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.
    Full-text · Article · Aug 2011 · PLoS ONE
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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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    • "It is worth mentioning that the two RNA structures are not mutually exclusive. It was therefore proposed that the binding of NasR to its target may either convert RNA polymerase in a terminator-resistant form as is known for phage λ antitermination or it might prevent the formation of the terminator in an as yet unknown way (Chai and Stewart 1999). "
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    ABSTRACT: During the past few years, our knowledge of gene regulation by RNA-binding proteins has greatly increased. RNA-binding proteins are involved in processes such as protection of RNAs from RNase degradation, prevention of ribosome binding to mRNA, control of formation of secondary structures of the mRNA that permit or prevent translation initiation, and termination/antitermination of transcription in response to external signals. Modulation of transcription termination by RNA-binding proteins involves the formation of alternative structures. One of the structures can act as a transcriptional terminator, while adoption of the alternative structure prevents formation of the terminator and does thus result in transcript elongation. Which of the two structures prevails under a given condition depends on two factors: the intrinsic stability of the alternative structures and the stabilization of one of both by an RNA-binding regulatory protein. Binding of a protein to the nascent mRNA may result in transcript elongation, as is the case for cold-shock proteins or in several catabolic operons. The RNA-binding ability of the RNA-binding proteins is modulated by direct interaction with the inducer, by protein-protein interactions with sensor proteins or by protein phosphorylation. In contrast, in the pyrimidine or tryptophan biosynthetic operons of Bacillus subtilis, the transcriptional terminators are stabilized by RNA-binding proteins resulting in the absence of expression of these operons.
    Full-text · Article · Jul 2002 · Archives of Microbiology
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