Purification and activities of the Rhodobacter capsulatus RpoN (sigma N) protein.
The rpoN-encoded sigma factors (sigma N) are a distinct class of bacterial sigma factors, with no obvious homology to the major sigma 70 class. The sigma N-containing RNA polymerase holoenzyme functions in enhancer-dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatus sigma N protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coll sigma N holoenzyme, and may represent a minor subclass of sigma N proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatus sigma 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 Klebsiella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatus sigma 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 sigma N to regulate sigma 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 sigma N.
Cannon
W
W
AFRC-IPSR Nitrogen Fixation Laboratory, University of Sussex, Falmer, Brighton, UK.
Missailidis
S
S
Austin
S
S
Moore
M
M
Drake
A
A
Buck
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54