title: Chemical modifications of the sigma subunit of the E. coli RNA polymerase.
abstract: The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000502:physiology; D001120:Arginine / Q000502:physiology; D003545:Cysteine / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004926:Escherichia coli; D005998:Glycine / Q000502:physiology; D012808:Sigma Factor / Q000502:physiology*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*
chemical_list: D000600:Amino Acids, Dicarboxylic; D012808:Sigma Factor; D014157:Transcription Factors; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine; D005998:Glycine