M O D I F I C A T I O N OF P O L Y U R I D Y L I C A C I D BY B I S U L F I T E . II: S T U D I E S ON R I B O S O M A L B I N D I N G A N D E N Z Y M A T I C H Y D R O L Y S I S

ROBERT SHAPIRO, BARBARA BRAVERMAN, AND WLODZIMIERZ SZER

Dept. of Chemistry, New York University, New York, N. Y. 10003 U.S.A. and Dept. of Biochemistry, New York University School of Medicine, New York, N.Y. 10016, U.S.A.

(Received 6 June, 1973) ABSTRACT. The reaction of polyuridylic acid with sodium bisulfite produces modified polymers in which up to 95 ~o of the uracil residues have been converted to uracil-6-sulfonate residues. A 91.6 ~ bisulfite-saturated polymer was found to resist hydrolysis by spleen phosphodiesterase and phosphorolysis by polynucleotide phosphorylase. Digestion by pancreatic ribonuclease was successful and gave the bisulfite adduct of uridine-3'-phosphate. Treatment of this nucleotide adduct with acid phosphatase afforded the bisulfite adduct of uridine. The ability of polyuridylic acid to bind to ribosomes, and to stimulate the binding of phenylalanine tRNA to ribosomes was abolished by progressive bisulfite saturation of the polymer. The rate of decline of these functionsf with increasing bisulfite content, was less sharp than the loss of phenyl-alanine coding ability o, the modified polymer.

Sodium bisulfite reacts rapidly with uracil derivatives under physiological conditions to give dihydrouracil-6-sulfonate derivatives [1, 2] (Scheme 1, I ~ II). We have been interested in this reaction as a potential contributor to the biological damage inflicted by sulfur dioxide [3]. In our previous paper we reported that the saturation of uracil residues in polyuridylic acid by bisulfite interfered with its ability to bind to polyadenylic acid, and eliminated its coding ability for polyphenylalanine [4]. We have now conducted studies on the effect of this modification on other messenger functions of poly (U): its ability to bind to ribosomes and to stimulate the binding

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Scheme 1 123 Molecular Biology Reports 1 (1973) 123-127. All Rights Reserved Copyright 9 1973 by D. Reidel Publishing Company, Dordrecht-Holland

of p h e n y l a l a n y l - t R N APh~ to ribosomes. Bisulfite saturation interferes with these functions. I n addition, it renders poly (U) resistant to hydrolysis by spleen phosphodiesterase a n d to phosphorolysis by polynucleotide phosphorylase, b u t n o t to digestion by pancreatic ribonuclease. A 91.6 ~ bisulfite-saturated sample of p o l y ( U ) was prepared, and freed of excess bisulfite, by methods already described [4]. The enzymatic hydrolyses that were carried out o n the modified polymer, a n d o n a poly (U) control, are s u m m a r i z e d i n Fig. 1. The selection of enzymes used, a n d the p H values employed, were limited by the tendency of the s a t u r a t i o n reaction to reverse (II ~ I)

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~ polynucleotidephosphorylasea or spleenphosphodiesterasea no reaction Fig. 1. Enzymatic digestion of bisulfite-saturated polyuridylic acid. a. TheconditionsofChouandSinger[5] were employed. The pH was 7.0 and the poly(U) concentration 1.5 • 10-aM (in monomer). The reaction was analyzed by thin layer chromatography on cellulose in 95 ~ ethanol-1 M ammonium acetate, pH 3.8 (75: 30). The plate was exposed to NHa before development. The ultraviolet-absorbingbands observed were eluted with 0.1 M Tris-HCl buffer, pH 7.0, and quantitated by ultraviolet spectrophotometry (260nm). b. Bisulfite was removed by dialysis against 0.02 M sodium borate buffer, pH 8.9, at 25~ for 16 hr. This 'reversed' polymer was digested by spleen phosphodiesterase and acid phosphatase under the conditions described in d and g below. A single spot was observed, of the same R~ as uridine, upon thin layer chromatography on cellulose in 2-propanol-conc. HC1-H~O (65:16.7:18.3), isopropanol-NHa-H20(70:10:20) and in the system described in f. The product, after elution, had ultraviolet spectra at pH 2 and 12 identical to those of uridine. c. Pancreatic ribonuclease (Worthington, 1500 traits) was allowed to react with poly(U), 3 • 10-a M, or with poly (U-HSOa-), 1.5 • 10-a M in 0.108 M sodium acetate buffer, pH 5.0 at 37~ for 22 hr (1 hr proved sufficient for poly(U) hydrolysis). d. Spleen phosphodiesterase (Worthington, 0.5 units) was allowed to react with 3 • 10-a M poly(U) for 4hr or with 1.5 • 10-a M poly(U-HSOa-) for 22 hr in 0.078 M sodium acetate buffer, pH 5.0, 37~ e. The bisulfite adducts of uridine and uridirte 3'-phosphate were prepared chemically by allowing the compounds (7.5 • 10-a M) to react with 1M NaHSOa, pH 7.0, for 18 hr at 25~ f. The product of reaction of uridine 3'-phosphate with bisulfite displayed the same RF (0.16) as the product of the ribonuclease digest of poly(U-HSOa-). The reaction mixtures were analyzed by thin layer chromatography on cellulose in 95 ~ ethanol - 1 M ammonium acetate, pH 5.0 (80:20). The plates were exposed to ammonia after development, for visualization of the adduct spots. If the plates were exposed to ammonia before development, only a spot of the same Rv (0.34) as uridine 3'-phosphate was observed. g. When digestion to the nucleoside level was desired, acid phosphatase (Worthington, 0.15 units) was added to the reaction mixtures described in c or d. h. The product of reaction of uridine with bisulfite displayed the same RF (0.34) as the product of the ribonucleaseacid phosphatase digest of poly(U-HSO3-). Analysis was performed by the method described in f. When the plates were exposed to ammonia before development, only a spot of the same RF (0.69) of uridine was observed. 124

in the absence of bisulfite at pH more alkaline than 7. The control poly(U) sample was completely digested to uridine-3'-phosphate by spleen phosphodiesterase, and by ribonuclease. When acid phosphatase was included in the reaction mixture, or added subsequently, uridine was the final product. In the phosphorolysis by polynucleotide phosphorylase at pH 7, poly(U) was converted to UDP to the extent of 42% in 1 hr and 67% in 3 hr. The 91.6% bisulfite-saturated poly(U) sample was digested only by pancreatic ribonuclease. The product of digestion was identified as the bisulfite adduct of uridine 3'-phosphate. It was prepared from uridine 3'-phosphate by reaction with bisulfite, and reconverted to uridine Y-phosphate by ammonia treatment. It was hydrolyzed to the bisulfite adduct of uridine by acid phosphatase. This latter adduct was prepared from uridine by reaction with bisulfite, and reconverted to uridine by ammonia treatment. As an additional control, a sample of bisulfite-saturated poly (U) was reconverted to poly(U) by dialysis at pH 9. This product was now completely digested to uridine by the combination of spleen phosphodiesterase and acid phosphatase. These results reinforce our earlier conclusion that the reaction of bisulfite with poly (U) follows the same course at the polymer level that it does at the monomer level. Our studies on the effect of varying degrees of bisulfite saturation on the various messenger functions of poly(U) are summarized in Fig. 2. The earlier study on the ability of the modified polymers to direct the incorporation of phenylalanine into polypeptides [4] has been included for comparison. It can be seen that all of the messenger functions studied are progressively abolished by increasing saturation of poly(U) by bisulfite. The fall off is most dramatic in the case of polyphenylalanine synthesis, where, for example, a 2.6 % saturated sample has lost 46 % of its activity. The ability of the modified messengers to bind to ribosomes [8] in the presence of Mg 2§ was measured with and without added phenylalanyl-tRNAPhe. The ribosomal binding ability in each case decreased with increasing bisulfite content, but the loss of binding activity for a given degree of saturation was less than the loss of coding ability. For each degree of saturation, binding ability was moderately higher in the presence of the added tRNA. When the bisulfite was removed from the most saturated sample by dialysis against 0.05 M sodium borate buffer at pH 8.9 for 21 hr, 25~, 90% of the binding activity of the control was restored. The ability of bisulfite-saturated poly(U) samples to stimulate the binding of phenylalanyltRNA phe to ribosomes [6] was also explored, and the results are included in Fig. 2. Once again, a gradual loss of function of the poly(U) was seen with increasing saturation. The curve generated was very close to that expected on the basis of probability, (X 3, where X is the fraction of unmodified bases) if an unmodified triplet was needed to effect the binding of a tRNA molecule. However, it was possible to increase the tRNA binding above the theoretical expectation by increasing the Mg 2+ concentration from 0.01 M to 0.015 M. It is interesting to compare the messenger activity ofpoly (U) samples containing the dihydrouracil 6-sulfonate, II, with that of samples containing the related analog, dihydrouracil. Dihydrouracil residues can be introduced into poly (U) by photoreduction in the presence of sodium borohydride [9]. The effects of both types of modification are quite similar in terms of polyphenylalanine coding and stimulation of phenylalanyl-tRNAphe binding to ribosomes. Although polydihydrouridylic acid and poly-N-3-methyluridylic acid do not form complexes with poly(A), they have been reported to bind to ribosomes as well as polyuridylic acid [10]. This observation supported the notion that ribosomal binding involved the phosphates of poly(U), and that the base was unimportant [10, 11]. Our present results suggest that the type of base modification can be important. The difference between dihydrouracil and the adduct II may be due to the extra negative charge of the latter. Alternatively, it may be due to a conformational change in the base. Dihydrouridine exists in the anti-conformation [12]. The nucleoside adduct II, with its bulky 125

6-substituent, is likely to be in the syn-conformation. This postulate would also explain the failure o f the bisulfite-saturated p o l y m e r to respond to polynucleotide phosphorylase, as that enzyme a p p a r a n t l y requires the a n t i - c o n f o r m a t i o n of its substrate [13, 14]. A final p o i n t of interest concerns the u n i q u e l y sharp decline of p o l y p h e n y l a l a n i n e coding ability, as compared to the other messenger functions, with increasing bisulfite saturation. This is partially

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Modification of polyuridylic acid by bisulfite. II: Studies on ribosomal binding and enzymatic hydrolysis.

The reaction of polyuridylic acid with sodium bisulfite produces modified polymers in which up to 95% of the uracil residues have been converted to ur...
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