NASR, A NOVEL RNA-BINDING PROTEIN, MEDIATES NITRATE-RESPONSIVE TRANSCRIPTION ANTITERMINATION OF THE KLEBSIELLA-OXYTOCA M5AL NASF OPERON LEADER IN-VITRO

In Klebsiella oxytoca (pneumoniae), enzymes required for nitrate assim ilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF opero n expression is determined by a transcriptional antitermination mechan ism. in the presence of nitrate of nitrite, the nasR gene product is h ypothesized to inhibit transcription termination at the factor-indepen dent terminator site located in the nasF operon leader region. To test this model in vitro, we first purified NasR as both a maltose binding protein fusion form (MBP-NasR) and a His(6)-tagged form (His(6)-NasR) . Templates for in vitro transcription contained the nasF operon leade r region, with a substitution of the sigma(70)-dependent tac promoter for the native sigma(N)-dependent promoter. We found that in vitro tra nscription of the leader template terminated at the terminator site, a nd that MBP-NasR and His(6)-NasR proteins both caused transcription re adthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 mu M ) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions id entified regions required for both negative regulation (the terminator ) and for positive regulation. Results from in vitro transcription of these deletion templates correlated fully with the in vivo analysis. F inally, electrophoresis mobility shift analysis revealed that His(6)-N asR bound specifically to nasF leader RNA. This binding was independen t of nitrate in vitro. These results strongly support the conclusions drawn from previous in vivo analysis, and establish that NasR mediates ligand-responsive transcription antitermination through interaction w ith nasF leader RNA. (C) 1998 Academic Press.