Abstract
A rapid procedure for the simultaneous purification of yeast RNA polymerases I, II, and III is described. The procedure involves direct fractionation of a yeast cell extract by heparin agarose affinity chromatography, followed by glycerol gradient centrifugation and DEAE-Sephadex chromatography. The purification can be completed in 3-4 days using 20-200 g of yeast cells. Two forms each of RNA polymerases I, II, and III are resolved after DEAE-Sephadex chromatography. In the cases of RNA polymerases I and II, these forms differ in subunit structure. The transcriptional properties of the isolated enzymes were determined using hybrid plasmid DNA templates containing yeast ribosomal and glycolytic structural genes. Both forms of RNA polymerases I and II transcribe plasmid DNA templates with low efficiency and no evidence for selective initiation of transcription was found for these enzymes using a wide variety of templates. Both forms of RNA polymerase III transcribe plasmid DNA templates with high efficiency and direct the synthesis of discrete transcripts. Sites for initiation and termination of transcription by RNA polymerase III within defined plasmid DNA templates were determined. The data show that RNA polymerase III-dependent synthesis of discrete transcripts from restriction endonuclease-digested plasmid DNA templates is initiated from selected ends of the templates and terminates at discrete sites downstream from the site of initiation. RNA polymerase III initiates synthesis at many sites within supercoiled plasmid DNA templates.
Highlights
A rapid procedurefor the simultaneous purification for these studies are the yeast RNApolymerases
Two forms each of RNA polymerases I, 11, and I11 are resolved afterDEAE-Sephadex chromatographyIn. the cases of RNA polymerases I and 11, these forms differ in subunit structure
The specific activity of the isolated B.subtiLisenzyme is significantly higher than obglycolytic structural genes. Both forms of RNA polym- served with other preparation procedures [13] suggestingthat erases I and I1 transcribe plasmid DNA templates with a higher proportion of the active enzyme molecules in the low efficiency and no evidence for selective initiation extract is recovered after heparin agarose chromatography
Summary
The specific activity of the isolated B.subtiLisenzyme is significantly higher than obglycolytic structural genes Both forms of RNA polym- served with other preparation procedures [13] suggestingthat erases I and I1 transcribe plasmid DNA templates with a higher proportion of the active enzyme molecules in the low efficiency and no evidence for selective initiation extract is recovered after heparin agarose chromatography. Of transcription was found for these enzymes using a We have used a similar approach for the isolation of the three wide variety of templates Both forms of RNA polym- yeast RNA polymerases fromsmall amounts of cells. Erase 111 transcribe plasmid DNA templates with high We report here a rapid procedure for the simultaneous efficiency and direct the synthesis of discrete tran- isolation of yeast RNA polymerases I, 11, and 111. Plasmid DNA templates is initiated from selected ends of the templates and terminates at discrete sites down-
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