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The Qi3-RNA Polymerase Reaction Contained Guanosine

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The Qi3-RNA Polymerase Reaction Contained Guanosine 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 reac- tion 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 purifi- cation 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 pre- cursor, 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 elec- trophoretic 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 mono- phosphates 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 deter- mined 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.
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