FNR, NARP, AND NARL REGULATION OF ESCHERICHIA-COLI K-12 NAPF (PERIPLASMIC NITRATE REDUCTASE) OPERON TRANSCRIPTION IN-VITRO

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in resp onse to nitrate and nitrite by the homologous response regulators, Nar L and NarP. Among these operons, the napF operon, encoding a periplasm ic nitrate reductase, has unique features with respect to its Fnr-, Na rL-, and NarP-dependent regulation. First, the Fnr-binding site is unu sually located compared to the control regions of most other Fnr-activ ated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In th is study, we used DNase I footprint analysis to confirm our previous a ssignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive contr ol mutations on nagF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptio nal regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP protein s. These in vitro transcription experiments demonstrate that, in the p resence of Fnr, the NarP protein causes efficient transcription activa tion whereas the NarL protein does not. This suggests that Fnr and Nar P may act synergistically to activate napF operon expression.;is obser ved in vivo, this activation by Fnr and NarP is antagonized by the add ition of NarL in vitro.