Initiation and Release of RNA by DNA-Dependent RNA Polymerase* Robert L

Proceedings of the National Academy of Sciences Vol. 66, No. 3, pp. 701-708, July 1970 Initiation and Release of RNA by DNA-Dependent RNA Polymerase* Robert L. Millettet and Carol D. Trotter DEPARTMENT OF PATHOLOGY, UNIVERSITY OF COLORADO MEDICAL CENTER, DENVER Communicated by James Bonner, December 19, 1969 Abstract. During in vitro transcription of T4 DNA by E. coli RNA polymerase, chain initiation stops coincidentally with synthesis at low ionic strength (0.11) with an average of one RNA chain initiated per 24S polymerase molecule. At high ionic strength (0.37), initiation as well as synthesis continues for several hours, with an average of four chains initiated per enzyme molecule in two hours. The RNA product is released from the T4 DNA template at both low and high ionic strength. At high ionic strength, however, RNA polymerase can repeatedly initiate, synthesize, and release RNA from the synthesis complex in vitro. Under both conditions, the synthesized RNA sediments at 25-44 S, has a number average chain length of 5000 to 7500 nucleotides, and a weight average chain length of 11,500 nucleotides. Introduction. We previously demonstrated that the cessation of RNA synthesis in vitro can be overcome by elevating the salt concentration.1 Thus, at an ionic strength of 0.37, RNA synthesis continues over many hours with only slightly decreasing rate. Preliminary experiments based on the incorporation of ly-32P-ATP indicated that high salt concentrations allow RNA polymerase to initiate RNA chains continuously.1 These observations suggested that the polymerase is able to terminate RNA chains under these conditions. We show here that at high ionic strength RNA polymerase continually ini- tiates and releases RNA of high molecular weight from the synthesis complex in vitro. In addition, we find that even at low ionic strength, where synthesis terminates by 1 hr, the bulk of the product is released. This work has been presented in preliminary form elsewhere.2 Materials and Methods. RNA polymerase was prepared from 250 g Escherichia coli K12 (3/ log phase, washed, from Grain Processing Corp.) as previously described1 but with the following modifications: The DNA-protein complex was precipitated from the crude extract with a 1-5% w/v solution of polyethylenimine (Polysciences, Inc.)4 in TMA buffer' containing 2.5 X 10-4 M EDTA. An amount determined to precipitate 95% of the polymerase activity was added slowly with stirring to the extract. The result- ing precipitate was centrifuged and washed with 195 ml of 0.2 M NH4Cl in TMA, and the enzyme eluted by homogenizing with 170 ml of 0.4 M NH4Cl in TMA and centrifuging. After ammonium sulfate fractionation of the eluate,3 the polymerase was chromatographed on a 3 X 33 cm DEAE-cellulose column as previously described.3 Using precipitation with 55% saturated (NH4)2S04 to concentrate the enzyme between steps, we further purified it by two zone centrifugations: the first at low ionic strength (10-30% w/v sucrose in TMA),3 and the second at high ionic strength (10-30% w/v sucrose in 0.4 M NH4Cl in TMA, 25,000 rpm for 27 hr at 20C in the SW-25.1 rotor) to separate the en- 701 Downloaded by guest on September 30, 2021 702 BIOCHEMISTRY: MILLETTE AND TROTTER PROC. N. A. S. zyme in the 13S form from larger contaminants. The precipitated enzyme was dissolved in 0.04 M potassium phosphate, 1 mM f3-mercaptoethanol, 1 mM EDTA, pH 7.0, dialyzed for 2 hr against the same buffer, and then chromatographed on hydroxylapatite according to the method of Richardson,5 but with 1 mM EDTA present in the chromatography buffer. Two peaks of polymerase activity elute from the hydroxylapatite column, one at about 0.13 M phosphate, the other at about 0.16 M phosphate. Each peak was precipitated with ammonium sulfate, dissolved in glycerol-2X TMA (1:1), pH 7.9, and stored at -20'C. The first eluting enzyme peak, which contained subunits B, O', a, a, and co6 was used in these studies. Its specific activity averaged 1800 enzyme units9/mg protein (determined from a specific absorbance at 280 mu of 0.65/mg/ml).' RNA synthesis was measured as published earlier' at either low ionic strength, 1A = 0.11 (50 mM NH4Cl, 8 mM magnesium acetate, 30 mM Tris, pH 7.9), or at high ionic strength, A = 0.37 (0.13 M NH4Cl, 0.07 M magnesium acetate, 0.03 M Tris, pH 7.9), with either UTP-3H (0.5 or 1 Ci/mole) or ATP-8-'4C (1 Ci/mole) used as the labeled substrate. Chain initiation was followed by the incorporation of y-32P-ATP or -GTP7 having specific activities of 26-58 cpm/pmole. To eliminate any artifacts owing to pos- sible polyphosphate kinase contamination, 0.02 mM ADP was included in the reaction mixture.8 Aliquots of 0.1 ml were pipetted into 1 ml ice-cold H20, then 0.05 ml unlabeled ATP (100 ,umoles/ml) plus 1 ml 0.12 M sodium pyrophosphate (pH 6-7) were added with mixing. 2.2 ml 10% trichloroacetic acid containing 0.02 M sodium pyrophosphate were added with mixing and the samples were filtered through glass filters (Whatman GF/C) presoaked in 5% trichloroacetic acid containing 0.1 M pyrophosphate. Filters were washed five times with 7 ml of 5% trichloroacetic acid-0.1 M pyrophosphate, three times with 5% trichloroacetic acid, and twice with ethanol, dried, and counted in a liquid scintillation counter. Determination of s20,W values and molecular weights of RNA from sucrose gradients: Zone centrifugation procedure is described in the legends. Individual gradients were calibrated by computer analysis to give s20o, as a function of r, with R17 phage and R17 RNA as internal standards (J. McConnell, to be published). Sedimenta- tion coefficients were converted to molecular weights using Spirin's equation,9 and molecular weight distributions were determined for each gradient. Template DNA: 3H-Thymidine-labeled T4 phage were prepared by the method of Richardson et al.'0 T4 phage were purified by sedimentation through sucrose gradients," dialyzed against NET (0.1 M NaCl, 1 mM EDTA, 0.01 M Tris, pH 7.5), and the DNA prepared by extracting with phenol and chloroform-octanol.' The free T4 DNA had an 820,w of 46 S in our sucrose gradients. 3H-R17 phage RNA: 3H-Labeled R17 phage RNA was prepared by the method of Strauss and Sinsheimer12 using, as host, E. coli Hfr Hayes (AB259, obtained from A. L. Taylor). Three min after infection 0.8 mCi of uridine-5-3H (20 Ci/mmole) was added to a 1 liter culture. Chemicals: y-32P-ATP and -GTP were synthesized enzymatically by the method of Glynn and Chappell"3 with a modification after the Dowex-1 chromatography step to yield the lithium salts.'4 Unlabeled nucleotides were obtained from P-L Biochemicals, Inc., 3H- and "4C-labeled substrates from Schwarz BioResearch, and hydroxylapatite from Bio-Rad. Ammonium sulfate (enzyme grade) and sucrose (enzyme grade, for polymerase purification; density gradient grade, for zone centrifugation of the reaction product) were purchased from Mann Research Labs., and DEAE-cellulose (Type 20) from Schleicher & Schuell. Results. Kinetics of chain initiation and synthesis at low and high ionic strength: In vitro transcription under the usual low ionic strength conditions (u = 0.11) terminates in about 30 min-." The stopping of synthesis at low ionic strength is accompanied by a parallel cessation in chain initiation as evi- Downloaded by guest on September 30, 2021 VOL. 66, 1970 BIOCHEMISTRY: MILLETTE AND TROTTER 703 50 SO p = 0.37 40 * ,L- 0.11 FIG. 1.-Kinetics of chain initiation and synthesis at low and high ionic strength. RNA synthesis was performed at .i low ionic strength, ju = 0.11, and > 30 15 at high ionic strength, s =/ 0.37, with 11 ug RNA poly- X/ merase/ml and 98 ug T4 DNA/ ml. Labeled substrates: 3H-/ UTP, 0.5 Ci/mole, oy..2P-ATP E 20 - 1 + (31 cpm/pmole) and _y-32P-GTP / / X (33.2 cpm/pmole). Duplicate reactions were run containing either 3H-UTP and 32P-ATP or 3H-UTP and 32P-GTP. 0.2- ml aliquots taken at various 10 -5 times were assayed as described 0 in Materials and Methods. QE 0 30 60 90 120 150 Ml nutes denced by the incorporation of y-32P-ATP and -GTP (Fig. 1). Similar findings have teen reported previously.7" 6 Synthesis at high ionic strength (0.37), however, continues for several hours with only slightly decreasing rate as we have earlier demonstrated.' Under these conditions, chain initiation shows a rapid initial rise during the first 10 min and then increases almost linearly throughout the course of transcription (Fig. 1). By 2 hr of synthesis, approximately three times as many RNA chains are initiated at high ionic strength as at low ionic strength. The ratio of ATP/GTP initiation is unaffected by the ionic conditions used in these experiments: At both low and high ionic strength, 67% of the RNA chains transcribed from T4 DNA are initiated with ATP. This agrees with values reported for initiation at low salt concentrations.8 Effect of ionic strength on chain length and number of RNA molecules synthesized per enzyme particle: From an average of five experiments, the number of TABLE 1. Effect of ionic strength on RNA chain length and number. Number of RNA Chains* Number-Averaget per Polymerase Molecule Chain Length (Nucleotides) Minutes , = 0.11 0.37 0.11 0.37 5 0.654 0.316 4335 4592 15 0.973 0.934 5274 5507 30 0.928 1.58 6116 7059 60 1.09 2.72 5116 7761 90 1.22 3.62 6311 7574 120 1.38 4.23 5413 7678 * Calculated from nmoles 7-32P-ATP + y-32P-GTP incorporated/nmoles enzyme; nmoles enzyme = ng enzyme/720,000.

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