Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase.
The function of lysyl residues of the sigma subunit of the RNA polymerase from Escherichia coli was investigated by chemical modification with trinitrobenzenesulfonic acid (TNBS). Following reaction with TNBS, analysis of the modified sigma indicated that trinitrophenylation was limited to the epsilon-amino groups of lysyl residues. Progressive loss in the activity of sigma followed increasing trinitrophenylation as assayed by the ability to stimulate RNA polymerase core enzyme in a reaction directed by T7 DNA. Modification of five lysyl groups resulted in the complete loss of sigma activity. Kinetic analysis indicated that one lysyl group is critical for the function of sigma. TNP-sigma was able to form a holoenzyme complex with a binding affinity comparable to that of sigma. Promoter recognition studies were done by using HindIII fragments from T5 DNA. The TNP-sigma core complex was unable to form a tight binary complex with the T5 promoters. Studies on RNA chain initiation were carried out by using d(A-T)n and T7 DNA templates. TNP-sigma was unable to stimulate RNA chain initiation by core polymerase. Limited proteolytic digests of TNP-sigma or sigma using Staphylococcus aureus V8 protease were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results suggested a change in the conformation of sigma following trinitrophenylation.
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D009578:Nitrobenzenes / Q000494:pharmacology*; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology*
D046911:Macromolecular Substances; D009578:Nitrobenzenes; D014302:Trinitrobenzenesulfonic Acid; D012321:DNA-Directed RNA Polymerases; D008239:Lysine