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 Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.

Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits

“…Micropurification of radioactive RNA polymerase. The procedure described in the accompanying paper (6) for micropurification was employed, including deoxyribonucleic acid-cellulose and diethylaminoethyl-cellulose column chromatography.…”

“…Samples were prepared and subjected to electrophoresis as described in the accompanying paper (6), except that 7.5% acrylamide gels were used and 1.5 ,ug of RNA polymerase holoenzyme and 0.5 ,ug of /-galactosidase were added to each sample to provide visible markers. The stained bands corresponding to the /3', /3, S-galactosidase, o, and a subunits were cut out of each destained gel and dissolved in hydrogen peroxide, and the radioactivity was determined in Scintisol (Isolabs) as described in the accompanying paper (6). The samples were counted in an Isocap liquid scintillation counter (Amersham-Searle) set for simultaneous counting of 3H and 14C and equipped for automatic quench and crossover correction.…”

“…For each culture triplicate samples were subjected to gel electrophoresis on two separate days. The 3H/35S ratios for /3', ,/, o, and a were determined by electrophoresis of RNA polymerase eluting from the diethylaminoethyl-cellulose column at 0.25 M NaCl (corresponding to step 5 in Table 1 of the accompanying paper [6]). The 3H/35S ratio for /3-galactosidase was determined on the supernatant fraction of the low-salt polyethylene glycol precipitation in step iii of the purification procedure described in the accompanying paper (6).…”

“…The 3H/35S ratios for /3', ,/, o, and a were determined by electrophoresis of RNA polymerase eluting from the diethylaminoethyl-cellulose column at 0.25 M NaCl (corresponding to step 5 in Table 1 of the accompanying paper [6]). The 3H/35S ratio for /3-galactosidase was determined on the supernatant fraction of the low-salt polyethylene glycol precipitation in step iii of the purification procedure described in the accompanying paper (6). This fraction contains /3-galactosidase but no other polypeptides of similar mobility on sodium dodecyl sulfate-polyacrylamide gels, since no radioactivity co-migrated with carrier /-galactosidase when similar samples from cells grown in the absence of the inducer IPTG were analyzed.…”

“…(When methionine is present, the 35S is incorporated only as cysteine; when methionine is absent, both cysteine and methionine become labeled.) The RNA polymerase is then purified from these labeled cells by the micromethod described in the accompanying paper (6), and polymerase subunits are separated from each other by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Stained gel bands are cut out and dissolved, and the radioactivity is determined by liquid scintillation counting to give the 3H/35S ratio for each subunit.…”