Purification and activities of the Rhodobacter capsulatus RpoN (σ N ) protein

The rpoN ‐encoded sigma factors (σ N ) are a distinct class of bacterial sigma factors, with no obvious homology to the major σ 70 class. The σ N ‐containing RNA polymerase holoenzyme functions in enhancer‐dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatus σ N protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coli σ N holoenzyme, and may represent a minor subclass of σ N proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatus σ N protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins. As first described for the Kleb‐siella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatus σ N protein and its response to core RNA polymerase was clearly different from that determined for the K. pneumoniae and E. coli proteins. Results are discussed in the context of a requirement to modulate the activity of the DNA‐binding surfaces of σ N to regulate σ N function. Circular dichroism was used to evaluate the structure of the R. capsulatus protein and revealed differences in the tertiary signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA‐binding domain of σ N