PURIFICATION AND ACTIVITIES OF THE RHODOBACTER-CAPSULATUS RPON (SIGMA(N)) PROTEIN

The rpoN-encoded sigma factors (sigma(N)) are a distinct class of bact erial sigma factors, with no obvious homology to the major sigma(70) c lass. The sigma(N)-containing RNA polymerase holoenzyme functions in e nhancer-dependent transcription to allow expression of positively cont rolled genes, We have purified the Rhodobacter capsulatus sigma(N) pro tein, which is distinctive in lacking an acidic region implicated in t he melting of promoter DNA by the Escherichia coli sigma(N) holoenzyme , and may represent a minor subclass of sigma(N) proteins. Assays of p romoter recognition and holoenzyme formation and function showed that the purified R. capsulatus sigma(N) protein is distinct in activity co mpared to the enteric proteins, but retains the broad functions descri bed for these proteins. As first described for the Klebsiella pneumoni ae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsula tus sigma(N) protein and its response to core RNA polymerase was clear ly different from that determined for the K. pneumoniae and E. coli pr oteins. Results are discussed in the context of a requirement to modul ate the activity of the DNA-binding surfaces of sigma(N) to regulate s igma(N) function, Circular dichroism was used to evaluate the structur e of the R. capsulatus protein and revealed differences in the tertiar y signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA-binding domain of sigma(N).