Sequences in sigma(N) determining holoenzyme formation and properties

Sigma subunits of bacterial RNA polymerases are closely involved in many st eps of promoter-specific transcription initiation. Holoenzyme formed with t he specialised minor sigma-N (sigma(N)) protein binds rare promoters in a t ranscriptionally inactive state and functions in enhancer-dependent transcr iption. Using competition and dissociation assays, we show that sigma(N)-ho loenzyme has a stability comparable to the major sigma(70)-holoenzyme. Puri fied partial sequences of sigma(N) were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigma(N) which both bind the core but with significantly different affinities were identified, demonstrating that the sigma(N) interface with core RNA polymer ase is extensive. The low affinity segment of sigma(N) included region I se quences, an amino terminal domain which mediates activator responsiveness a nd formation of open promoter complexes. The higher affinity site Lies with in a 95 residue fragment of region III. We propose that the fore to region I contact mediates properties of the sigma(N)-holoenzyme important for enha ncer responsiveness. Heparin is shown to dissociate sigma(N) and core, indi cating that disruption of the holoenzyme is involved in the heparin sensiti vity of the sigma(N) closed complex. (C) 1999 Academic Press.