Aminoacids and the anticodon: Anticodon interaction: A test of the stereochemical hypothesis?

BIOCH1M1E, 1981, 63, 77-81.

Aminoaeids and the anticodon: Anticodon interaction" A test of' the stereochemical hypothesis .9 Damian LABUDA + and Henri GROSJEAN <>.

(Refue le 12-09-1980, acceptde aprks rdvision le 13-11-1980).

+ Department oJ Biochemistry, Adam Mickiewicz University, 67-701 Poznan, Fredry 10, Poland and + Max-Planck-Institut liir Biophysikalische Chemie, Nikolausberg, D 3400 GOttingen, Germany. Department of Molecular Biology, University oJ Brussels, 1640 Rhode-Saint-GenOse, 67, rue des Chevaux, Belgium.

Mots-el6s : antieodon / code g6n~tique / hypoth~se

st6r6ochimique / 6volution.

A stereochemical relationship between the codon or complementary anticodon triplet and a particular aminoacid has been presented as a plausible explanation for the origin of aminoacid assignments and a rational basis for the currently observed vocabulary of the genetic code. This so called <~stereochemical hypothesis >>was proposed in several versions, most of which result from theoretical predictions and model building studies (for reviews see 1-3, also 4 and ref. therein). Efforts have been made to test the stereochemical hypothesis experimentally by a variety of methods [5, ll],none of which has revealed a particularly strong affinity nor high specificity in the interaction between a nucleic acid and a free aminoacid that could be taken to support conclusively the above hypothesis. However, appreciable interaction between certain aminoacids and nucleic acids have been detected in organic solvents or with aminoacids in short peptides [see 12-13 and ref. therein]. Therefore the possibility exists that all the measurements with free aminoacids in aqueous solution were performed under conditions too dissimilar from those in the primitive decoding system. It might be also, as speculated by several authors [4, 14-15], that the interaction of an amp noacid with the corresponding RNA triplet depends not only upon the triplet sequence but also upon a specific structural context or architecture of the nucleic acid moiety in the interacting system. The importance of structural parameters of this kind for the binding of two nucleic acids is particularly

Key-words : anticodon / genetic code / stereochemical hypothesis / evolution.

well illustrated in the case of the anticodon : anticodon interaction; the affinity between complementary triplets being enhanced by five to six orders of magnitude when they are built into the anticodon loops of two RNAs [16-17]. Considering this fact, we thought that the use of two tRNAs interacting with their complementary anticodons might be a valuable approach for testing the ability of certain free aminoacids to interact with the anticodon or anticodon-anticodon complex. Such a system may be used as a model for the codonanticodon interacting systems, half of the complex being the biologically relevant one, the other half serving as a model of a structured codon for the first tRNA, and vice-versa. We used the temperature-jump relaxation technique which has proved to be very useful in studies such as anticodon-anticodon interaction [17], and of the conformational dynamics of the anticodon loop of tRNA [18-19]. Also, we have taken advantage of the presence of a natural highly fluorescent label, the Wye base [20], in the position adjacent to the anticodon triplet of yeast tRNA Phe. The luminescence of this Wye base is very sensitive to any change in its environment [21-22]. Thus using fluorescence detected temperaturejump measurements we checked the effect of J.-phenylalanine, L-glutamic acid, z.-phenylalanylamide and L-lysine, at 10 mM concentration, either on the interaction between yeast tRNA phe (anticodon GmAA) and E. coli tRNA~ ~u (anticodon

78

D. Labuda and H. Groslean.

s2UUC) or E. coli tRNA r~r~ (anticodon S2UUU) [17], or on the transconformation in the anticodon loop of yeast tRNA Phe that follows the temperature jump [18] (Gin stands for 2'O-methylguanisine and s2U for S-methylaminomethyl-2-thiouridine). Figures 1A, 1B and 1C illustrate these experiments. It is clear that in both kinds of experiment the relaxation curves may be fitted by a single or by two exponential decay curves (solid line represents fitted curve), which are exactly the same whether or not any of the aminoacid is present in the reaction mixture. In no case we were able to detect any separate relaxation process due to the aminoacid binding to the anticodon of tRNA in the time range from microseconds to seconds. The time range longer than milliseconds was separately tested using stopped-flow measurements (results not shown). Table I summarizes the relaxation times, T and, the relative relaxation amplitudes (i.e. the ratio of the observed amplitude to the total fluorescence intensity of final temperature) calculated from the relaxation curves obtained for different experiments. Each value is an average of 5 to 8 successive temperature-jump measurements on the same solution ; in each case standard deviations have been carefully estimated. Results indicate that for each couple of tRNAs, or with tRNA ph~ alone, the experimental values either for time or for amplitude of the relaxation process are the same in the absence or in the presence of selected aminoacids, at least within the range of values expected from statistical fluctuations between successive measurements on the same solution and on independent mixtures of the same tRNAs and aminoacid samples. Thus clearly, an anticodon loop of a contemporary tRNA or a system of two interacting anticodons are not in priviledged conformations permitting exceptionally strong binding of an aminoacid. In order to evaluate the range of association constant of the putative binding of aminoacids either to the anticodon of one tRNA or to tRNAtRNA complex we took the maximum standard deviations obtained in each series of measurements (see table I) as a reflection of an upper limit of such a binding rather than as a statistical fluctuation. We assume the following simple reaction models in order to explain such putative interference of aminoacids on the measured system: (i) the competitive inhibitory binding of aminoacid to one of the anticodons, thus lowering the free reactant concentration; and (ii) the stabilizing binding of aminoacid to the anticodon-anticodon complex, thus displacing the equilibrium in favor of complex formation (an alternative model where

BIOCHIMIE, 1981, 63, n ° 1.

the aminoacid binding destabilizes the complex would be equivalent). For these two models the apparent binding constant of anticodon-anticodon complex formation can be expressed as follows: (i) K,pp = KtRNA-tIINA (1 + KAAC'AA)-1 and (ii) Kapp = KtRNA-tRNA (1 + KAACA.~) where Kttt~A-tI~NA denotes the equilibrium binding constant of the anticodon-anticodon complex, KAA the equilibrium binding constant of the aminoacid, and Caa the concentration of free aminoacid which is almost 10 mM since Ca~t > > CtRNA. The maximum deviations in relaxation times (ATm,~) as indicated in table I can be related to a change in free reactant concentration as follows [23]: 1 -

k. ~'~'tm,'A

/~Tma x

w h e r e CtRNA" is

the sum of the free concentrations of both tRNA species. The kinetic association rate constant, k., together with corresponding KtRNA-tRNA is known from independent measurements [17] (for the tRNAPhe-tRNA~lu pair at 16.2 °, k , = 3 × 106 M -1 s -1 and KtRNA-tRNA = 1.9 × 106 M -1, whereas for the tRNAVhe-tRNA Lrs pair at 4.5°C k , = 1 × 106 M -1 s-1 and KtltNA-tRNA = 0.24 × 106 M-0. Knowing these values and taking maximal deviations given in table I one may calculate K,pp and extract KAA from equations (i) and (ii). On the basis of ATm,x we found that KAa is about 35 M -~ and 50 M -1 for both the tRNAVh~-tRNA2 Gh' pair at 16.2°C and the tRNAr~-tRNALys pair at 4.5°C, respectively. Independently we can estimate the same KAA from the maximum deviations in relaxation amplitudes (table I). For small perturbations, as in our measurements, and because the fluorescence of the complex is 0.1 that of free yeast tRNAm'L we can consider for our purposes that the deviations in relaxation amplitudes are proportional to the changes in complex concentration after the temperature-jump [23]. KAA calculated on this basis was found to be about 25 M -1 and 5 M -~ for the tRNArh~-tRNA2 G~u pair and the tRNA vhotRNA Ly~ pair, respectively. Similar calculations for the putative binding of aminoacids to the anticodon of yeast tRNA P~e alone, as shown in figure 1C and table I, were not possible since we do not know the equilibrium constant and the relative quantum yields of the two tRNA m'e conformers under our experimental conditions. Since the above calculations of KAA for aminoacid binding to the anticodon or anticodon : anticodon complex were based on the assumption that

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Typical oscilloscope traces are shown of fluorescence detected temperature jump experiments with a 1 : 1 mixture o] yeast t R N A Phe and coli t R N A ~ lu in the presence of 10 m M L-phenylalanylamide (1A), with a I : 1 mixture ol yeast tRNAP~e and E. coli tRNAL~ 8 in the presence o] 10 m M L-phenylalanine (1B) and with yeast t R N A Phc in the presence o] 10 m M L-phenylalanine (lc), Each signal (an average of 3 measurements) is displayed on two times scales ; the lower curve corresponds to the time scale indicated on the abscissa, the upper curve is a 5 X magnification in time scale of the first part of the curve. Amplitudes are given in arbitrary units. The solid line passing through the relaxation signal is the best exponential fitting curve, calculated using the procedure based on non linear analysis using Simplex method (Striker, to be published). In 1C the curve is fitted with two exponentials ; the shortest relaxation corresponds to the very fast unspecific fluorescence quench by temperature which follow here the <~machine time ,, the slower relaxation process is due to the transconformation in the anticodon loop of yeast t R N A 1'he [18-19]. In 1A and 1B the very first part of the curve, corresponding to the effects seen in 1C (compare the time scales) was cut off electronically, and the long single relaxation observed is due to bimolecular reaction between two complementary anticodons [171. Under certain conditions the tRNAPhe-tRNAr,r s pair gives rise to two very close relaxation times, never observed in the case of the tRNAP~,~'-tRNA.~ ]" (unpublished results). AU t R N A samples were from Boehringer Mannheim ; their concentration was 1.32 I~M~ based on specific aceptor activity (about 1.3 nmoles per A2oo unit). Concentration of aminoacid was 0 or 10 m M in each case. Buffer: 8 mM Na-cacedylate, pH 6.9, 8 mM MgSO, and 80 mM Na._,SO,. For experiments A and C the final temperature of the solution, after the temperature jump, was 4.5°C, and for B, 16.2°C. The temperature jump was 3.2°C. The excitation wavelength was 312 nm and emission was measured at wavelength higher than 385 nm (Schott curt off filter gg385). The instrument has been described elsewhere [25].

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D. Labuda and H. Grosjean.

80

TABLE I.

Relaxation times (~) and relative relaxation amplitudes (AMP) measured in the presence and in the absence ol amino acids. Experim e n t (*)

Sample

(**)

Amino acid (***)

Temp.

-L-Phe L-GIu L-PheNH_. L-Lys

16.2 >> >> >> >>

~

°C t R N A m~e A

+ t R N A ~ lu >> >> >>

Mean value Max. deviation t R N A Phe

B

+ >> >> >> >>

t R N A Lys

-L-Phe L-Glu L-PheNH~ L-Lys

4.5 >~ >> >> >>

Mean value Max. deviation t R N A l'he a l o n e >> >> >> >>

C

Mean value Max. deviation

(*) T y p i c a l

oscilloscope traces are shown

ms 192.8 196.2 192.3 190.8 190.5

± _+ ± ± _+

4.5 >> >> >> >>

% 2.2 3.2 2.0 2.1 4.2

10.58 9.90 9.47 9.35 9.70

± ± ~ ± -4-

0.1 0.1 0.1 0.1 0.5

1 9 2 . 5 -4- 4 . 0 ± 4.0

9.8

+ ±

0.5 0.8

160.6 159.0 153.6 151.7 163.8

3.3 3.0 2.2 1.2 2.4

5.99 5.89 5.81 5.69 5.92

___ ± ± ___ ___

0.08 0.12 0.12 0.19 0.14

5.0 6.0

5 . 8 6 ___ 0 . 1 2 ± 0.2

± + ± ± _+

158.0 ± ± -L-Phe L-GIu L-PheNH.~ L-Lys

AMP (***)

0.76 0.70 0.74 0.71 0.73

± ± ~ -4-+-

0.08 0.05 0.09 0.08 0.05

0.96 1.08 1.11 1.02 0.99

___ -+___ ± ±

0.73

± ±

0.024 0.03

1.03 ± ±

0.05 0.08 0.08 0.09 0.08 0.06 0.08

o n f i g u r e 1.

(**) tRNA concentrations were 1.32 ~M each. (***) Concentrations of amino acids and phenylalanylamide were 10 mM. (****) Relative relaxation amplitude is the ratio of the observed amplitude to the total fluorescence intensity at final temperature. Conditions of measurements are those of legend in figure 1. Temperature refer to final temperature after 3.2°C temperature-jump.

the deviations observed in different experiments resulted only from interference due to this putative binding and not to statistical fluctuations between measurements, we may conclude with confidence that the binding of aminoacid to the present day anticodon, if any, is not exceptionally important ; the order of magnitude we give for such putative binding being even an upper limit of what we can expect. No apparent effect of aminoacid addition was observed either on the interaction between t R N A T M (anticodon VAC) and t R N A ~yr (anticodon QUA) from E. coli, studied in the absorption mode with the same technique, with different aminoacids and at various concentrations of Mg 2+ and Na + (results not shown). Therefore, our resul,ts do not support the <> of the stereochemical hypothesis. However,

BIOCHIMIE,

1 9 8 1 , 63, n ° 1.

they cannot be taken either as a definitive argument against this theory, since we will never know what were the chemical constraints and the architecture that existed for the codon-anticodon interaction in the very primitive translation machinery. It might be that the large variety of modified bases existing in almost all anticodon loops of present day tRNAs [24] constitute a severe constraint against binding an aminoacid to present day anticodons. The next best suited technique for studying such weak inteactions in solution with tRNAs having or not the modified bases in their anticodons would be N M R spectroscopy ; with this technique, relatively high concentration of materials could be used and valuable information on the environment of precise atoms should be obtained.

Aminoacids

and the a n t i c o d o n : a n t i c o d o n interaction.

Acknowledgements. We would like to thank Prof. Eigen for the opportunity to work in his Laboratory and particularly Dr. POrschke for his help and advice during the experiments. We are also indebted to Dr. Striker ]o1" giving us his curve fitting program, to Dr. R. H. Buckingham and to the referee's of this paper [or their comments on the manuscript. D.L. was a post-doctoral ]ellow of MP1. H.G. received a short-term fellowship from E M B O to visit M P I in G6ttingen and grant from F.N.R.S. in Belgium.

REFERENCES. 1. Woese, C. R. (1967) in <>(Harper and Row, Ed. New York) pp. 150-178. 2. Rohlfing, D. L. & Saunders, M. A. (1978) J. Theor. Biol., 71, 487-503. 3. Ninio, J. (1979) in <>, Ed. Masson, Paris, pp. 72-75. 4. Balasubramanian, R., Seetlaramolu, P. & Raghunathan, G. (1980) Origins of lile, 10, 15-30. 5. Zubay, G. 8, Doty, P. (1958) Biochim. Biophys. Acta, 29, 47-58. 6. Wcese, C. R., Dugre, D. H., Saxinger, W. C. & Dugre, S. A. (1966) Proc. Natl. Acad. Sci. USA, 55, 966973. 7. Saxinger, C. & Ponnamperuma, C. (1972) Origins of lile, 5, 189-200. 8. Raszka, M. & Mandel, M. (1972) J. Mol. Evol., 2, 3843.

BIOCH1MIE, 1981, 63, n ° 1.

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9. Nakashima, T. ~: Fox, S. W. (1972) Proc. Natl. Acad. Sci. USA, 69, 106-111, 10. Weber, A. L. 8~ Lacey, J. C. (1978) J. Mol. Evol., 11, 199-210. 11. Reuben, J. & Polk, F. E. (1980) J. Mol. Evol., 15, 103-112. 12. Lancelot, G., Mayer, R. & H~l~ne, G. (1979) Biochim. Biophys. Acta, 564, 181-190. 13. P6rschke, D. (1979) Biophys. Chemistry, 10, 1-16. 14. Hopfield, J. J. (1978) Proc. Natl. Acad. Sci. USA, 75, 4334-4338. 15. Woese, C. R. (1979) in < (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M.) pp. 357-373, University Park Press, Wisconsin, USA. 16. Eisinger, J. (1971) Biochem. Biophys. Res. Comm., 43, 854-860. 17. Grosjean, H., de Henau, S. & Crothers, D. M. (1978) Proc. Natl. Acad. Sci. USA, 75, 610-614. 18. Urbanke, C. & Maass, G. (1978) Nucl. Acids Res., 5, 1551-1560. 19. Labuda, D. & P6rschke, D. (1980) Biochemistry, 19, 3799-3805. 20. RajBandary, U. L., Chang, S. H., Stuart, A., Faulkner, R. H., Hoskinson, R. N. & Khorana, H. G. (1967) Proc. Natl. Acad. Sci. USA, 57, 751-762. 21. Beardsley, K., Tao, T. & Cantor, C. R. (1970) Biochemistry, 9, 3514-3532. 22. Labuda, D., Haertl6, T. & Augustyniak, J. (1977) Eur. J. Biochem., 79, 293-307. 23. Bernasconi, C. R. (1976) in <, Academic Press, New York. 24. Nishimura, S. (1979) in <>(Schimmel, P. $611, D. G. & Abelson, J., Ed.) Cold Spring Harbor Monograph 9A, p. 547. 25. Rigler, R., Rabl, C. R. ,~ Jovin, T. M. (1974) Rev. Sci. lnstrum., 45, 580-590.