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T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides (RNA Synthesis/RPC-5 Chromatography/Gradient Sievorptive Chromatography) GRAHAM C

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T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides (RNA Synthesis/RPC-5 Chromatography/Gradient Sievorptive Chromatography) GRAHAM C Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 122-126, January 1975 T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides (RNA synthesis/RPC-5 chromatography/gradient sievorptive chromatography) GRAHAM C. WALKER*, OLKE C. UHLENBECK, ELLIOTT BEDOWSt, AND RICHARD I. GUMPORTt Department of Biochemistry, School of Chemical Sciences and School of Basic Medical Sciences, and t Department of Microbiology, School of Life Sciences, University of Illinois, Urbana, Ill. 61801 Communicated by N. J. Leonard, October 9, 1974 ABSTRACT RNA ligase isolated from Escherichia coli hydroxyl groups on both the 3'- and 5'-termini. In this paper infected with bacteriophage T4 will catalyze the formation we present evidence for such an intermolecular reaction with of an intermolecular 3' -- 5' phosphodiester linkage be- tween an oligoribonucleotide with a free 3'-hydroxyl and T4 RNA ligase and examine the effect of a variety of parame- another oligoribonucleotide with a 5'-phosphate. Upon ters on the efficiency of the reaction. Since high yields of reaction with (Ap)5C, nearly quantitative conversion of product may be obtained, RNA ligase should be useful in the the hexamer [5'-"2Plp(Up)iU to the dodecamer (Ap)5C[3' - synthesis of oligoribonucleotides of defined sequence. 5'-"Plp(Up)5U was observed. The product was identified by its mobility on RPC-5 column chromatography, its MATERIALS AND METHODS resistance to alkaline phosphatase, and the appearance of the expected radiolabeled products on hydrolysis with Materials. Nucleoside 5'-diphosphates were purchased from alkali, ribonuclease A, snake venom phosphodiesterase, Sigma Chemical Co., and all other nucleotides were obtained and spleen phosphodiesterase. The coupling of other pairs of single-stranded oligoribonucleotides has also been from P-L Biochemicals, Inc. (Ip)5I and endonuclease from demonstrated. The intermolecular joining reaction is Neuroepora crassa were purchased from Miles Laboratories. probably mechanistically similar to the intramolecular Bacterial alkaline phosphatase, ribonuclease A, ribonuclease cyclization activity previously reported for T4 RNA ligase. T1, and spleen phosphodiesterase were purchased from It is expected that this enzyme will be useful for the syn- thesis of RNA fragments of defined sequence. Worthington Biochemical Corp. Polynucleotide kinase was a gift from C. Richardson. 32Pi was purchased from New RNA ligase from Escherichia coli infected with bacteriophage England Nuclear Corp. ['y-'2P]ATP was prepared by the T4 catalyzes the ATP-dependent formation of phosphodiester method of Glynn and Chappell (5). Bacteriophage and bac- bonds in RNA (1-3). Two structurally distinct classes of terial strains were obtained from J. Drake. molecules have been reported to be polynucleotide substrates Preparation of Oligoribonucleotides. The polyribonucleotides for this enzyme. First, RNA ligase has been shown to form a poly(U), poly(A), poly(A,C), poly(A,U), and poly(A,G) were phosphodiester bond between the 3'-hydroxyl and the 5'- synthesized from the corresponding ribonucleoside diphos- phosphate termini of an oligoribonucleotide, producing a phates with polynucleotide phosphorylase from Micrococcus single-stranded circular product (1, 2). Second, in analogy to luteus. Oligomers with 3'-phosphate termini and number T4 DNA ligase (4), T4 RNA ligase also appears capable of average chain length (in) = 5-10 were prepared from the joining the 3'-hydroxyl end of one ribo-oligomer to the 5'- polynucleotides by controlled alkaline hydrolysis or by RNase phosphate of another when the two molecules are aligned in a A or RNase T1 digestion (6). Poly(A) was digested with double helix by hydrogen-bonding to a third, complementary Neurospora crassa endonuclease to give a solution of oligo- "splint" strand (3). Although the enzyme has not been puri- riboadenylates with a 5'-terminal phosphate and ii = 6 (7). fied to homogeneity, it appears likely that the two activities Oligonucleotides in each homologous series were separated are catalyzed by the same protein. The intramolecular cycli- on DEAE-Sephadex A-25 (in the bicarbonate form) by a zation reaction of RNA ligase, which occurs in the absence of a. linear gradient (0.1-1.2 M) of triethylammonium bicarbonate splint strand, suggests that the critical requirement for ac- (pH 7.5). When an oligonucleotide was needed without a tivity of the enzyme is not double helical structure, but simply terminal phosphate, it was treated with bacterial alkaline a high concentration of 3'-hydroxyl with respect to 5'- phosphatase and repurified on a DEAE-Sephadex A-25 phosphate groups. If one end of an oligonucleotide binds to column. the enzyme and the chain is not too long, the other end would Oligomers were labeled on the 5'-terminus with [32P]phos- be in very high local concentration, facilitating an intra- phate, using polynucleotide kinase as described by Silber molecular reaction. This suggests that an intermolecular reac- et al. (1). The labeled products were isolated by thin-layer tion without a splint strand might be achieved (a) by pre- chromatography on cellulose-coated glass plates, followed by venting the intramolecular reaction through the use of a 5'- gradient elution from DEAE-Sephadex columns with tri- phosphate terminated oligomer too short to cyclize and (b) ethylammonium bicarbonate. by using a very high concentration of another oligomer with Separation of Oligoribonucleotides by RPC-6 Column Chro- matography. The resin was prepared by mixing Plaskon with * Current address: Department of Biochemistry, University of Andogen 464 in chloroform as described by Pearson et al. (8). California, Berkeley, Calif. 94720. The resin was washed extensively with 0.2 M KC1, 0.01 M t To whom correspondence should be addressed. imidazole*HCl (pH 7.0), and 0.7 X 10-cm jacketed columns 122 Downloaded by guest on September 27, 2021 Proc.PT4Nat. Acad. Sci. USA 72 (1975) RNA Ligase Intermolecular Reaction 123 were packed at 370 under pressure from a peristaltic pump. Columns were eluted with 150-ml, 0.2-1.0 M KCl linear gradients in 0.01 M imidazole-HCl (pH 7.0) at the fastest possible flow rate (about 40 ml/hr), and the effluent was monitored at 260 nm on a LKB Uvicord system. As may be seen in Fig. 2B, these conditions are adequate to resolve individual members of a homologous series of oligoribonucleo- tides to a chain length of twenty. If the gradient is extended to 2.0 M KCl, more than sixty peaks may be resolved. For determination of [82P]-labeled products, fractions of 1.8 ml were collected into plastic vials and the Cerenkov irradiation for the whole fraction was determined. Assay of RNA Ligase. The intramolecular cyclization reac- tion catalyzed by RNA ligase was used as the routine method of assay during the purification of the enzyme. The assay mixture was that of Silber et al. (1), except that about 0.02 1.Ci of [5'-32P]poly(A) (a mixture of oligomers from 22 to 27 nucleotide residues in length) at a concentration of from 0.02 to 0.74 MAM in termini was the substrate and the total FRACTION NUMBER assay volume was 50 ,l. The reaction mixtures were incubated FIG. 1. Purification of RNA ligase. (A) Gradient sievorptive at 370 for 30 min and at 1000 for 2 min, and then cooled. profile of the first column. The A2s0 peak (solid curve) appearing Water (50 ul) and 0.1 unit of bacterial alkaline phosphatase at the void volume is primarily protein, the second A28o peak ap- were added, and the mixtures were incubated at 650 for 30 pearing at the total column volume is primarily nucleic acid. min. A 50-IAI aliquot of the cooled reaction mixture was trans- RNA ligase activity (dotted curve) was assayed by the intra- ferred to a 2-cm-square piece of Whatman DEAE-paper molecular cyclization assay described in Materials and Methods. (DE81). The papers were dried under an infrared lamp, The KC1 concentration (dashed curve) was monitored by conduc- washed three times by gentle agitation in 50 ml of 0.3 M tivity. (B) Sephadex G-75 column profile. The major A28o peak ammonium formate (pH 8.2), and processed as described (9). (solid curve) appears at the void volume. RNA ligase activity is One unit is arbitrarily' defined as 1 pmol of [5'-32P]terminus assayed by the intramolecular cyclization (dotted curve) and inter- rendered resistant to phosphatase in 30 min at a substrate molecular joining (-) assays described in Materials and Methods. concentration of 0.025,uM. The intermolecular reaction of RNA ligase was assayed in a (11), was removed by centrifugation at 27,000 X g for 10 mixture (25 ul) containing 50 mM Tris HCl (pH 7.5), 0.15 min at 4°. The protein in the supernatant was precipitated mM ATP, bovine serum albumin (10 Mg/ml), 10 mM MgCl2, with 70% ammonium sulfate. 1.3 mM dithiothreitol, RNA ligase (about 1 pug), and various Gradient sievorptive chromatography (12) was performed RNA oligomers as substrates. A hexaribonucleotide with a at room temperature by applying a 27.5-ml linear gradient 5'-terminal [82Pjphosphate (5-20 Ci/mmol) was present in of 0.i75-0.400 M KCl in 0.1 M Tris - HCl (pH 7.5), 1 mM 2- the reactions at a concentration of about 1 MAM and was mercaptoethanol, 0.1 mM EDTA to a 1.8 X 38-cm (100 ml) called the donor molecule. The other RNA oligomer, called DEAE-Sephadex A-25 column that had been equilibrated the acceptor molecule, varied from three to six residues in with 0.175 M KCl in the same buffered mixture. The ammo- length, was unlabeled, had a 5'-hydroxyl group, and was nium sulfate pellet, which had been dissolved in 3.0 ml of 0.75 present at a concentration of 3-5 mM. Both oligomers had M KCl, was then applied to the column, washed on with 5 3'-hydroxyl termini unless otherwise stated.
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