Involvement of the anticodon region of Escherichia coli tRNAGln and tRNAGlu in the specific interaction with cognate aminoacyl-tRNA synthetase

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Biochirnica et Biophysica Acta, 349 ( 1 9 7 4 ) 3 2 8 - - 3 3 8 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98001

I N V O L V E M E N T OF THE ANTICODON R E G I O N OF ESCHERICIIIA COLI t R N A 6 In and t R N A G lu IN THE SPECIFIC INTERACTION WITH COGNATE AMINOACYL-tRNA SYNTHETASE A L T E R A T I O N OF THE 2-THIOURIDINE D E R I V A T I V E S LOCATED IN THE ANTICODON O F THE t R N A s BY BrCN O R S U L F U R D E P R I V A T I O N

T A K E S H I S E N O a, P A U L F. AGRIS *b and D I E T E R S O L L b

aBiology Division,National Cancer Center Research Institute,Tsukiji5-chome, Chuo-ku, Tokyo (Japan) and b Department of Molecular Biophysics and Biochemistry, Yale University,N e w Haven, Conn. 06520, (U.S.A.) (Received December 27th, 1973)

Summary

Treatment of unfractionatedEscherichia coli t R N A with BrCN produces a decrease in glutarnine and glutamate acceptance. This decrease may be explained by the specificcyanation of the 2-thiouridinederivative,located at the 5'-terminal of the anticodon of the tRNAs corresponding to these amino acids; the modification could prevent the accurate fit between the tRNAs and their cognate aminoacyl-tRNA synthetases. The activityof t R N A was measured in two ways, by aminoacylation and by ATP--PPi exchange in the presence of tRNA. Transfer R N A G lu and t R N A ~ In showed a decrease in affinity for their cognate synthetases following BrCN treatment, indicated b y a 10-fold increase in the apparent Kin. t R N A ~ In and other t R N A s not possessing a BrCN-reactive group in the anticodon were n o t affected b y BrCN treatment. Transfer R N A G lu or t R N A G In prepared from a cysteine-requiring relaxed m u t a n t of E. coli grown in sulfur
* Present address: Division of Biological Science, College of Arts and Science, University of MissouriColumbia, Columbia, Mo. 65201 (U.S.A.)

329 Introduction As part of our studies on the recognition of tRNA molecules by their cognate aminoacyl-tRNA synthetases, we have shown in a previous paper [1 ] the interaction of the anticodon region of Escherichia coli tRNA ° ' u with glutamyl-tRNA synthetase. This was achieved through two types of alterations of 5-methylaminomethyl-2-thiouridine, the modified nucleoside, located in the 5'-terminal position of the anticodon of tRNA Glu [2]. BrCN treatment of normal tRNA Glu decreased the affinity of the tRNA for glutamyl-tRNA synthetase as indicated by a six-fold increase in the apparent Kin. However, the 2-thiouridine derivative itself was not necessary for the aminoacylation of tRNA Glu since tRNA Gl~ purified from a cysteine-requiring relaxed mutant of E. coli grown in sulfur
Preparation o f tRNA Crude tRNA was isolated from E. coil B according to Zubay [7] with the addition of a DEAE~cellulose step at the end [8]. tRNA~ In and tRNA~ In were separated from each other by subjecting crude E. eoli B tRNA to DEAESephadex A-50 column chromatography as described by Nishimura [9]. The fractions containing tRNA~ 'n were pooled and used without further purifica-

330

tion. The tRNA6:ln-containing fractions were further purified by column chromatography on DEAE-Sephadex A-50 at pH 4 [10]. These tRNA fractions accepted 145 pmoles and 640 pmoles of glutamine, respectively, per A 260 ~ unit of tRNA and were generously supplied by Dr S. Nishimura. Pure tRNA~ lu, which accepted 1450 pmoles of glutamate per A260 nm unit of tRNA, was isolated from E. coli B and a gift from Dr A.D. Kelmers of the Oak Ridge National Laboratory. Sulfur-deficient tRNA was isolated from a cysteine-requiring mutant of E. coli 58-161 rel- (obtaine d by Mr Y. Taya of this Institute in Tokyo) grown under conditions of sulfur starvation [11] and a kind gift of Mr Y. Taya. This unfractionated sulfur-deficient tRNA preparation showed an absorbance at 336 nm which was equal to 0.6% of that at 260 nm. This ratio of absorbance was about 1/3 of the value obtained from the normal tRNA and was consistent with the value reported previously [ 1,11 ]. This indicates that 2/3 of the tRNAs in the sulfur
Preparation o f aminoacyl-tRNA synthetases Crude aminoacyl-tRNA synthetase was obtained from E. coli B as described previously [12]. Glutaminyl-tRNA synthetase was isolated from the above crude enzyme preparation according to Folk [13] except that the final gel filtration on Sephadex G-200 column was omitted. Glutamyl-tRNA synthetase was purified from E. coli K12(CA244) as described previously [14]. The enzymes were stored at --20°C in buffer solution containing 50% glycerol. A TP--PPi exchange reaction The ATP--PPi exchange was assayed as described previously [15] with slight modifications: the reaction (0.1 ml) contained 2.5 mM amino acid as specified, 2 mM ATP, 2 mM [32 P]pyrophosphate (about 1.104 cpm), 10 raM Tris--HCl (pH 7.5)~ 4 mM magnesium acetate, 10 mM NaF, 3 raM ~-mercaptoethanol, aminoacyl-tRNA synthetase and tRNA as specified. Incubation was at 37°C for 5 min or 30 min as specified in the legend to the figures. ATP was adsorbed to charcoal, filtered and counted in a Beckman wide-beta gas-flow counter. Control values (minus amino acid) were subtracted from the experimental values to give those presented in the figures. Amino acid acceptor activity o f tRNA Amino acid acceptor activity of tRNA was assayed as described previoualy [12], except that sodium cacodylate buffer (pH 7.0) was used in the reaction in place of Tris--HC1 buffer (pH 7.5). BrCN treatment o f t R N A BrCN treatment of t R N A was carried out in 0.05 M sodium carbonate buffer (pH 8.9) at room temperature for 10 min according to Saneyo~_i and Nishimura [16]. Control t R N A received the same treatment except that BrCN

331 was omitted in the reaction buffer. The BrCN-treated t R N A sh6wed an absorbance at 336 nm which was equal to only 5% of that obtained from the untreated t R N A . The minor peak at 336 nm of the ultraviolet spectrum due t6 the presence of 4-thiouridine in the t R N A [17] was n o t detectable in the BrCN-treated tRNA. Both BrCN-treated and sham-treated tRNAs were heatrenatured in the presence 6f Mg 2÷ [18] prior to use, 6therwise these tRNAs inhibited the enzymic reaction. It sh6uld be mentioned that this inhibition seems still to be amino acid-specific, as BrCN- or sham-treated pure t R N A Pbe without the heat treatment, for example, inhibited only phenylalanine-dependent ATP--PPi exchange (unpublished observations). Results BrCN-treated t R N A BrCN treatment of unfractionated E. coil t R N A causes a decrease in the acceptor activity of glutamine and glutamate alone among the 20 amino acids [ 1 9 ] . BrCN is k n o w n to react specifically with thiolated nucleosides in t R N A molecules [ 1 6 ] . It is k n o w n that one of the E. coli t R N A 61n species possesses the 2-thiouridine derivative at the 5'-terminal position of the anticodon [3] similar to the case of E. coli t R N A 61u [2]. The glutamyl-, glutaminyl- and arginyl-tRNA synthetases from E. coli require the presence of cognate t R N A for amino acid activation as measured b y the ATP--PPi exchange reaction [4,5]. Thus it is possible to examine the involvement of the thiolated nucleosides in these tRNAs in the ATP--PPi exchange reaction as well as in aminoacylation. Unfractionated t R N A from E. coil B was treated with BrCN and was tested for its ability to p r o m o t e the ATP--PPi exchange reaction using crude i

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Fig. 1. S t i m u l a t i o n o f t h e A T P - - P P i e x c h a n g e r e a c t i o n b y B r C N - t r e a t e d t R N A . I n i t i a l v e l o c i t y o f t h e ATP--'PPi excha~e r e a c t i o n d e p e n d e n t o n (a) g I u t a m i n e , ( b ) g l u t R m a t e (c) a~_tnlne o r (d) lys~ne w a s m e a s u r e d i n t h e p r e s e n c e o f v a r y i n g a m o u n t s o f t h e B r C N - t r e a t e d (o) o r t h e u n t r e a t e d ( e ) t R N A . A b o u t 10 #g of crude aminoacyl-tRNA synthetase preparation was used in the reaction (0.1 ml). Incubation was at 3 7 ° 0 f o r 3 0 r a i n . F o r d e t a i l s see M a t e ~ a l R a n d M e t h o d s .

332

TABLE I AMINO ACID-ACCEPTOR A C T I V I T Y OF THE B r C N - T R E A T E D t R N A The r e a c t i o n m i x t u r e (0.1 rnl) c o n t a i n e d a b o u t 3 A250 n m u n i t s of the t R N A a n d 10 p g of c r u d e arninoa c y l - t R N A s y n t h e t a s e p r e p a r a t i o n f o r Arg, Lys, Aia, L e u a n d 1 0 0 p g f o r Gln a n d Glu. I n c u b a t i o n w a s at 3 7 ° C f o r 1 0 rain. F o r o t h e r c o n d i t i o n s see ref. 12.

Amino acid

Gin Glu Arg Lys Ala Leu

A m i n o acid accepted (pmoles/A260 n m unit) BrCN-treated R N A

Untreated R N A

0.07 0.05 18 11 5 4

0.9 0.6 16 12 6 3

Relativeacceptor activity (% of untreated control) 8 8 110 92 83 130

aminoacyl-tRNA synthetases (Fig. 1). Table I shows some amino acid acceptor activities which were consistent with the values reported previously [19]. The ATP--PPi exchange reactions, dependent on arginine and lysine, were chosen as controls for the glutamine- and glutamate~iependent reaction for the following reasons: (i) Arginyl-tRNA synthetase also requires cognate tRNA to promote the exchange reaction [5]. One tRNA Arg species possesses 2-thiouridine in the anticodon loop region [20]. (ii) E. coil tRNA Lys may possess a 2-thiouridine derivative in the anticodon as inferred by the cases of tRNA L y s from rat liver [21] and yeast [22]. In the case of the glutamine~iependent ATP--PPi exchange reaction (Fig. la), the BrCN-treated tRNA stimulated the reaction to a considerable extent. This is in contrast with its apparent loss of glutarnine acceptor activity (Table I). With glutamate the BrCN-treated tRNA lost activities both for stimulation of the ATP--PPi exchange reaction (Fig. lb) and aminoacylation (Table I). This was further confirmed using BrCN-treated pure tRNA~ lu and pure glutamyltRNA synthetase (Fig. 2, for aminoacylation see ref. 1). With arginine the BrCN-treated tRNA was able to promote the ATP--PPi exchange reaction but less efficiently (Fig. lc) while the amino acid-acceptor activity was normal (Table I). With lysine both activities were not affected following treatment of the tRNA with BrCN (Fig. l d and Table I). It seemed curious that BrCN treatment of the crude tRNA did not result in a significant loss of its ability to stimulate the glutamine-dependent ATP-PPi exchange reaction (Fig. la), while it led to almost complete loss of the glutamine-charging activity (Table I and ref. 19). In order to clarify this discrepancy the experiments were repeated using both glutaminyl-tRNA synthetase and tRNA GIn which had been partially purified. In E. coil two isoaccepting tRNAs for glutamine are known and their prim~T structure have been determined [3]. tRNA~ m responding to the codon CAA possesses a 2-thiouridine derivative, which is reactive with BK~N, at the 5'-te~ainal position of anticodon, while tRNA~ In responding to the codon CAG has cytidine at the

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Concentrcltion ot tRN/k (A2ElCnm,lml) Fig. 2. S t i m u l a t i o n of the ATP--PP i exchange reaction by BrCN-treated t R N A p lu. The initial velocity of the ATP--PP i exchange reaction d e p e n d e n t on glutamat e ~ w a s measured in t h e p r e s e n c e of varying a m o u n t s of u n t r e a t e d (o) o r B r C N t r e a t e d (o) pure t R N A ~ lu. The re a c t i on (0.1 ml) contained 3 ~tg of g l u t a m y l - t R N A synthetase. The i n c u b a t i o n was at 37°C for 30 rain. For details see Materials and M e t h ods.

corresponding site. Both tRNAs possess 4-thiouridine, which is also reactive with BrCN, at the 8th position from the 5'-end. It is, therefore, possible that u p o n BrCN treatment only one of the t w o t R N A G~n isoacceptors lost the ability to stimulate the ATP--PPi exchange reaction, thus resulting in a partial loss of the stimulation (Fig. la). Two t R N A ~ n fractions were prepared as described in Materials and Methods, treated with BrCN and then their aminoacylation and ability to p r o m o t e the ATP--PPi exchange reaction were examined. As shown in Figs 3a and 3b, the aminoacylation of t R N A ~ in was preferentially inactivated by the BrCN treatment while t R N A ~ In was rather insensitive to BrCN in these experiments. The BrCN-treated t R N A ~ in showed a decreased affinity for glutaminyl-tRNA synthetase as indicated b y a 10-fold increase in the apparent Km (Fig. 3b). However, one can not directly compare the activities of the t w o isoaccepting tRNAs, since glutamine acceptor activity in the t R N A ~ ~u preparation was 4--5 times higher than in the t R N A ~ In preparation (see Materials and Methods). The small increase of Km in the case of BrCN-treated t R N A ~ TM may be caused by contaminating t R N A ,Gin in the t R N A ~ In preparation or due to the reaction of BrCN with 4-thiouridine. As for the ability to stimulate the ATP--PPi exchange reaction, again the t R N A ,G'n was much more susceptible to the BrCN treatment, while the BrCN-treated t R N A ~ TM was able to p r o m o t e the reaction almost to a normal extent (Fig. 4). It should be emphasized that t R N A ~ In stimulated the ATP--PPi exchange reaction to the same extent as t R N A ,G in It seems that t R N A ,~ In lost both the glutamine-acceptor and the ATP--PPi exchange promoting activities u p o n BrCN treatment by the modification of the 2-thiouridine derivative located in the anticodon, thus the accurate fit of the modified t R N A with glutaminyl-tRNA synthetase was disturbed as in the case of t R N A Glu [1].

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Sulfur-deficient tRNA The sulfur
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Fig. 4. S t i m u l a t i o n of the ATP--PP i exchange reaction b y t h e B ~ N - t r e a t e d t R N A ~ In and t R N A 2Gin. The initial v e l o c i t y o f t h e ATP--PP i exchange r e a c t i o n d e p e n d e n t on gl ut a mi ne was measured in the presence of varying a m o u n t s o f BrCN-treated t T N A IGin (~) u n t r e a t e d t R N A IGIn (A), BrCN-treated t R N A 2Gin (o) o r u n t r e a t e d t R N A 2Gin ( •) . The reaction (0.1 ml) contained 50 Dg of g l u t a m i n y l - t R N A synthetase. The i n c u b a t i o n was at 37°C for 5 min. For details see Materials and M e t h o d s .

manner as described above for BrCN-treated tRNA. In contrast to the BrCN treatment which selectively impaired the g l u t a m i n e - a n d glutamate-acceptor activity through specific modification of the thiolated nucleosides in the t R N A molecules, sulfur deprivation of t R N A did n o t affect the amino acid-accepter activity. Unfractionated sulfur-deficient t R N A accepted glutamine, and glutamate as well as other amino acids to a normal extent (data n o t shown) in agreement with the values reported previously [ 1 ]. Fig. 5 shows the stimulation of the ATP--PPi exchange reaction by the sulfur
336

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Fig. 5. S t i m u l a t i o n o f t h e A T P - - P P i e x c h a n g e r e a c t i o n b y s u l f u r - d e f i c i e n t t R N A . T h e initial v e l o c i t y o f t h e A T P - - P P i e x c h a n g e r e a c t i o n d e p e n d e n t o n (a) g l u t s m i n e , (b) g l u t a m a t e , (c) a r g t n i n e o r (d) l y ~ n e w a s m e a s u r e d i n t h e p r e s e n c e o f v a r y i n g a m o u n t s o f c r u d e s u l f u r - d e f i c i e n t t R N A (o) o r n o r m a l t R N A ( e ) . About 10/~g of crude Amlnoacyl-tRNA synthetase preparation was used in the reaction (0.1 ml). The i n c u b a t i o n w a s a t 3 7 ° C f o r 3 0 r a i n . (e) T h e g l u t a m i n e - d e p e n d e n t A T P - - P P i e x c h a n g e r e a c t i o n as s h o w n i n (a) w a s r e p e a t e d u s i n g p u r i f i e d g l u t a m i n ¥ 1 - t R N A s y n t h e t a s e ( 5 0 # g p e r 0 . 1 m l r e a c t i o n ) i n p l a c e o f t h e c r u d e a m i n o a c y l - t R N A s y n t h e t a s e . I n c u b a t i o n w a s a t 3 7 ° C f o r 5 m i n . (f) T h e g l u t a m i c a c i d - d e p e n d e n t A T P - - P P i e x c h a n g e r e a c t i o n as s h o w n i n ( b ) w a s r e p e a t e d u s i n g p u r e g l u t a m y l - t R N A s y n t h e t u e (3 ~ g p e r 0.1 m l r e a c t i o n ) a n d s u l f u r - d e f i c i e n t t R N A o l u i n s t e a d o f c r u d e a m i n o a c y l - t R N A s y n t h e t a s e a n d c r u d e t R N A . I n c u b a t i o n s w e r e a t 3 7 ° C f o r 3 0 r a i n . F o r d e t a i l s see M a t e r i a l s a n d M e t h o d s .

for these tRNAs to be recognized by the cognate aminoacyl-tRNA synthetase as measured by aminoacylation or by the stimulation of the ATP--PPI exchange reaction. Discussion The preferential decrease in the affinity of glutaminyl-tRNA synthetase for E. coli tRNA~ TM following the BrCN treatment of the tRNA could be explained in terms of the specific cyanation of the 2-thiouridine derivative

337 located in the 5'-terminal position of the anticodon of tKNA~ In. The other isoacceptor, tRNA~ In, which contains cytidine in place of the modified nucleoside in the anticodon did not lose the activity following the BrCN treatment. Since 4-thiouridine, which is also reactive with BrCN, is located at the 8th position from the 5'-end of both isoaccepting species of tRNA GI" [3], involvement of the 4-thiouridine in tRNA~ 1, in the inactivation is unlikely. This suggests that the accurate fit of the cognate aminoacyl-tRNA synthetase with tRNA GI n requires a defined steric relationship around the anticodon region of the tRNA, just as it has been suggested previously for the inactivation of tRNA ~lu by BrCN [1]. The involvement of the ~_nticodon region of tRNA has been also implicated by means of other experimental approaches for tRNA ~Met [23,24], tRNA ne [25,26], tRNA Gly [27], and su7 ÷ tRNA [28] from E. coli, and tRNA Phe [29] and tRNA val [30,31] from yeast. It is unlikely that the/2-thiouridine derivative itself located in the 5'-terminal position of anticodon of tRNA~ ~" or tRNA G~u is involved in the ability of the tRNA to promote the ATP--PPi exchange reaction or to accept amino acids, because tRNA Gl~ or tRNA ~ in purified from E. coli cells grown under sulfur-deficiency, or normal tRNA~ In which does not possess the 2-thiouridine derivative in the anticodon of the tRNA, functions normally in both reactions. In this context, it is to be noted that the ATP--PPi exchange reactions dependent on lysine (Figs l d and 5d) and phenylalanine (data not shown), were stimulated by the presence of tRNA, although the reactions proceeded to a considerable extent even in the absence of tRNA in these cases. When the glutamine-acceptor activities of the BrCN-treated tRNA~ In and tRNA~ In were separately measured using crude aminoacyl-tRNA synthetase, both tRNAs showed less than 10% of the normal activity (data not shown). This was consistent with the results obtained for the unfractionated tRNA which was modified by BrCN (Table I). However, it was not consistent with the results obtained when purified glutaminyl-tRNA synthetase was used instead of crude aminoacyl-tRNA synthetase (Fig. 3). The BrCN-treated tRNA~ 1" gave almost normal aminoacylation kinetics, while the BrCN-treated tRNA~ in did not. This discrepancy was not further investigated in the present study. However, the discrepancy in results following the use of crude aminoacyl-tRNA synthetase rather than purified glutaminyl-tRNA synthetase was not observed when the ATP--PP~ exchange reaction was measured even though both activities were assayed under similar conditions of tRNA and aminoacyl-tRNA synthetase concentration (data not shown). If any modification of a tRNA molecule inactivates only one of the two functions of the tRNA, i.e., either the aminoacylation or the promotion of the ATP--PPi exchange reaction, it should be a powerful tool to elucidate the steps of the recognition of the tRNA molecule by cognate aminoacyl-tRNA synthetase. In this respect, it seems interesting that the BrCN-treated tRNA gave a rather poor promotion of the arginine-dependent ATP--PPi exchange reaction (Fig. lc) while it gave a normal charging of arginine (Table I). However, further discussion should be suspended until the analysis has been made with each of the isoaccepting species of tRNA Arg [32] and with pure arginyl-tRNA synthetase.

338

Acknowledgements We are indebted to Drs S. Nishimura, A.D. Kelmers, K. Imahori, T. Ohta and Mr Y. Taya for their valuable gifts of materials without which this work was impossible. We also thank Ms M. Kobayashi for her excellent technical assistance. The work was supported by grants from the Japanese Ministry of Education, the United States Public Health Service and the National Science Foundation. References 1 Agris, P., $511, D. and Seno, T. (1973) Biochemistry 12, 4331--4337 2 0 h a s h i , Z., Saneyoshi, M., Harada, F., Hara, H. and Nishin~ura, S. (1970) Biochem. Biophys. Res. Commun. 40, 866--872 3 Folk, W.R. and Yanlv, M. (1972) Nat. New Biol. 237,165--166 4 Ravel, J.M., WavE, S.F., Heinemeyer, C. and Shive, E. (1965) J. Biol. Chem. 240, 432--438 5 Mitra, S.K. and Mehler, A.H. (1966) J. Biol. Chem. 241, 5161--5164 6 Berg, P. (1958) J. Biol. Chem. 233, 601--607 7 Zubay, G. (1962) J. Mol. Biol. 4, 347--356 8 S~II, D., Chezayil, J.D. and Book, R.M. (1967) .L MoL Biol. 29, 97--112 9 Nishimura, S. (1971) in Proc. in Nucl. Acid Res. (Cantoni, G.L. and Davies, D.R., eds), Vol. 2, pp. 542--564, Harper and Row, New York 10 Yoshida, M., Takeishi, K. and Ukita, T. (1971) Biochim. Blophys. Acta 228, 153--166 11 Harris, C.L., Titchener, E.B. and Cline, A.L. (1969) J. Baet. 100, 1322--1327 12 Nishimura, S., Harada, F., Naxushima, U. and Seno, T. (1967) Biochim. Biophys, Acta, 142, 133--148 13 Folk, W.R. (1971) Biochemistry 10, 1728--1732 14 Lapolnte, J. and $511, D. (1972) J. Biol. Chem. 247, 4966--4972 15 Calender, R. and Berg, P. (1966) Biochemistry 5, 1681--1690 16 Saneyoshi, M. and Ni,hlmuxa, S. (1970) Bioehim. Biophys. Acta 204, 389--399 17 Lipsett, M.N, (1965) Bioehcm. Biophys. Res. Commun. 20, 224--229 18 Hirsh, D. (1971) J. Mol. Biol. 58, 4 3 9 - 4 5 8 19 Saneyoshi, M. and Nishimura, S. (1971) Biochim. Biophys. Acta 246, 123--131 20 Murao, K., Tanabe, T., Ishii, F., Namlki, M. and N4Rhlmum, S. (1972) Biochem. Biophys. Res. Commun. 47, 1332--1337 21 Kimuza-Harada, F., Saneyoshi, M. and Nishlmuxa, S. (1971) FEBS Lett. 13, 335---338 22 Madison, J.T., Boguslawski, S.J. and Teefor, G.H. (1972) Science 176,687--689 23 Schulman, L.H. and Goddard, J.P. (1973) J. Biol. Chem. 248, 1341--1345 24 Dube, S.K. (1973) Nat. New Biol. 243, 103--105 25 Schimmel, P.R., Uhlenbeek, O.C., Lewis, J.B., Dickson, L.A. Eldred, E.W. and Schrei~, A.A. (1972) Biochemistry, 1 1 , 6 4 2 - - 6 4 6 26 Harada, F. and Nishimuxa, S. (1974) Biochemistry 13, 300---307 27 Squires, C. and Carbon, J. (1971) Nat. New Biol. 233,274--277 28 Crothers, D.M., Seno, T. and SSII, D.O. (1972) Proc. Natl. Aead. Sci. U.S. 69, 3063--3067 29 Thiebe, R. and Zachau, H.G. (1968) Bioehem. Biophys. Res. Commun. 33, 260--265 30 Mirzabekov, A.D., Lastity, D., Levina, E.S. and Bayer, A.A. (1971) Nat. New Biol. 229, 21--22 31 Chambers, R.W., Aoyagi, S., Furukawa, Y., Zawadzka, H. and Bhanot, O.S. (1973) J. Biol. Chem. 248, 5549--5551 32 Celis, T.F.R. and Maas, W,K. (1971) J. Mol. Biol. 62, 179--188