A New Method for the Large Scale Purification of Escherichia coli Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

Purification and Base Composition Analysis of Phage Lambda Early Promoters

“…(a) Enzymes and DNA. RNA-polymerase was extracted from E. coli A 19 (RNase) -according to Babinet (12) and further purified by two cycles of glycerol gradient centrifugation (13). Stock solutions of the enzyme were kept at -20'C in Burgess' storage buffer (pH 7.9) (13).…”

“…RNA-polymerase was extracted from E. coli A 19 (RNase) -according to Babinet (12) and further purified by two cycles of glycerol gradient centrifugation (13). Stock solutions of the enzyme were kept at -20'C in Burgess' storage buffer (pH 7.9) (13). Acrylamide gel electrophoresis in 0.1% SDS (14) revealed the presence, in addition to the regular a, fl, p3', and a subunits, of r band and traces of uL band (15).…”

Lambda transducing bacteriophage carrying deletions of the argCBH-rpoBC region of the Escherichia coli chromosome

“…Partially purified E. coli RNA polymerase was generously supplied by K. O'Hare. This was further purified by phosphocellulose chromatography (5). The RNA polymerase core enzyme was judged by SDS gel analysis to be greater than 98% pure after this treatment.…”

What is in the black box? The discovery of the sigma factor and the subunit structure of E. coli RNA polymerase

“…Centrifuging at low salt (about 0.05 M KCl), reveals the dimer at 23S (a measure of its rate of sedimentation that indicated a size of around 900 kDa), which provided an effective means to remove most other proteins that are smaller in size. Fractions containing the polymerase activity were then pooled, the salt increased to 0.3 M KCl, and the protein re-centrifuged in a high-salt glycerol gradient where it dissociated to a 13S monomer (about 450 kDa) removing protein contaminants that were larger (see Fig 1, left and middle panels) (9). This use of low and high salt sizing steps in a sequential manner gave active, reasonably pure RNA polymerase, referred to as glycerol gradient (GG) enzyme (later called holoenzyme).…”

“…To our great surprise, this peak had all the RNA polymerase activity. We had removed tremendous amounts of unwanted proteins and obtained an excellent yield of polymerase activity in this one small peak (see Fig 1, right panel) (9). In hindsight, this worked because RNA polymerase bound tightly, presumably because of its positively charged groove where negatively charged DNA would normally bind.…”