Promoter selectivity of the Bradyrhizobium japonicum RpoH transcription factors in vivo and in vitro.
Expression of the dnaKJ and groESL1 heat shock operons of Bradyrhizobium japonicum depends on a sigma32-like transcription factor. Three such factors (RpoH1, RpoH2, and RpoH3) have previously been identified in this organism. We report here that they direct transcription from some but not all sigma32-type promoters when the respective rpoH genes are expressed in Escherichia coli. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH3 was insoluble. The purified proteins were recognized by an anti-E. coli sigma32 serum. While RpoH1 and RpoH2 productively interacted with E. coli core RNA polymerase and produced E. coli groE transcript in vitro, RpoH3 was unable to do so. B. japonicum core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH1 and RpoH2 were active, and they initiated transcription at the B. japonicum groESL1 and dnaKJ promoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the B. japonicum dnaKJ and groESL1 promoters were suboptimal for transcription by RpoH1- or RpoH2-containing RNA polymerase from B. japonicum. In a mixture of different templates, the E. coli groESL promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward B. japonicum rpoH-dependent promoters. We conclude that the primary function of RpoH2 is to supply the cell with DnaKJ under normal growth conditions whereas RpoH1 is responsible mainly for increasing the level of GroESL1 after a heat shock.
Narberhaus
F
F
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland. fnarber@micro.biol.ethz.ch
Kowarik
M
M
Beck
C
C
Hennecke
H
H
D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012230:Rhizobiaceae / Q000235:genetics*; D012808:Sigma Factor; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; C106850:RpoH1 protein, Bradyrhizobium japonicum; C106851:RpoH2 protein, Bradyrhizobium japonicum; C106852:RpoH3 protein, Bradyrhizobium japonicum; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32