The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis

Abstract

The spoIIA operon codes for an alternative sigma factor which appears in the early stages of a sigma factor expression cascade during sporulation in Bacillus subtilis. We have used a single round in vitro transcription assay to probe requirements for transcription initiation at the spoIIA promoter. Core RNA polymerase or holoenzyme containing sigma A was reconstituted with sigma H protein and used to transcribe the spoIIA promoter. Formation of heparin resistant transcription initiation complexes required that the spoIIA template be supercoiled. Topoisomers of the spoIIA template were created and transcribed at various temperatures. Changes in the superhelicity of template DNA had a significant influence on the amount of transcription complexes formed at the spoIIA promoter.