). Subsequent site-directed mutagenesis of some of the putative methionine-binding-site residues led to identification of Asp-52 and Arg-233 as playing an important role in stabilization of the transition state for Met-AMP formation, possibly by interacting with the a-amino and carboxyl groups, respectively, of the methionine substrate (Fig. SB). The Asp-52 residue of MetRS corresponds to Asp-78 of TyrRS, and all but one of class I aminoacyl-tRNA synthetases have an aspartic acid residue at a site corresponding to Asp-52 in MetRS (58). The synthetases most closely related to MetRS (such as IleRS, LeuRS, and ValRS) also have a conserved arginine at the position corresponding to Arg-233. The specificity site for methionine contains Trp-305. Substitution of Ala for Trp-305 leads to a 200-fold decrease in efficiency of methionine activation (58). The Trp-305 -- Ala substitution does not affect interaction of the synthetase with the noncognate substrate homocysteine, since mutant methionyl-tRNA synthetase with an alanine residue at position 305 misactivates and edits homocysteine as efficiently as the wild-type enzyme does (76a).

CONCLUSIONS In this review, we have described and compared the pathways and consequences of editing of errors in selection of amino acids by aminoacyl-tRNA synthetases, which is a crucial quality control point in maintaining the accuracy of reading the genetic code. Several synthetases have to distinguish between the correct substrate and a homolog differing by just one methyl group; this binding energy has been estimated to contribute only a factor of 100 to selectivity, whereas synthetases distinguish such closely related substrates by a factor of 104 to 105, by editing. Although the early information about editing by aminoacyl-tRNA synthetases was developed through in vitro studies, new approaches taking advantage of the unique chemical products of some editing reactions have enabled editing to be studied in vivo as well. The in vivo studies have established the importance of editing in living cells, assessed the energy cost of editing (which can be reflected in the growth rate), and added new perspectives on the necessity of cells to carefully regulate both synthetase levels (to limit energy waste from excessive editing) as well as amino acid biosynthetic pathways, which might otherwise create selectivity problems for aminoacyl-tRNA synthetases and the protein-synthesizing apparatus of the cell in general. In this regard, high-level expression of recombinant proteins stresses the protein synthesis machinery, exacerbating misincorporation, which is in part a consequence of misactivation by aminoacyltRNA synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA charging, adding a new perspective to future studies of such disorders. The molecular basis for selectivity, including editing function, of aminoacyl-tRNA synthetases at the physical-chemical level is also beginning to be elucidated in some systems, by combining X-ray crystallographic structures and site-directed mutagenesis with functional assays or phenotypes. A considerable body of knowl-


425

edge and understanding has now evolved about this important biological phenomenon of editing of errors by aminoacyl-tRNA synthetases, which is a universal property of all living cells. ACKNOWLEDGMENT We gratefully acknowledge support by a research grant (GM27711) from the National Institutes of Health.

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37. Fersht, A. R., and C. Dingwall. 1979. Establishing the misacylation/deacylation of tRNA pathway for the editing mechanism of prokaryotic and eukaryotic valyl-tRNA synthetases.

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