Characterization of the alternative sigma factor sigmaG in Streptomyces coelicolor A3(2).
Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.
Sevcíková
B
B
Institute of Molecular Biology, Center of Excellence for Molecular Medicine, Slovak Academy of Sciences, 845 51 Bratislava, Slovakia.
Mazuráková
V
V
Kormanec
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D020459:Genes, rRNA; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012808:Sigma Factor