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 assimilation are encoded by the nasFEDCBA operon. Previous genetic studies led to the conclusion that nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism. In the presence of nitrate or nitrite, the nasR gene product is hypothesized to inhibit transcription termination at the factor-independent 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 His6-tagged form (His6-NasR). Templates for in vitro transcription contained the nasF operon leader region, with a substitution of the sigma70-dependent tac promoter for the native sigmaN-dependent promoter. We found that in vitro transcription of the leader template terminated at the terminator site, and that MBP-NasR and His6-NasR proteins both caused transcription readthrough of this site in response to nitrate or nitrite. Half-maximal antitermination required nitrate or nitrite at moderate (1 to 10 microM) concentrations, and several other anions tested, including chlorate, were without effect. Previous in vivo analysis of leader deletions identified 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. Finally, electrophoresis mobility shift analysis revealed that His6-NasR bound specifically to nasF leader RNA. This binding was independent 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 with nasF leader RNA.
Chai
W
W
Section of Microbiology, Cornell University, Ithaca, NY, 14853-8101, USA.
Stewart
V
V
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007709:Klebsiella / Q000235:genetics* / Q000502:physiology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009566:Nitrates / Q000502:physiology; D009876:Operon; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D015534:Trans-Activators / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; C110969:NasF protein, Klebsiella oxytoca; D009566:Nitrates; D033902:Periplasmic Binding Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C088711:nasR protein, Klebsiella pneumoniae