Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.
The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(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 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.
Scott
D J
DJ
Department of Biology, University of York, Heslington, York YO10 5YW, U.K.
Ferguson
A L
AL
Gallegos
M T
MT
Pitt
M
M
Buck
M
M
Hoggett
J G
JG
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D011485:Protein Binding; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D014461:Ultracentrifugation
D012808:Sigma Factor; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases