Characterisation of holoenzyme lacking σ(N) Regions I and II

Abstract

The sigma-N (σ(N)) protein associates with bacterial core RNA polymerase to form a holoenzyme that is silent for transcription in the absence of enhancer-binding activator proteins. Here we show that the acidic Region II of σ(N) from Klebsiella pneumoniae is dispensable for polymerase isomerisation and transcription under conditions where the inhibited state of the holoenzyme is relieved by removal of σ(N) Region I sequences. Holoenzymes lacking Region I or Regions I+II were equally susceptible to the order of addition-dependent inhibition or stabilisation of DNA binding afforded by in trans Region I sequences. Region I+II-deleted σ formed a holoenzyme with a DNA-binding activity more susceptible to inhibition by non-specific DNA than that lacking Region I. Region II sequences appear more closely associated with formation of a holoenzyme and σ proficient in DNA binding than with changes in holoenzyme conformation needed for unmasking a single-strand DNA-binding activity used for open complex formation. Region II may therefore function to optimise DNA interactions for an efficient σ cycle.