Proc. Nati. Acad. Sci. USA Vol. 74, No. 6, pp. 2246-2250, June 1977

Biochemistry

Experimental evidence for kinetic proofreading in the aminoacylation of tRNA by synthetase (stoichiometry of energy coupling/aminoacyl tRNA synthetase/error rate in biosynthesis)

T. YAMANE* AND J. J. HOPFIELD*t * Bell Laboratories, Murray Hill, New Jersey 07974; and t Department of Physics, Princeton University, Princeton, New Jersey 08540

Contributed by J. J. Hopfield, March 7, 1977

and B, which, for the sake of simplicity, form enzyme complexes at similar rates. Let us furthermore define a branching ratio for an enzyme-substrate complex, g = Vmax forward/k dissociation rate. Then the discrimination between A and B at equal concentrations will be given by the discrimination factor or error fraction fo = gB/gA. A given overall discrimination factor, f~n, could be achieved by a sequence of n similar Michaelis-Menten reactions requiring n enzymes. As proposed by the kinetic proofreading theory, the same order of discrimination could be attained with one enzyme and a series of "irreversible" steps with side reactions or rejection paths. The necessary irreversibility could be achieved by coupling to the hydrolysis of a high-energy compound such as ATP. Thus, the cleavage of pyrophosphate from ATP in the presence of inorganic pyrophosphatase would provide a thermodynamic "push" in the cell and ensure irreversibility of the reaction. Selectivity would be achieved by providing a series of such driven steps or a single very high energy intermediate cascading down to lower intermediates, some or all of which have access to a rejection path. For high selectivity, the rate of a rejection path for incorrect substrates should be much higher than the forward rate, whereas for correct subtrates it may be comparable. The essential features of kinetic proofreading are then (i) initial complex formation, (ii) hard-driven (irreversible) step(s), and (iii) one or more rejection paths constituting additional discriminating processes. The generally accepted mechanism for aminoacylation of tRNA shown in the scheme below can accommodate the three essential elements of the kinetic proofreading theory: ---o- (AA.AMP).E AA + ATP + E (a) = AA-ATPE (b)

ABSTRACT The enzymatic aminoacylation of tRNA can be viewed as a means of proofreading either the amino acid or the tRNA or both. We have conducted further experimental tests of kinetic proofreading in discriminating between cognate and noncognate amino acids and tRNAs as follows: (i) Thr + tRNAVal y

tR

synthetase

Thr-tRNAval;

tyIoy-tRNA

(ii) D-Tyr + tRNATYr tyros

(iii) Ile + tRNAPhe

synthetase isoleucyl-tRNA

(iv) Ie + tRNA3fMet i

>

synthetase

ioleucyl-RNA synthetase

TyrtRNATYr;

Ile-tRNAPhe; IletRNAfMet;

Met isoleucyl-tRNA Met 30 (v) Val + tRNA3Mt Val-tRNA3fe synthetase In cases (i) and (ii) the amino acids are proofread, in cases (iii) and (iv) the tRNA is proofread, and in case (v), both the amino acid and the tRNA are proofread. ATP consumed per acylation was 400, 1.5, 40, 25, and 1000, respectively. High ATP/ aminoacylation ratios are diagnostic for kinetic proofreading.

According to our general understanding, the primary amino acid-specific step in the biosynthesis of protein is the enzymatic aminoacylation of tRNA, with the specificity of subsequent steps residing in codon-anticodon interactions. It has been estimated that the maximum frequency of replacement of a correct amino acid (as encoded in the genetic material) by a sterically similar incorrect one is less than 1 in 104 (1-3). On the basis of energetic considerations it has been calculated that in a Michaelis-Menten enzyme-substrate interaction with one recognition step, discrimination against smaller isosteric compounds is no more than 1 in 20 (4-6)-e.g., isoleucyl-tRNA synthetase [IRS; L-isoleucine:tRNAIIeligase (AMP-forming), EC 6.1.1.5] with a binding site for isoleucine cannot completely exclude the smaller valine which has a hydrogen atom in place of methyl group. This calculation leads one to conclude that there may be a succession of discriminating processes holding error to the observed, biologically tolerable level. The kinetic proofreading theory (7) provides an explanation for how this might be achievedt. In a Michaelis-Menten scheme, the enzyme-substrate complex either goes "irreversibly" on to completion or dissociates to give the initial reactants. Let us consider two substrates, A

+

'I

PP,

-

2Pi tRNA

111

(AA-AMP).E tRNA AMP E + AA + tRNA '-

(C)

I-1

E-(tRNAAA)

It AA-tRNA + E

in which (a) is an initial discriminatory Michaelis-Menten complex (AA-ATP-E), (b) is an energy-coupled irreversible step, and (c) is one or more intermediate complexes which, in addition to the main path, can proceed along rejection or side path(s). To test the present hypothesis, it would suffice to measure the number of energy-coupled steps per aminoacylation. The chemical form in which erroneous material escapes or is rejected and the detailed nature of intermediates are not important to the kinetic proofreading concept. The basically equivalent models of chemical proofreading (10) and

Abbreviations: IRS, isoleucyl-tRNA synthetase [L-isoleucine:tRNAIle ligase (AMP-forming), EC 6.1.1.5]; VRS, valyl-tRNA synthetase [Lvaline:tRNAVal ligase (AMP-forming), EC 6.1.1.91: TRS, tyrosyl-tRNA synthetase [L-tyrosine:tRNATYr ligase (AMP-forming), EC 6.1.1.1]; Tu, elongation factor Tu. f However, this does not preclude the existence of alternative or additional mechanisms for control of abnormal proteins (e.g., refs. 8 and 9).

2246

Biochemistry:

Yamane and Hopfield

Proc. Natl. Acad. Sci. USA 74 (1977)

?the aminoacylation and the quantitation of ATP hydrolyzed were carried out as previously described (12, 23).§ The amount of AMP produced was measured according to Remy et al. (24, 25). Other experimental details are given in legends to the figures. RESULTS With VRS. These reactions were:

hydrolytic editing (11) are special casesof the mo4i;ral notion of kinetic proofreading. In a previous communication (12), direct experimental evidence for kinetic proofreading in the aminoacylation of tRNAIle was presented. The stoichiometry between ATP hydrolyzed and tRNAIle acylated by IRS with the cognate amino acid isoleucine or the noncognate one valine was shown to be 1.5 or 270, respectively. These ratios demonstrate that kinetic proofreading results in a reduction of error to the extent of 1.5/270. Because the Michaelis constants for formation of the synthetase-amino acid complex with Val and lie differ by a factor of 100 (13), an overall error rate of about 10-4 is obtained in this first step of protein synthesis. The aminoacylation reaction scheme can be viewed as a means of proofreading either the amino acid or the tRNA or both. We have conducted further experimental tests of kinetic proofreading by synthetase in discriminating between cognate and noncognate substrates as follows:

Val + tRNAVal

Thr + tRNAVal

vlyl-tRNA

synthetase

valyl-tRNA 3

synthetase

Val-tRNAVa1 Thr-tRNAVal

(11).

The present case is essentially identical to the case of valylization of tRNAIle by IRS studied before (12), in which, in order to observe misacylation, Tu-GTP was added to the reaction mixture. This factor binds aminoacylated tRNA strongly and might therefore be expected to protect acylated tRNA from enzymatic hydrolysis (27, 28). This expectation was borne out. Acylation was very fast in the case of the cognate amino acid and the concomitant hydrolysis of ATP ceased with the acylation. The reaction with threonine showed a slow but continuous acylation, reaching only 1% in 30 min of incubation accompanied by simultaneous hydrolysis of ATP. The stoichiometry of the reactions can be taken directly from the slopes of the plot of the amount of acylation versus the quantity of ATP hydrolyzed, without the necessity of considering kinetic detail. The values obtained were 1.5 for valine and 400 for threonine. With TRS. These reactions were:

synthetase

3 (b) D-Tyr + tRNATYrTrtyrosyl-tRNA DTyr-tRNATyr

synthetase

IetRNAPhe

synthetase

fMtisoleucyl-tRNAfMe . (d) Ile + tRNA3fMet Ile-tRNA3fMet synthetase

fMtisoleucyl-tRNA ) (e) Val + tRNA3fMet Val-tRNA3fMet fe

synthetase

In cases (a) and (b) the amino acid is proofread, in cases (c) and (d) the tRNA is proofread, and in case (e) both the amino acid and the tRNA are proofread. MATERIALS AND METHODS Isoleucyl-tRNA synthetase from Escherichia coli B was isolated as described-by Baldwin and Berg (14) and modified by Eldred and Schimmel (15), valyl-tRNA synthetase [VAS; L-valine: tRNAVal ligase (AMP-forming), EC 6.1.1.91 according to Yaniv et al. (16, 17), and tyrosyl-tRNA synthetase [TRS; L-tyrosine: tRNATYr ligase (AMP-forming) EC 6.1.1. 1] according to Calendar and Berg (18-20). All were stored at -20° in 50% glycerol (vol/vol). The preparations were free of ATPase activity and the TRS preparation showed no D-tyrosyl-tRNA deacylase activity (18-20). Elongation factor Tu, prepared as described by Miller and Weissbach (21), was supplied by D. Miller. Tu-GTP complex was prepared as described before (12). tRNA3fMet (batch no. 18-290) from E. coli K12 was obtained from the National Institute of General Medical Sciences-sponsored purification program at Oak Ridge National Laboratory. tRNAPhe, tRNATYr, and tRNAVal from E. coli MRE 600 were purchased from Boehringer-Mannheim. The tRNA3fMet and tRNAPhe samples were found to contain about 1.5% and 0.5% tRNAIle, respectively, and these preparations were further purified through an.RPC-5 column (1 X 25 cm) (22). All radiochemicals were obtained from New England Nuclear: ['-32P]ATP (3.95 Ci/mmol); [a-32P]ATP (36.97 Ci/ mmol); L-[3H]valine (1.3 Ci/mmol); L-[3Hlisoleucine (83.4 Ci/mmol); L-[3H]threonine (2.0 Ci/mmol); L-[ '4C]tyrosine (420 mCi/mmol); D-[14C]Tyr (47.6 mCi/mmol). Unlabeled ATP, GTP, L-valine, L-tyrosine, D-tyrosine and L-threonine were purchased from Calbiochem. Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) was obtained from Worthington and pyruvate kinase (ATP: pyruvate 20-phosphotransferase, EC 2.7.1.40) from Calbiochem.

v

It has been known from earlier work by Bergmann et al. (26) that E. coli VRS forms threonyladenylate (Thr-AMP) with Vmax of about 30% the rate of valyladenylate formation but fails to yield Thr-tRNAVal. Similar kinetic studies and observations with VRS from Bacillus stearothermophilus have also been reported

(a) Thr + tRNAVal ThrvtRNA Y

(c) Ile + tRNAPhe isoeucy-tR

2247

L-Tyr + tRNATvr

t% rosv l-tRNA

L-Tyr-tRNATyr

ss nthetase

D-Tvr + tRNAT>r

tSrosvi-tRNA ''

sN.i1thetase

D-Tvr-tRNAT'r

Calendar and Berg (18-20) reported the surprising observation that D-tyrosine was esterified to tRNATYr by E. coli and B. subtilis TRS to the same extent as L-tyrosine and also that it was incorporated into peptide linkage. They reported values for Vmax of 2.6 and 0.11 for the acylation of L- and D-tyrosine, respectively. Under our experimental conditions, these amino acids behaved as described by Calendar and Berg and the amount of ATP hydrolyzed was practically identical in both cases, slopes being 1.3 and 1.5 for L- and D-tyrosine, respec-

tively. With IRS. The reaction was:

Ile + tRNAPI'e

isoletivS I-tR NA sV llthet{ase

ie-tIRNAPtle

A detailed study of Yarus (29) on the misacylation of tRNAPhe with isoleucine by IRS showed unequivocally that there is a measurable amount of misacylation under normal conditions and that it can be greatly enhanced by the addition of methanol. The association constant of IRS for tRNAPhe is 20

< 0.05

_

O

CO

'5 _

0 >

-

C

ot 30 I

20

-0

o 20

C-

0 /2 0

[

101-

slope = 40

-


~~~~~~5

0