Purification and properties of the .sigma. subunit of Escherichia coli DNA-dependent RNA polymerase

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Abstract
An improved purification procedure is described for the .sigma. subunit of E. coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70 and finally Ultragel AcA44. The .sigma. factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical-physical properties of .sigma. are presented. A MW of 82,000 was determined by phosphate buffered sodium dodecyl sulfate-polyacrylamide gel electrophoresis. UV absorption spectra were used to determine an E280nm1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of .sigma. were determined. The isoelectric focusing properties of .sigma. are presented. Denaturation-renaturation studies indicate that .sigma. is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000 dalton fragment is generated from .sigma. by mild trypsin treatment.