The Qi3-RNA Polymerase Reaction Contained Guanosine

Home , Cytidine monophosphate, Guanosine, Guanosine monophosphate, Guanosine triphosphate, Ribonucleoside

IDENTIFICATION OF GUANOSINE TRIPHOSPHATE AS THE 5'-TERMINUS OF RNA FROM BACTERIOPHAGE Qj# AND R23* BY MIAMORU WATANABEt AND J. THOMAS AUGUSTt

DEPARTMENT OF MOLECULAR BIOLOGY, ALBERT EINSTEIN COLLEGE OF MEDICINE, NEW YORK, NEW YORK Communicated by B. L. Horecker, December 8, 1967 It was previously found in this laboratory that Qi3-RNA synthesized in vitro in the Qi3-RNA polymerase reaction contained guanosine triphosphate (GTP) at the 5'-terminus.' This discovery appeared to have considerable relevance to many problems concerning phage RNA structure and function. An immediate ques- tion was how this finding correlated with the 5'-terminus of natural phage RNA. It has been reported by Takanami that RNA of phage f2 contained a triphosphate group at the 5'-terminus and the 5'-terminal nucleoside appeared to be adenosine.2 We have now examined RNA from phages Q3 and R23 and have found that the 5'-terminus in both cases contained GTP. Recently, Roblin has identified GTP at the 5'-terminus of R17 RNA.3 Identification of the 5'-terminus in this study was based on the isolation and characterization of products of alkaline digestion of the phage RNA. Because the 5'-triphosphate structure is resistant to mild alkali, alkaline hydrolysis releases the terminal structure as the tetraphosphate, ribonucleoside 5'-tri, 2',3'-monophosphate (pppRp). As described in this report, this component has been isolated after alkaline hydrolysis of RNA from phages QB and R23 and identified as guanosine tetraphosphate (pppGp). Materials and Methods.-M4aterials: Cytosine-5-HI (18.0 c/mmole), adenine-8-H3 (7.3 c/mmole), guanine-8-H3 (9.8 c/mmole) and uracil-6-H3 (15.0 c/mmole), were purchased from Schwarz BioResearch, Orangeburg, N.Y.; p,32 from International Chemical and Nuclear Corporation, City of Industry, Calif.; unlabeled ribonucleotides from Mann Research Laboratories, New York, N.Y.; ribose-5-phosphate from Calbiochem, Los Angeles, Calif.; alkaline phosphatase (BAPF) from Worthington Biochemical Corpora- tion, Freehold, N.J.; and Dowex-50 (H+) from Bio-Rad Laboratories, Richmond, Calif. For comparison with the natural product, y-P'2-guanosine tetraphosphate (p32ppGp) was prepared by alkaline hydrolysis of RNA synthesized in the QB-RNA polymerase reaction using Y-P32-GTP as the labeled substrate, as previously described,' and was kindly supplied by A. K. Banerjee of this department. Organisms and growth conditions: RNA bacteriophage R23, isolated in this laboratory, has been described in a previous publication.4 RNA bacteriophage Q3 5, 6 was provided by S. Spiegelman of the University of Illinois. The bacterial host used in these studies was Escherichia coli K38,7 and the media and growth conditions employed for bacteria and bacteriophages were as previously described.4 Preparation and purification of labeled phage: Bacteria were grown in medium A at 370 in a gyrotory shaker to a density of 5 X 108 cells/ml and infected with the RNA bacteriophage R23 or Q,3 at a multiplicity of infection of 10. Fifteen min after infection, one of the labeled RNA precursors was added. HI-labeled cytosine, adenine, guanine, or uracil was added to a final concentration of 0.5-1 /Lc/ml or p32 to a final concentration of 5-10 ,c/ml. Usually a total volume of 2 liters was employed. The bacteria were lysed 2 hr after infection by the addition of 0.1 vol of lysing medium and 0.01 vol of chloro- form,4 and the phage were purified by liquid polymer phase fractionation and density equilibrium centrifugation in CsCl.8 513 Downloaded by guest on September 26, 2021 514 BIOCHEMISTRY: WATANABE AND AUGUST PROC. N. A. S.

Isolation and alkaline hydrolysis of RNA: The purified phage suspension was gently shaken with redistilled phenol that had been previously equilibrated with 0.5 M potassium phosphate buffer at pH 6.8. The aqueous phase was collected and sodium acetate was added to a final concentration of 0.3 M. Two vol of cold 95% ethanol were added and the sample was stored at -20° for 2 hr, after which the precipitated RNA was collected by centrifugation at 11,000 rpm for 10 min. The precipitate was dissolved in standard saline-citrate (0.15 M NaCl plus 0.015 M sodium citrate) and the ethanol precipitation repeated. The final precipitate was dissolved in water. Alkaline hydrolysis was carried out at 370 for 24 hr in 0.3 N KOH. The hydrolysate was neutralized to pH 7.0 with Dowex-50 (H+); the resin was then removed by filtration through glass wool and the effluent concentrated in vacuo prior to analysis by high-voltage electrophoresis. High-voltage electrophoresis: Electrophoresis was carried out on Whatman no. 3MM paper. The paper had previously been washed with 0.01 N NH40H, followed by de- ionized water until neutral, and dried. Usually the electrophoresis was carried out in pyridine, acetic acid, water (1:10:89), pH 3.5, at 50 v/cm for 2 hr. For additional purification of P32labeled pppGp, a second electrophoresis in 0.04 31 sodium citrate buffer, pH 4.0, at 50 v/cm for 2 hr, was employed. Appropriate nucleotide markers were applied with the samples prior to electrophoresis, since the migration characteristics of material present in small quantities varied in the absence of carrier compounds. After the paper was dried, the nucleotide markers were visualized by ultraviolet light. Radioactivity was counted with a Vanguard 880 strip scanner or in a liquid scintillation spectrometer. For the latter procedure, 1-cm-wide strips were cut longitudinally from the center of the paper and divided into 1-cm squares; these squares were placed in the bottom of counting vials containing 2 ml of toluene phosphor (0.4% p-bis-(2-(5-phenyloxazolyl)-benzene and 0.01% 2,5-diphenyloxazole). Radioactive compounds were eluted from the paper with 0.01 N NH40H and concentrated for additional electrophoresis or counting. Inorganic phosphate was detected by the Hanes-Isherwood test.9 Hydrolysis of guanosine tetraphosphate: Alkaline phosphatase treatment was carried out in (0.1 ml) 50 mM Tris-HCl buffer, pH 8.4, with 1 unit of enzyme (capable of hydrolyz- ing 1 ,mole of p-nitrophenol/min at 250) at 370 for 30 min. Hydrolysis at pH 4.0 was carried out in 1.0 M ammonium formate buffer, at 100° for 2 hr. Acid hydrolysis was carried out in 1 N HC1 at 1000 for 30 min. Results.-Isolation of pppGp: To identify the putative triphosphate terminus, RNA of phages R23 and Q,3 was labeled in separate experiments with H3 or p32. The use of H3-labeled bases allowed relatively specific labeling of the phage RNA (Tables 1 and 2). When H3-cytosine or H3-uracil was the labeled precursor, radioactivity after alkaline hydrolysis was recovered in both the 2',3'- cytidine monophosphate (Cp) and 2',3'-uridine monophosphate (Up) regions of the electropherogram. With H3-adenine and H3-guanine the major radioactive components migrated with the markers 2',3'-adenosine monophosphate (Ap) and 2',3'-guanosine monophosphate (Gp), respectively. However, RNA labeled with H3-guanine also yielded 0.12-0.16 per cent of the total radioactivity as a compound which migrated beyond GTP, with the same mobility as pppGp. With the other three labeled precursors, no H3 radioactivity possessing the electrophoretic mobility of a tetraphosphate was detected. After alkaline hydrolysis, P32-labeled R23 or Q,3-RNA yielded as many as three radioactive peaks which migrated more rapidly than the ribonucleoside monophosphates in high-voltage electrophoresis at pH 3.5. These peaks were eluted and further purified by electrophoresis at pH 4.0. Three distinct components were characterized (Table 3). Peak 1, which migrated more slowly than pppGp, corresponded to ribose-5-phosphate (R-5-P) in its migration characteristics and the radioactivity associated with this peak was only partially charcoal-(Norit) Downloaded by guest on September 26, 2021 VOL. 59, 1968 BIOCHEMISTRY: WATANABE AND AUGUST . 515

TABLE 1. Alkaline hydrolysis products of H3-labeled Q(3-RNA. Products of Alkaline Hydrolysis (% of total radioactivity) Component Precursor of migrating labeled RNA Cp Ap Gp Up beyond pppG H3-Cytosine 56.5 0.5 N.D. 43.0 N.D. HM-Adenine 2.5 96.2 1.2 0.1 N.D. HM-Guanine N.D. 1.1 98.8 N.D. 0.12 H3-Uracil 43.0 N.D. N.D. 57.0 N.D. Q#-RNA labeled with H3 was prepared as described in the Method8 section, and the products of alkaline hydrolysis were separated by high-voltage electrophoresis at pH 3.5. The radioactive compounds were eluted with 0.01 N NH4OH and radioactivity was determined in a liquid scintillation spectrometer. N.D. signifies "not detectable."

TABLE 2. Alkaline hydrolysis products of HI-labeled R23 RNA. Product of Alkaline Hydrolysis (% of total radioactivity) Component Precursor of migrating labeled RNA Cp Ap Gp Up beyond pppG H3-Cytosine 61.5 N.D. N.D. 38.5 N.D. H3-Adenine N.D. 98.7 1.3 N.D. N.D. HI-Guanine (1) N.D. N.D. 99.8 N.D. 0.16 (2) N.D. N.D. 99.8 N.D. 0.12 H3-Uracil 49.8 N.D. N.D. 50.2 N.D. H3-labeled R23 RNA was prepared and analyzed as described in Table 1 for Q,-RNA.

adsorbable (30-35%). The radioactivity in peak 2 migrated together with pppGp, while that in peak 3 was noncharcoal-adsorbable and migrated with carrier inorganic phosphate (Pi). The amount of Pi detected in the alkaline hydrolysates was variable and was present in the hydrolysates of QB as well as of R23 RNA. The radioactivity in peak 2, representing 0.11 per cent of the total alkaline hydrolysis product, was completely adsorbed by charcoal and was further characterized as guanosine tetraphosphate (see below). The compounds in peaks 1 and 2 (ribose phosphate and guanosine tetraphosphate) were not readily separable on electrophoresis at pH 3.5 but could be separated on electrophoresis in citrate buffer at pH 4.0.

TABLE 3. Alkaline hydrolysis products of Q( and R23 RNA labeled uith P32. Radioactivity Radioactive (% of total Phage peaks Migration characteristics hydrolysate) R23 1 Ribose-5-phosphate 0.19 2 Guanosine tetraphosphate 0.11 3 Inorganic phosphate 0.24 Q,# 1 Ribose-5-phosphate 0.07 2 Guanosine tetraphosphate 0.11 Alkaline hydrolysis products of R23 and QB-RNA labeled with P32 was prepared as described in the Methods section and separated on high-voltage electrophoresis at pH 3.5. The radioactive compounds migrating beyond the ribonucleoside monophosphates were eluted with 0.01 N NH40H and further purified by second electrophoresis at pH 4 in 0.04 M sodium citrate buffer. Radioactivity was assayed in a liquid scintillation spectrometer following elution from paper. Downloaded by guest on September 26, 2021 516 BIOCHEMISTRY: WATANABE AND AUGUST Puoc. N. A. S. Characterization of ppp~p: (a) Electrophoretic mobility: The radioactive products migrating beyond GTP from both H3- and P32-labeled RNA (Tables 1, 2, and 3) were eluted and rerun with authentic p32ppGp. The mobilities of both the H3-labeled and P32-labeled hydrolysis products were identical to that of p32ppGp, all three migrating faster than GTP added as marker (Fig. 1). The

bWHYDROLYSISPom-E> -aME Lmm N 2w Cp AP Gp Up WppGPPG p

FIG. mobility of hy- WHYDROEp32 PROD=2 ^drolysis 1.-Electrophoreticproducts of phage RNA. Degrada- > 20°in 11 tion products of alkaline hydrolysis of phage hi81lRNA labeled with H3-guanine or P32 were separated on high-voltage electrophoresis at J A pH 3.5 as described in the Method8 section. 0 1 TheS radioactive compounds migrating be- 400 - IETRAPHOSFE 8yond GTP were eluted with 0.01 N NH40H and concentrated and rerun in high-voltage electrophoresis at pH 3.5 at 50 v/cm for 2 hr. Radioactivity was determined in a liquid scintillation spectrometer. 20 40 60 Do)^-#GU^No61>DISTANCE FROM ORIGN (cmi) hydrolysis product of the P32-labeled phage RNA, obtained from electrophoresis on pH 3.5, showed, three components. The most rapidly migrating component was Pi. The other two components migrating more rapidly than GTP (R-5-P and pppGp) were separated in high-voltage electrophoresis at pH 4 (Table 3), R-5-P migrating somewhat less rapidly than pppGp in electrophoresis at pH 3.5. The p32ppGp reference sample was contaminated with GTP, a small amount of which remained from the Q13-RNA polymerase reaction mixture where 'yP32- GTP (p32ppG) was used as substrate for the synthesis of RNA labeled at the 5'-terminus. (b) Degradation products: Radioactivity from the P32-labeled phage RNA hydrolysis product corresponding to guanosine tetraphosphate in its migration characteristics was completely charcoal-adsorbable. After treatment with 1 N HCO at 1000 for 30 minutes or after incubation with alkaline phosphatase, the p32 became completely charcoal nonadsorbable. This charcoal nonadsorbable p32 radioactivity after acid hydrolysis or alkaline phosphatase treatment was identified as Pi by electrophoresis. Mild acid hydrolysis at pH 4.0 yielded pre- dominantly guanosine 3',5'-diphosphate (pGp) as well as smaller quantities of guanosine 5'-monophosphate and Pi. pGp is the expected major product since the # and y phosphate groups of nucleoside 5'-triphosphates would be preferen- tially hydrolyzed,10 although partial cleavage of the 3'-phosphate would also occur under these conditions." The H3-guanine-labeled material migrating as pppGp gave rise to guanosine on incubation with alkaline phosphatase. Discussion.-Alkaline hydrolysis of phage RNA would be expected to yield a ribonucleoside 5'-tri, 2',3'-monophosphate (pppRp) if the RNA contained a Downloaded by guest on September 26, 2021 VOL. 59, 1968 BIOCHEMISTRY: WATANABE AND AUGUST 517

5'-triphosphate at a terminus. In the case of P32-labeled bacteriophage RNA, the tetraphosphate moiety would account for approximately 4/3300 of the total p32 activity, the RNA containing approximately 3300 Aucleotides.12 With RNA labeled in one of the four bases, approximately 1/825 of the total radioactivity should be recovered in the tetraphosphate form since the four bases are almost equally represented in both R23 and Q3-RNA.6, 8 Thus, approximately 0.12 per cent of the radioactivity should appear with the ribonucleoside tetraphosphate when the RNA is labeled with p32 or the appropriate base. We have observed that approximately 0.12-0.16 per cent of the total radioactivity migrated beyond GTP when either R23 or Q#-RNA -was labeled with H3-guanine, but not with the other three labeled bases. A similar amount of radioactivity migrated beyond GTP, as pppGp, when either R23 or Q13-RNA was labeled with p32 Both the H3- and p32-labeled products were plectrophoretically identical with authentic p32ppGp prepared from RNA synthesized in vitro in the QP- polymerase reaction.' The H3- or P32-labeled guanosine tetraphosphate yielded guanosine 3',5'-diphosphate and guanosine 5'-monophosphate after mild acid hydrolysis, and the H3-labeled material was converted by treatment with alkaline phosphatase to guanosine. It thus appeared that guanosinle triphosphate was present at the 5'-terminus of both R23 and QB-RNA. As indicated earlier, Roblin has also found R17 RNA to contain GTP at the 5'-terminus.3 One of the remarkable features of the Q,-RNA polymerase reaction is its specificity for template RNA;13 R17 or R23 RNA will not substitute for Qi6-RNA in the reaction.'4 Since Qf3, R17, and R23 RNA all contain guanosine triphosphate at the 5'-terminus, this terminal structure cannot of itself be the critical structure which determines the specificity of the QP-RNA polymerase for RNA template. Ribonucleoside 5'-triphosphate termini have previously been found with RNA synthesized in vitro, both with the DNA-dependent RNA polymerasel5' 16 and the RNA-dependent Q,3-RNA polymerase.' The discovery of the triphosphate terminus of bacteriophage RNA in these studies, as well as those of Roblin3 and Takanami,2 establishes the natural occurrence of this structure. The ques- tion may thus be raised as to whether the 5'-triphosphate terminus has any functional significance, either in the replication of RNA or in the messenger function of this RNA. In this respect, it may bQ relevant to compare the 5'- terminus of phage RNA with that of the bacterial messenger RNA. It is note- worthy that TMV-RNA does not appear to possess phosphoryl groups at the 5'-hydroxyl end.2' 17 Summary.-Alkaline hydrolysis of RNA of the RNA phages QB and R23 yielded a compound which has been identified as guanosine 5'-tri, 2',3'-monophosphate (pppGp). This material was present in a 1:1 molar ratio with RNA labeled either with H3-guanine or p32, and was detected in experiments with specific-labeled bases only with H3-guanine. The electrophoretic mobility of the compound was identical to that of authentic p32ppGp both at pH 3.5 and 4.0. The degradation products guanosine and guanosine 3',5'-diphosphate plus guanosine 5'-monophosphate were obtained after treatment with alkaline phosphatase or after mild acid hydrolysis at pH 4.0, respectively. Downloaded by guest on September 26, 2021 518 BIOCHEMISTRY: WATANABE AND AUGUST PROC. N. A. S.

* This investigation was supported in part by grants from the National Institutes of Health (GM-11936 and GM-11301) and the National Science Foundation (GB-5082). This is com- munication No. 86 from the Joan and Lester Avnet Institute of Molecular Biology. t Recipient of the Research Fellowship of the American College of Physicians, 1964-1967. Present address: Departments of Medicine and Biochemistry, University of Alberta, Edmon- ton, Alberta. $ Career Scientist of the Health Research Council of the City of New York under contract I-346. 1 Banerjee, A. K., L. Eoyang, K. Hori, and J. T. August, these PROCEEDINGS, 57, 986 (1967). 2 Takanami, M., in Cold Spring Harbor Symposia on Quantitative Biology, vol. 31 (1966), p. 611. 3 Roblin, R., J. Mol. Biol., in press (1968). 4Watanabe, M., H. Watanabe, and J. T. August, J. Mol. Biol., in press (1968). 5 Overby, L. R., G. H. Barlow, R. H. Doi, M. Jacob, and S. Spiegelman, J. Bacteriol., 91, 442 (1966). 6 Ibid., 92, 739 (1966). 7Zinder, N. D., and S. Cooper, Virology, 23, 152 (1964). 8 Watanabe, M., and J. T. August, in Methods in Virology, ed. K. Maramorosch and H. Koprowski (New York: Academic Press, 1967), vol. 3. 9 Hanes, C. S., and F. H. Isherwood, Nature, 164, 1007 (1949). 10 Li6becq, C., Arch. Intern. Physiol. Biochim., 65, 141 (1957). "Baddiley, J., J. G. Buchanan, and R. Letters, J. Chem. Soc., 1000 (1958). 12Sinha, N. K., R. K. Fujimura, and P. Kaesberg, J. Mol. Biol., 11, 84 (1965). 13 Haruna, I., and S. Spiegelman, these PROCEEDINGS, 54, 579 (1965). 14August, J. T., unpublished observations. 15 Maitra, U., and J. Hurwitz, these PROCEEDINGS, 54, 815 (1965). 16Bremer, H., M. W. Konrad, K. Gaines, and G. S. Stent, J. Mol. Biol., 13, 540 (1965). 17Sugiyama, T., and H. Fraenkel-Conrat, Biochemistry, 2, 332 (1963). Downloaded by guest on September 26, 2021