Some physical properties of RNA polymerase.

purchased from Worthington Biochemical Corp., were dialyzed overnight against a solution containinlg 0.2 M KCI and 0.01 M potassium phosphate buffer, pH 7.0, before use. Solutions: 0.1 M KCl buffer: 0.1 M KCI, 0.01 M potassium phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol and 0.5 mM EDTA; 0.5 M KCl buffer: 0.5 M KCI, 0.01 M potassium phosphate buffer, pH 7.0, 1 mM 2-mercaptoethanol, and 0.5 mM EDTA. Miscellaneous: Hydroxylapatite prepared by the method of Tiselius, Hljerten, and Levin5 was a generous gift from Dr. Bruce Alberts. Dialysis tubing was boiled twice for 15 mi in 5%/ Na2CO3, rinsed thoroughly with water, then boiled for 15 min in 0.05 M EDTA, pH 7. Boiled dialysis tubing was stored at 2?C in 0.05 M EDTA, pH 7. Calf thymus DNA assay: The calf thymus DNA assay reactioln mixture, in 0.25 ml, con-tained 0.04 M tris-HCl buffer, pH 7.9, 0.05 KCI, 5 mM MgCl2, 2 mMI MnCl2, 0.16 mM each of UTP, CTP, and GTP, 0.12 mM ATP-8-C14, 120 jig/ml calf thymus DNA (350 mgtmoles/ml in DNA phosphorus), 4 mM 2-mercaptoethanol, 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin (BSA), and enzyme. After incubation for 10 or 20 min at 37?C, the reaction was stopped by adding 3 ml of cold 5% trichloroacetic acid. After 10 min at 0?C, the precipitate was collected and washed two times with 3-ml portions of cold 2% trichloroacetic acid on a Millipore HA filter. The filter was affixed to an aluminium planchet with a drop of 1% BSA, dried and counited in an end-window, low background, gas-flow counter. T7 DNA assay: The T7 DNA assay was the same as the calf thymus DNA assay except that 60 gig/ml of T7 DNA was used in place of calf thymus DNA, and MnCl2 was omitted. With purified enzyme, the T7 DNA assay gave from two- to threefold higher rates of incolporation than the calf thymus DNA assay. A unit of enzyme activity is defined as the inlcorporatiorl of 1 /hmole of total nucleotide into an acid-insoluble product in 1 min at 37?C. Sedimentation velocity measurements of RNA polymerase activity on sucrose gradients: Sedimen