The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) σ N -holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)σ 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 7AWσ N retained its biological activity in stimulating transcription from σ 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 σ N were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the σ N -dependent nifH promoter in vivo , indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWσ N to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315nm (which reports selectively on the distribution of free and bound 7AWσ N ) established that a 1:1 complex was formed, with a dissociation constant lower than 2 µ M. The kinetics of the interaction between 7AWσ N and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280nm, whereas only the slower of the two phases was observed at 315nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of σ N with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between σ N and the major sigma factor, σ 70 , in Escherichia coli are discussed.