Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis

Abstract

A rapid micromethod is described for the preparation of nucleic acid-free extracts from E. coli that involves precipitation with polyethylene glycol. Extracts are prepared from growing cells in 75 min by 3 short, low-speed centrifugations. The extract did not inhibit added purified RNA polymerase [EC 2.7.7.6], suggesting that major inhibitors of RNA synthesis were removed. This extract is ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from DNA- and DEAE-cellulose columns resulted in high yields of substantially pure RNA polymerase. This technique was used to purify 35S-labeled RNA polymerase. This system is applicable for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.