Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Properties, purification, and subunit structure of class III RNA polymerases.

Abstract

The class III RNA polymerases present in Xenopus laevis tissues were solubilized and partially purified by ion exchange chromatography. Single chromatographic species were detected in extracts from ovary, liver, and a kidney-derived cultured cell, whereas two chromatographic forms were detected in liver extracts. All class III enzymes analyzed exhibited characteristic responses to ionic strength changes, synthetic polynucleotide templates, and alpha-amanitin. These chromatographic and catalytic properties readily distinguished all the class III enzymes from the corresponding class I and II enzymes but clearly failed to reveal any significant differences between the various ovarian and somatic class III enzymes (with the possible exception of one of the liver enzyme forms). The ovarian RNA polymerase III was completely soluble in low ionic strength buffers and was subjected to further purification via standard procedures involving differential centrifugation, ammonium sulfate fractionation, and ion exchange chromatography. Polyacrylamide gel electrophoresis under denaturing conditions revealed at least 10 distinct polypeptides (designated subunits a-j) which were consistently present in near equimolar amounts and which ranged in size from 155,000 to 19,000 daltons. Several smaller polypeptides (less than 19,000) were also evident in all preparations but were not further characterized. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms, although subsequent analysis on denaturing gels failed to reveal any differences in polypeptide composition. From the present data, it is estimated that there are about 4 X 10(9) molecules of RNA polymerase III per oocyte nucleus. This extraordinary level of soluble RNA polymerase III in part explains the ability of oocyte nuclei (or extracts) to support the active transcription of exogenous tRNA and 5 S RNA genes. The present and previous data concerning the properties and structures of the oocyte versus somatic cell enzymes also suggest that the RNA polymerase III present in the oocyte is functionally equivalent to a somatic cell enzyme and support the previous suggestion that this enzyme is conserved and used later in (somatic) embryonic cells.