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. 2012 Sep 28;287(40):33153-62.
doi: 10.1074/jbc.M112.391755. Epub 2012 Aug 1.

The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply

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The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply

Marion Eisenhut et al. J Biol Chem. .

Abstract

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

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Figures

FIGURE 1.
FIGURE 1.
Noncoding RNAs connected with the flv4-2 operon. A, schematic presentation of chromosomal arrangement of the genes flv4, sll0218, and flv2 in the flv4-2 operon; ctpB; the antisense transcribed sections for as1_flv4, as2_flv4, and as_flv2; and the gene for ncRNA, ncr0080. B, verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M, molecular mass marker; R, total RNA after electrophoresis. C, classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface, and the transcription start for flv4, according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow. Predicted −10 and −35 elements are underlined. The chromosomal positions are according to Cyanobase.
FIGURE 2.
FIGURE 2.
Accumulation of flv4-2 operon-related ncRNAs, mRNA, and proteins after shift of Synechocystis WT cells from HC to LC conditions. Cells were precultivated under HC conditions and then shifted to LC. Samples were collected 0, 1, 3, 4.5, 6, 12, and 24 h after the LC shift and analyzed as described under “Experimental Procedures.” A, results of blotting experiments. 5 S rRNA and AtpΒ were used as loading controls for RNA and protein, respectively. B, quantification of signal intensities. The strongest signal intensity for each probe was set to 100%, and the other signal intensities were related accordingly. Shown are the results of one representative experiment.
FIGURE 3.
FIGURE 3.
Impact of artificial modulation of As1_flv4 transcript amount on the expression of the flv4-2 operon after shift from HC to LC conditions. For overexpression, the as1_flv4 coding sequence was fused with the petJ promoter, which is induced in the absence of Cu2+. For construction details, see “Experimental Procedures.” A mutant in spkA served as control strain. To reach maximal petJ promoter activity, cultures were precultivated under HC conditions and in the absence of Cu2+ for 48 h. After precultivation, LC-induced expression patterns of the asRNA As1_flv4 and the mRNAs of flv4, sll0218, and flv2 were monitored as described previously. As a loading control, rnpB was used. In this experiment, cultures were aerated by continuous bubbling.
FIGURE 4.
FIGURE 4.
Activities of as1_flv4 and flv4-2 operon promoters after shift from HC to LC conditions. Promoter sequences of the as1_flv4 gene and the flv4-2 operon were fused with the luxAB genes. The generated mutant strains MpILA-as1_flv4 and MpILA-flv4-2 were verified by PCR and used for luminescence measurement. The promoterless construct MpILA served as a negative control. Cells were cultivated under HC conditions and shifted to LC. At the given time points, samples were taken, and bioluminescence was measured as described under “Experimental Procedures.” The bioluminescence was normalized to the OD750 and corrected by subtraction of luminol autoluminescence and MpILA luminescence. Each sample was measured in triplicates. Given are the means and S.D. (error bars) of triplicates of a representative time course. Pflv4-2, promoter of the flv4-2 operon; Pas1_flv4, promoter of the as1_flv4 gene. A.U., arbitrary units.
FIGURE 5.
FIGURE 5.
Expression of as1_flv4 and the flv4-2 operon in the mutant ΔndhR and the WT. Shown is a representative time course of transcript abundances of the asRNA As1_flv4 and the mRNA of the flv4-2 operon after LC shift. 5 S rRNA was used as a loading control.
FIGURE 6.
FIGURE 6.
Expression of as1_flv4 and the flv4-2 operon in the mutant Δsll0822 and the WT. Shown is a representative time course of transcript abundances of the asRNA As1_flv4 and the mRNA of the flv4-2 operon after LC shift. 16 S rRNA was used as a loading control.
FIGURE 7.
FIGURE 7.
Models of the Ci-controlled expression of the flv4-2 operon in Synechocystis. A, schematic representation of changes in the abundances of the asRNA As1_flv4 and flv4-2 operon transcripts upon shift of Synechocystis cells from HC to LC conditions. Given are relative abundances of the asRNA As1_flv4 (red line) and the flv4-2 operon mRNA (blue) in WT (solid line) and As1_flv4 overexpression strains (dashed lines). For a detailed description, see the text. The kinetics of the transcriptional response is strongly dependent on the experimental implementation and the resulting Ci levels in the medium. B, hypothetical model on the integrated function of transcriptional regulators NdhR and Sll0822 and the asRNA As1_flv4 in Ci-controlled expression of the flv4-2 operon. For a detailed description, see the text. 2PG, 2-phosphoglycolate.

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