Analysis of wheat-germ RNA polymerase II by trypsin cleavage. The integrity of the two largest subunits of the enzyme is not mandatory for basal transcriptional activity.
When wheat-germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220,000-Mr and 140,000-Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172,800, 155,000, 143,000, 133,800, 125,000 and 115,000. The use of affinity-purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220,000-Mr and 140,000-Mr subunits of wheat-germ RNA polymerase II and their breakdown products generated in the presence of trypsin. None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones. Furthermore, in transcription of a poly[d(A-T)] template, the catalytic activity of the proteolyzed form of wheat-germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template-directed catalysis of phosphodiester bond formation.
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Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
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D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014908:Triticum / Q000201:enzymology*; D014357:Trypsin
D007106:Immune Sera; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012319:RNA Polymerase II; D014357:Trypsin