Interaction of sigma factor sigma(N) with Escherichia coli RNA polymerase core enzyme

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA p olymerase (RNAP) sigma (N)-holoenzyme has been investigated using biosynthe tically labelled 7-azatryptophyl-(7AW)sigma (N). The spectroscopic properti es of such 7AW proteins allows their absorbance and fluorescence to be moni tored selectively, even in the presence of high concentrations of other try ptophan-containing proteins. The 7AW sigma (N) retained its biological acti vity in stimulating transcription from sigma (N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherich ia coli. Furthermore, five Trp --> Ala single mutants of sigma (N) were sho wn to support growth under conditions of nitrogen limitation, and showed co mparable efficiency in activating the sigma (N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for ac tivity. The equilibrium binding of 7AW sigma (N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments , absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AW sigma (N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 muM. The kinetics of the interac tion between 7AW sigma (N) and core RNAP was investigated using stopped-flo w spectrofluorimetry. A biphasic decrease in fluorescence intensity was obs erved when samples were excited at 280 nm, whereas only the slower of the t wo phases was observed at 315 nm. The kinetic data were analysed in terms o f a mechanism in which a fast bimolecular association of sigma (N) with cor e RNAP is followed by a relatively slow isomerization step. The consequence s of these findings on the competition between sigma (N) and the major sigm a factor, sigma (70), in Escherichia coli are discussed.