Purification and Physical Characterization of T7 RNA polymerase from T7-infected escherichia coli B

A procedure for the purification of T7 RNA polymerase from large quantities of phage-infected cells is described. Chromatography of the infected cell extract on phosphocelluiose resolves three activity forms which elute at about 0.2, 0.28, and 0.38 M KC1. Characterization of the three T7 RNA polymerase forms indicates that they are only slightly different in activity and the physiological role of the three forms, if any, is not clearly understood. The 0.38 M KCl form which constitutes about 80% of the T7 RNA polymerase activity has been purified to homogeneity. It behaves in solution as a monomer of about 110, 000 molecular weight as determined by equilibrium sedimentation, analytical Sephadex G-200 chromatography, analytical sedimentation velocity, and glycerol gradient sedimentation velocity. The amino acid composition of the protein is presented. T7 RNA polymerase asymmetrically transcribes in vitro from a T7 DNA template six major size classes of RNA (I—VI), which can be separated by polyacrylamide gel electrophoresis. Three of these RNAs, III, IV, and V, appear to comigrate in a polyacrylamide gel with in vivo late T7 RNAs, but there are no apparent in vivo counterparts for RNAs I, II, and VI. The apparent molecular weights of the in vitro synthesized T7 late RNAs have been estimated by polyacrylamide gel, electrophoresis under “native” conditions, by electrophoresis after formaldehyde treatment of the RNA, and by electrophoresis of the RNA in the nonaqueous denaturing solvent, formamide. Values for the molecular weights of RNAs III—VI are: 1.92, 0.92, 0.51, and 0.26 × 10⁶ daltons, respectively, and are constant when measured under “native” or “denaturing” conditions. RNAs I and II exhibit apparent molecular weights of 5.2 and 4 × 10⁶ daltons when measured by “native” gel electrophoresis, but these RNAs appear as ribopolymers of about 3.1 and 2.6 × 10⁶ daltons when measured as formylylated RNAs, or when they are denatured in formamide. In vitro, the same six RNAs are synthesized by the enzyme at four different stages of purification, demonstrating that the specificity necessary for the transcription of these unique RNA species resides in the 110, 000 molecular weight polypeptide, and that there is no additional protein effector which is necessary for specific transcription in vitro.