RNA sequence requirements for NasR-mediated, nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader

Section of Microbiology Cornell University, Ithaca, NY 14853-8101, USA
Journal of Molecular Biology (Impact Factor: 4.33). 10/1999; 292(2):203-216. DOI: 10.1006/jmbi.1999.3084


In Klebsiella oxytoca, enzymes required for nitrate assimilation are encoded by thenasFEDCBA operon. Nitrate and nitrite induction of nasF operon expression is determined by a transcriptional antitermination mechanism, in which the nasR gene product responds to nitrate or nitrite and overcomes transcription termination at the factor-independent terminator site located in the nasF upstream leader region. Previous studies led to the hypothesis that the NasR protein mediates transcription antitermination through interaction with nasF leader RNA. Here, we report a DNA sequence comparison that reveals conserved 1:2 and 3:4 RNA secondary structures in the nasF leader RNAs from two Klebsiella species. Additionally, we found that specific binding of the NasR protein to nasF leader RNA was stimulated by nitrate and nitrite. We combined mutational analysis, in vivo and in vitro antitermination assays, and an RNA electrophoretic mobility shift assay to define regions in the nasF leader that are essential for antitermination and for NasR-RNA interaction. Formation of the 1:2 stem structure and the specific sequence of the 1:2 hexanucleotide loop were required for both nitrate induction and for NasR-RNA interaction. Mutations in the 1:2 stem-loop region that abolished nitrate induction also interfered with NasR-leader RNA interaction. Finally, nucleotide alterations or additions in the linker region between the 1:2 and 3:4 stem-loops were deleterious to nasF operon induction but not to NasR-leader RNA interaction. We hypothesize that NasR protein recognizes the 1:2 stem-loop structure in the nasF leader RNA to mediate transcription antitermination in response to nitrate or nitrite.

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    • "To further reduce basal expression levels, an additional regulatory circuit was also incorporated into this system, consisting of two elements from Klebsiella oxytoca involved in nitrate assimilation, nasF, a transcriptional attenuator [10], [11], [12], [13] and nasR, which encodes the corresponding antiterminator protein that prevents nasF transcriptional termination in the presence of nitrate or nitrite. Incorporation of nasF downstream of the Pm promoter reduces basal transcriptional levels from the expression module [3]. "
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    ABSTRACT: In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.
    PLoS ONE 08/2011; 6(8):e23055. DOI:10.1371/journal.pone.0023055 · 3.23 Impact Factor
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    • "RNA secondary structure comparison is essential for : • identification of highly conserved structures during evolution, non detectable in the primary sequence which is often slightly preserved. These structures suggest a significant common function for the studied RNA molecules [11] [15] [9] [7], • RNA classification of various species (phylogeny)[4] [3] [16], • RNA folding prediction by considering a set of already known secondary structures [18] [10], • identification of a consensus structure and consequently of a common role for molecules [17] [5]. "
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