Structural Relationships Among the Multiple Forms of DNA-Dependent RNA Polymerase II from Cultured Parsley Cells

DNA-dependent RNA polymerase II (or B) was purified from cultured parsley cells, and its molecular structure was examined in detail. Upon centrifugation through glycerol gradients, RNA polymerase II sediments as a single band with an apparent sedimentation constant of 15 S. No contamination with RNA polymerases I or III could be detected when the activity of purified RNA polymerase II was assayed in the presence of high concentrations of α-amanitin. Analysis of purified RNA polymerase II by nondenaturing and denaturing polyacrylamide gel electrophoresis revealed that this enzyme exists in multiple forms. They were designated IIo, IIA, and IIB. It is suggested that each form has a subunit of Mr= 140 000 as well as smaller polypeptides in common. They differ, however, in the molecular weights of their largest subunits which is 220 000 in form IIo, 200 000 in form IIA, and 180 000 in form IIB. These large subunits were labelled with 125I, digested with trypsin, and tryptic digests were compared by two-dimensional analysis on thin-layer plates [Elder et al. (1977) J. Biol. Chem. 252, 6510–6515]. Fingerprints of tryptic digests from the polypeptides with Mr= 220 000, Mr= 200 000, and Mr= 180 000 were similar. It is, therefore, suggested that these subunits are structurally related. A tryptic digest was also produced from the subunit with Mr= 140 000. Its fingerprint was found to yield a considerably different distribution of peptides as compared to those from the three large subunits.