Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.
An improved purification procedure is described for the sigma subunit of escherichia 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 molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% 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 have been 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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012808:Sigma Factor / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014157:Transcription Factors / Q000302:isolation & purification*
D000596:Amino Acids; D046911:Macromolecular Substances; D012808:Sigma Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases