Purification of the alternative sigma factor, sigma 54, from Salmonella typhimurium and characterization of sigma 54-holoenzyme

Abstract

The alternative sigma factor sigma 54 of enteric bacteria, or its homologue in other purple bacteria, is required for transcription of genes whose products have diverse physiological roles. Previous studies have indicated that sigma 54 confers on core RNA polymerase the ability to recognize a specific class of promoters but not the ability to isomerize from closed to open complexes. Isomerization requires ATP and one member of a family of activator proteins, it being different activator proteins that allow this form of polymerase to respond to different physiological signals. We have developed a strategy for overproducing and purifying sigma 54 from Salmonella typhimurium and have studied several biochemical properties of reconstituted sigma 54-holoenzyme. The initial binding constant KB for the formation of closed complexes between this holoenzyme and the ginA promoter in our transcription buffer is approximately 3 x 10(8) M-1, which was determined from DNaseI protection assays at 37 degrees C. After the formation of open complexes, several properties of sigma 54-holoenzyme appear to be similar to those of sigma 70-holoenzyme. We have determined the complete nucleotide sequence of the gene encoding sigma 54 (ntrA) in Salmonella.