ABSTRACT
Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000.
Subject(s)
DNA-Directed RNA Polymerases/isolation & purification , Plants/enzymology , Chromatography , Chromatography, DEAE-Cellulose , DNA , DNA-Directed RNA Polymerases/analysis , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Peptides/analysis , Templates, Genetic , Zea mays/enzymologySubject(s)
Cell Nucleus/enzymology , Cell Nucleus/metabolism , Chloroplasts/enzymology , Chloroplasts/metabolism , DNA-Directed RNA Polymerases/metabolism , Plant Proteins/metabolism , Plants/metabolism , Protein Biosynthesis , Ribosomal Proteins/biosynthesis , Ribosomes/metabolism , Transcription, Genetic , Biological Evolution , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry, Physical , Chromatography, DEAE-Cellulose , Chromatography, Gel , DNA/metabolism , DNA Replication , DNA-Directed RNA Polymerases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Erythromycin/metabolism , Genes , Genotype , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Mutation , Plants/enzymology , Protein Biosynthesis/drug effects , RNA/biosynthesis , Rifamycins/pharmacology , Transcription, Genetic/drug effects , Zea mays/enzymologyABSTRACT
Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH(4))(2)SO(4), respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg(++) than Mn(++) and are inhibited by (NH(4))(2)-SO(4) or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by alpha-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA.
