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Stimulation of amino acid incorporation into protein by polyuridylic-8-azaguanylic acid
BIOCHIMICA ET BIOPHYSICA ACTA
581
BBA 95399
STIMULATION OF AMINO ACID INCORPORATION INTO PROTEIN BY POLYURIDYLIC-8-AZAGUANYLIC ACID
DEZIDER
GRI3NBERGER*,
CHARLES
O'NEAL
AND
MARSHALL
NIRENBERG
Department o] Pharmacology, The George Washington University School o/ Medicine, Washington, D. C. (U.S.A.) and National Heart Institute, National Institutes o] Health, Bethesda, Md. (U.S.A.) (Received September i6th, 1965)
SUMMARY
Polynucleotides containing uracil and guanine, and uracil and 8-azaguanine were prepared with polynucleotide phosphorylase (EC 2.7.7.8), and their template activities in stimulating [14C]amino acid incorporation into protein in Escherichia coli extracts were investigated. Both polynucleotides stimulated the incorporation of [14C]valine, E~4C]leucine and [14C]phenylalanine into protein, but had no effect upon incorporation of [a4C]tyrosine. The results suggest that 8-azaguanine may replace guanine in an RNA codon corresponding to valine.
INTRODUCTION
The purine base analogue 8-azagnanine is incorporated into the RNA of different organisms and partly replaces guanine 1. In each case protein synthesis is inhibited soon after the addition of the analogue to the growing cells2,3. Such results have led to the suggestion that the analogue alters the coding properties of messenger RNA containing azaG (ref. 4)LEVlN has shown that polynucleotide phosphorylase (EC 2.7.7.8) catalyses the polymerization of azaGDP (ref. 5). The present investigation was designed to test this hypothesis by preparing poly (U-azaG) and studying its ability to direct [14C]amino acids into protein in Escherichia coli extracts. METHODS AND MATERIALS
Poly (U) was obtained from Miles Chemical Co. The synthesis of poly (UG) and poly (U-azaG) was catalyzed by Micrococcus lysodeikticus polynucleotide phos" Present address: I n s t i t u t e of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.
Biochim. Biophys. Aeta, 119 (1966) 581-585
582
D. GRUNBERGER, C. O'NEAL, M. NIRENBERG
phorylase by methods described previously 8. The input ratio of UDP to GDP was 4 to I and the ratio of UDP to azaGDP was 4 to I also. AzaGDP was obtained by courtesy of Cancer Chemotherapy National Service Center, prepared by Pabst Laboratories; UDP and GDP from Calif. Biochem. Corporation. Base ratios of each polynucleotide were obtained by Dr. R. BRIMACOMBE. Polynucleotides were digested with T, phosphodiesterase as previously described 7. Digestion products were separated by electrophoresis at IO kV on Whatman No. 40 paper with 0.035 M ammonium formate (pH 2.7). Each ultraviolet-absorbing spot was eluted with o.i M HC1 and its spectrum was determined against an appropriate blank eluate in a Cary recording spectrophotometer. The base-ratio of poly (UG) was found to be 0.74 2'(3')-UMP/o.26 2'(3')-GMP; the base-ratio of poly (U-azaG) was 0.82 2' (3')-UMP/o.I8 2'(3')-azaGMP. Extracts prepared from E. coli W3Ioo were treated with deoxyribonuclease (EC 3.1.4.5) and were preincubated and dialyzed as described previously s. Each reaction contained 0.84 mg of protein in the S-3o fraction, 15/zg of polynucleotide, as specified, and other components previously described 8. Other methods, such as deproteinization, washing of protein precipitates, and determination of radioactivity have been described elsewhere s. The source and specific radioactivity of each ~14C]amino acid was: L-~l~C]leucine, 237/zC//~mole, New England Nuclear; L- I14C]tyrosine, 217/~C/#mole, New England Nuclear; and L-~4CJphenylalanine, 3o2/~C//~mole, Schwarz BioResearch, Inc.
TABLE I Effect of poly (U), poly (UG) and poly (U-azaG) upon the incorporation into protein of [14CJphenylatanine, [14Clleucine, [laC]valine and [14C]tyrosine. Reactions were incubated for 3 ° min at 37 °.
Polynucleotide
None Poly (U-azaG) Poly (UG) Poly (U)
#tzmoles o/ E14C~amino acid incorporated into protein [14C!Phenylalanine E14C]Leucine
E14C]Valine
[14C!Tyrosine
0.90 22.19 33-55 35.02
0.32 4.94 i1.49 0.42
0.063 0.084 o. i i o --
0.84 6.76 12.24 lO.O4
RESULTS AND DISCUSSION
In Table I is shown the effect of poly (U-azaG), poly (UG), and poly (U) upon the incorporation of [x4C]phenylalanine, ~14C]leucine, Lx4CJvaline and ~14C]tyrosine into protein in E. coli extracts. Both poly (U-azaG) and poly (UG) markedly stimulated incorporation of [x4C]phenylalanine, [14C]leucine and [14CJvaline into protein. The template activity of polv (U-azaG) was approximately one-half that of poly (UG). Poly (U) stimulated the incorporation of [14CJphenylalanine and [14C]leucine into protein, but had no effect upon [14C]valine incorporation. The effect of poly (U) upon leucine incorporation was observed during the early phase of such work 9,1°. Thus, the template activity of poly (U-azaG) for leucine may be due to sequences of U only. However, the effect of poly (U-azaG) upon incorporation of valine into Biochim. Biophys. Acta, 119 (1966) 581-585
POLYURIDYLIC-8-AZAGUANYLIC ACID AND PROTEIN SYNTHESIS
583
protein clearly demonstrates that the azaG residues are capable of replacing guanine in the polynucleotide template. The nucleotide sequences, UpApU and UpApC, serve as RNA codons for tyrosinen. To determine whether azaG could replace A during codon recognition, the effect of poly (U-azaG) upon the incorporation of [14Cltyrosine into protein was investigated. Poly (U-azaG) and poly (UG) had little effect upon the incorporation of [14Cltyrosine into protein. These results suggest that azaG may not often be recognized as A during protein synthesis. In addition, the results are in accord with the calculations of the strength of the interaction between C and G and C and azaG by NASH AND BRADLEY12. They indicate that these two base pairs have within 1-2 % of the same interaction energy. Therefore azaG should behave very similarly to G in any reaction where the formation of G-C base pairs is involved. In Fig. I is shown the effect of different concentrations of poly (UG) and poly (U-azaG) upon the incorporation of [14Clvaline into protein. Both polynucleotides stimulated [14Clvaline incorporation into protein at each polynucleotide concentration tested. However, poly (UG) was approx. 3 times more efficient a template for valine than poly (U-azaG).
1."
1(
5
10
~g
15
20
Fig. I. Effect of p o l y (UG) a n d poly (U-azaG) u p o n [14C]valine incorporation, into p r o t e i n in p r e i n c u b a t e d S-3 o E. coli e x t r a c t s . 0 - 0 , poly (UG); Q-(2), poly (U-azaG). Reaction.s w e r e i ~ c u b a t e d for 15 m i n at 37 ° .
The pK of azaG has been shown to be approx. 7, whereas, the pK of guanine is 9.2. Since the pH optimum for template activity might reflect, in part, the pK of each base, the relation between template activity of poly (UG) and poly (U-azaG) and pH was investigated (Fig. 2). Both poty (UG) and poly (U-azaG) stimulated the incorporation of [14C]valine into protein at each pH tested (pH 6-9). E14ClValine incorporation was stimulated maximally by both polynucleotides at approx, pH 8. As was shown in the previous experiments, poly (U-azaG) was a less efficient template than poly (UG). Biochim. Biophys. Acta, I19 (1966) 581-585
584
D. GRUNBERGER, C. O'NEAL, M. NIRENBERG m
35 3O
2520 .
~
13t4 Fig. 2. The relation between the pH of reactions and the template activity of poly (UG) and poly (U-azaG) for [l~C]valine incorporation into protein. 0 - 0 , poly (UG); (D-O, poly (U-azaG). Reactions were incubated for 15 min at 37 °.
Because poly (UG) and poly (U-azaG) may differ in secondary structure, it was of interest to determine the effect of Mg 2+ concentration on the template activity of each polymer for [14C]valine. As shown in Fig. 3, the Mg 2+ concentration has a profound influence on the incorporation of [14C]valine into protein. Maximal incorporation of [14C]valine was found when the concentration of Mg 2+ in reactions was O.Ol 7 M. m
J
20
15
N~nesuiocet rn o(xl te2'0O3M)
3'0
Fig. 3. The relation between the concentration of Mg ~+ and the template activity of po]y (UG) and poly ( U - a z a G ) f o r [14C]valine incorporation into protein. Q - O , poly (UG); G-(D, poly (U-azaG). Reactions were incubated for 15 mill at 37 °.
Biochim. Biophys. Acta, 119 (t966)
581
585
POLYURIDYLIC-8-AZAGUANYLIC ACID AND PROTEIN SYNTHESIS
585
Although the difference between the template efficiency of poly (U-azaG) and poly (UG) may reflect differences between the recognition of azaG and G during protein synthesis, other factors also deserve consideration. For example, chain length, secondary structure, and presence of terminal phosphate would be expected also to influence the template efficiency of polynucleotides. These results indicate that azaG may replace G in a valine RNA codon which still corresponds to valine during protein synthesis. These results agree with the findings of GRUNBERGERAND MANDELla that messenger RNA synthesized by growing Bacillus cereus cells in the presence of azaG can serve as templates for the incorporation of [14C]valine into protein in E. coli extracts.
ACKNOWLEDGEMENTS
It is a pleasure to acknowledge the interest and support of Dr. H. G. MANDEL in whose laboratory some of the experiments were performed. Also to be acknowledged is the excellent technical assistance of Miss M. RIIs. This research was partly supported by grant CA-o2978 from the National Cancer Institute, U.S. Public Health Service, Bethesda, Md.,
REFERENCES I 2 3 4 5 6 7 8
9 io Ii 12 13
E. F. MATTHEWS AND J. D. SMITH, Nature, 177 (1956) 271. G. MANDEL, Arch. Biochem. Biophys., 76 (1958) 23o. GRUNBERGER AND F. SORM, Collection Czech. Chem. Commun., 28 (1963) lO44. A. CHANTRENNE, J. Cellular Comp. Physiol., Suppl. i, 64 (1964) 149. H. LEVlN, Biochim. Biophys. Acta, 61 (1962) 75. F. SINGER AND J. K. G u s s , J. Biol. Chem., 237 (1962) 182. W . RUSHIZKY AND H. A. SOBER, J. Biol. Chem., 238 (1963) 371. W . lXTIRENBERG, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic, New York, 1964, p. 17. M. S. BRETSCHER AND M. GRUNBERG-MANAGO, Nature, 195 (1962) 283. J. H. MATTHAEI, O, W. JONES, R. W. MARTIN AND M. W. NIRENBERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 666. J. TRUPIN, •. M. ROTTMAN, R. L. C. BRIMACOMBE, P. LEDER, M. R. BERNFIELD AND M. W . NIRENBERG, Proc. Natl..4cad. Sci. U.S., 53 (1965) 8o7. H. NASH AND D. F. BRADLEY, in preparation. D. GRUNBERGER AND H. G. MANDEL, in the press.
R. H. D. H. D. i. G. M.
Biochim. Biophys. Acta, 119 (1966) 581-585
581
BBA 95399
STIMULATION OF AMINO ACID INCORPORATION INTO PROTEIN BY POLYURIDYLIC-8-AZAGUANYLIC ACID
DEZIDER
GRI3NBERGER*,
CHARLES
O'NEAL
AND
MARSHALL
NIRENBERG
Department o] Pharmacology, The George Washington University School o/ Medicine, Washington, D. C. (U.S.A.) and National Heart Institute, National Institutes o] Health, Bethesda, Md. (U.S.A.) (Received September i6th, 1965)
SUMMARY
Polynucleotides containing uracil and guanine, and uracil and 8-azaguanine were prepared with polynucleotide phosphorylase (EC 2.7.7.8), and their template activities in stimulating [14C]amino acid incorporation into protein in Escherichia coli extracts were investigated. Both polynucleotides stimulated the incorporation of [14C]valine, E~4C]leucine and [14C]phenylalanine into protein, but had no effect upon incorporation of [a4C]tyrosine. The results suggest that 8-azaguanine may replace guanine in an RNA codon corresponding to valine.
INTRODUCTION
The purine base analogue 8-azagnanine is incorporated into the RNA of different organisms and partly replaces guanine 1. In each case protein synthesis is inhibited soon after the addition of the analogue to the growing cells2,3. Such results have led to the suggestion that the analogue alters the coding properties of messenger RNA containing azaG (ref. 4)LEVlN has shown that polynucleotide phosphorylase (EC 2.7.7.8) catalyses the polymerization of azaGDP (ref. 5). The present investigation was designed to test this hypothesis by preparing poly (U-azaG) and studying its ability to direct [14C]amino acids into protein in Escherichia coli extracts. METHODS AND MATERIALS
Poly (U) was obtained from Miles Chemical Co. The synthesis of poly (UG) and poly (U-azaG) was catalyzed by Micrococcus lysodeikticus polynucleotide phos" Present address: I n s t i t u t e of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.
Biochim. Biophys. Aeta, 119 (1966) 581-585
582
D. GRUNBERGER, C. O'NEAL, M. NIRENBERG
phorylase by methods described previously 8. The input ratio of UDP to GDP was 4 to I and the ratio of UDP to azaGDP was 4 to I also. AzaGDP was obtained by courtesy of Cancer Chemotherapy National Service Center, prepared by Pabst Laboratories; UDP and GDP from Calif. Biochem. Corporation. Base ratios of each polynucleotide were obtained by Dr. R. BRIMACOMBE. Polynucleotides were digested with T, phosphodiesterase as previously described 7. Digestion products were separated by electrophoresis at IO kV on Whatman No. 40 paper with 0.035 M ammonium formate (pH 2.7). Each ultraviolet-absorbing spot was eluted with o.i M HC1 and its spectrum was determined against an appropriate blank eluate in a Cary recording spectrophotometer. The base-ratio of poly (UG) was found to be 0.74 2'(3')-UMP/o.26 2'(3')-GMP; the base-ratio of poly (U-azaG) was 0.82 2' (3')-UMP/o.I8 2'(3')-azaGMP. Extracts prepared from E. coli W3Ioo were treated with deoxyribonuclease (EC 3.1.4.5) and were preincubated and dialyzed as described previously s. Each reaction contained 0.84 mg of protein in the S-3o fraction, 15/zg of polynucleotide, as specified, and other components previously described 8. Other methods, such as deproteinization, washing of protein precipitates, and determination of radioactivity have been described elsewhere s. The source and specific radioactivity of each ~14C]amino acid was: L-~l~C]leucine, 237/zC//~mole, New England Nuclear; L- I14C]tyrosine, 217/~C/#mole, New England Nuclear; and L-~4CJphenylalanine, 3o2/~C//~mole, Schwarz BioResearch, Inc.
TABLE I Effect of poly (U), poly (UG) and poly (U-azaG) upon the incorporation into protein of [14CJphenylatanine, [14Clleucine, [laC]valine and [14C]tyrosine. Reactions were incubated for 3 ° min at 37 °.
Polynucleotide
None Poly (U-azaG) Poly (UG) Poly (U)
#tzmoles o/ E14C~amino acid incorporated into protein [14C!Phenylalanine E14C]Leucine
E14C]Valine
[14C!Tyrosine
0.90 22.19 33-55 35.02
0.32 4.94 i1.49 0.42
0.063 0.084 o. i i o --
0.84 6.76 12.24 lO.O4
RESULTS AND DISCUSSION
In Table I is shown the effect of poly (U-azaG), poly (UG), and poly (U) upon the incorporation of [x4C]phenylalanine, ~14C]leucine, Lx4CJvaline and ~14C]tyrosine into protein in E. coli extracts. Both poly (U-azaG) and poly (UG) markedly stimulated incorporation of [x4C]phenylalanine, [14C]leucine and [14CJvaline into protein. The template activity of polv (U-azaG) was approximately one-half that of poly (UG). Poly (U) stimulated the incorporation of [14CJphenylalanine and [14C]leucine into protein, but had no effect upon [14C]valine incorporation. The effect of poly (U) upon leucine incorporation was observed during the early phase of such work 9,1°. Thus, the template activity of poly (U-azaG) for leucine may be due to sequences of U only. However, the effect of poly (U-azaG) upon incorporation of valine into Biochim. Biophys. Acta, 119 (1966) 581-585
POLYURIDYLIC-8-AZAGUANYLIC ACID AND PROTEIN SYNTHESIS
583
protein clearly demonstrates that the azaG residues are capable of replacing guanine in the polynucleotide template. The nucleotide sequences, UpApU and UpApC, serve as RNA codons for tyrosinen. To determine whether azaG could replace A during codon recognition, the effect of poly (U-azaG) upon the incorporation of [14Cltyrosine into protein was investigated. Poly (U-azaG) and poly (UG) had little effect upon the incorporation of [14Cltyrosine into protein. These results suggest that azaG may not often be recognized as A during protein synthesis. In addition, the results are in accord with the calculations of the strength of the interaction between C and G and C and azaG by NASH AND BRADLEY12. They indicate that these two base pairs have within 1-2 % of the same interaction energy. Therefore azaG should behave very similarly to G in any reaction where the formation of G-C base pairs is involved. In Fig. I is shown the effect of different concentrations of poly (UG) and poly (U-azaG) upon the incorporation of [14Clvaline into protein. Both polynucleotides stimulated [14Clvaline incorporation into protein at each polynucleotide concentration tested. However, poly (UG) was approx. 3 times more efficient a template for valine than poly (U-azaG).
1."
1(
5
10
~g
15
20
Fig. I. Effect of p o l y (UG) a n d poly (U-azaG) u p o n [14C]valine incorporation, into p r o t e i n in p r e i n c u b a t e d S-3 o E. coli e x t r a c t s . 0 - 0 , poly (UG); Q-(2), poly (U-azaG). Reaction.s w e r e i ~ c u b a t e d for 15 m i n at 37 ° .
The pK of azaG has been shown to be approx. 7, whereas, the pK of guanine is 9.2. Since the pH optimum for template activity might reflect, in part, the pK of each base, the relation between template activity of poly (UG) and poly (U-azaG) and pH was investigated (Fig. 2). Both poty (UG) and poly (U-azaG) stimulated the incorporation of [14C]valine into protein at each pH tested (pH 6-9). E14ClValine incorporation was stimulated maximally by both polynucleotides at approx, pH 8. As was shown in the previous experiments, poly (U-azaG) was a less efficient template than poly (UG). Biochim. Biophys. Acta, I19 (1966) 581-585
584
D. GRUNBERGER, C. O'NEAL, M. NIRENBERG m
35 3O
2520 .
~
13t4 Fig. 2. The relation between the pH of reactions and the template activity of poly (UG) and poly (U-azaG) for [l~C]valine incorporation into protein. 0 - 0 , poly (UG); (D-O, poly (U-azaG). Reactions were incubated for 15 min at 37 °.
Because poly (UG) and poly (U-azaG) may differ in secondary structure, it was of interest to determine the effect of Mg 2+ concentration on the template activity of each polymer for [14C]valine. As shown in Fig. 3, the Mg 2+ concentration has a profound influence on the incorporation of [14C]valine into protein. Maximal incorporation of [14C]valine was found when the concentration of Mg 2+ in reactions was O.Ol 7 M. m
J
20
15
N~nesuiocet rn o(xl te2'0O3M)
3'0
Fig. 3. The relation between the concentration of Mg ~+ and the template activity of po]y (UG) and poly ( U - a z a G ) f o r [14C]valine incorporation into protein. Q - O , poly (UG); G-(D, poly (U-azaG). Reactions were incubated for 15 mill at 37 °.
Biochim. Biophys. Acta, 119 (t966)
581
585
POLYURIDYLIC-8-AZAGUANYLIC ACID AND PROTEIN SYNTHESIS
585
Although the difference between the template efficiency of poly (U-azaG) and poly (UG) may reflect differences between the recognition of azaG and G during protein synthesis, other factors also deserve consideration. For example, chain length, secondary structure, and presence of terminal phosphate would be expected also to influence the template efficiency of polynucleotides. These results indicate that azaG may replace G in a valine RNA codon which still corresponds to valine during protein synthesis. These results agree with the findings of GRUNBERGERAND MANDELla that messenger RNA synthesized by growing Bacillus cereus cells in the presence of azaG can serve as templates for the incorporation of [14C]valine into protein in E. coli extracts.
ACKNOWLEDGEMENTS
It is a pleasure to acknowledge the interest and support of Dr. H. G. MANDEL in whose laboratory some of the experiments were performed. Also to be acknowledged is the excellent technical assistance of Miss M. RIIs. This research was partly supported by grant CA-o2978 from the National Cancer Institute, U.S. Public Health Service, Bethesda, Md.,
REFERENCES I 2 3 4 5 6 7 8
9 io Ii 12 13
E. F. MATTHEWS AND J. D. SMITH, Nature, 177 (1956) 271. G. MANDEL, Arch. Biochem. Biophys., 76 (1958) 23o. GRUNBERGER AND F. SORM, Collection Czech. Chem. Commun., 28 (1963) lO44. A. CHANTRENNE, J. Cellular Comp. Physiol., Suppl. i, 64 (1964) 149. H. LEVlN, Biochim. Biophys. Acta, 61 (1962) 75. F. SINGER AND J. K. G u s s , J. Biol. Chem., 237 (1962) 182. W . RUSHIZKY AND H. A. SOBER, J. Biol. Chem., 238 (1963) 371. W . lXTIRENBERG, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic, New York, 1964, p. 17. M. S. BRETSCHER AND M. GRUNBERG-MANAGO, Nature, 195 (1962) 283. J. H. MATTHAEI, O, W. JONES, R. W. MARTIN AND M. W. NIRENBERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 666. J. TRUPIN, •. M. ROTTMAN, R. L. C. BRIMACOMBE, P. LEDER, M. R. BERNFIELD AND M. W . NIRENBERG, Proc. Natl..4cad. Sci. U.S., 53 (1965) 8o7. H. NASH AND D. F. BRADLEY, in preparation. D. GRUNBERGER AND H. G. MANDEL, in the press.
R. H. D. H. D. i. G. M.
Biochim. Biophys. Acta, 119 (1966) 581-585