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Purification of Active RNA Polymerase I from Yeast

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1276)

Abstract

Eukaryotic cells employ at least three nuclear, DNA-dependent RNA polymerase systems for the synthesis of cellular RNA. RNA polymerases I, II, and III primarily produce rRNA, mRNA, and tRNA, respectively. In a rapidly growing cell, most RNA synthesis is devoted to production of the translation machinery, with rRNA synthesis by RNA polymerase I representing more than half of total cellular transcription. The fundamental connection between ribosome biogenesis and cell growth is clear; furthermore, recent studies have identified transcription by RNA polymerase I as a key target for anticancer chemotherapy. Thus, efficient methods for characterizing transcription of the ribosomal DNA and its regulation are needed. In order to describe enzymatic features of an enzyme, in vitro assays are critical. Here we describe a method for purifying RNA polymerase I. This approach yields enzyme of sufficiently high quantity and activity for an array of experiments directed at describing the enzymatic properties of RNA polymerase I in detail.

Key words

RNA polymerase I Transcription rRNA Ribosome Protein purification 

Notes

Acknowledgements

The authors thank the UAB Fermentation Facility for continual support in cell growth and harvesting. This work is supported by the National Institutes of Health grant #GM84946.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular GeneticsUniversity of Alabama at BirminghamBirminghamUSA
  2. 2.Department of Biochemistry and Molecular GeneticsUniversity of Alabama at BirminghamBirminghamUSA

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