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Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

Sakai Y, Abe K, Nakashima S, Ellinger JJ, Ferri S, Sode K, Ikebukuro K - Microbiologyopen (2015)

Bottom Line: Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp.PCC 6803.The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

No MeSH data available.


Related in: MedlinePlus

Scaffold-fused taR*2’s in Synechocystis sp. PCC 6803. (A) Schematic representation of the construct to evaluate the taR*2’s in Synechocystis sp. PCC 6803. (B) The taR*2’s were evaluated in Synechocystis sp. PCC 6803. The transcription of taR*2’s was induced in Ni2+-inducible nrsB promoter. The cellular fluorescence of taR*2 in the presence of NiSO4 was normalized to 1.0. The expression-fold representing the ratio of GFPuv expression levels in the presence and absence of NiSO4 are shown. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate. (C) Northern blot analysis of taR*2’s. The transcription was stopped by adding rifampicin and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for taR*2s and 16S rRNA, respectively.
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fig02: Scaffold-fused taR*2’s in Synechocystis sp. PCC 6803. (A) Schematic representation of the construct to evaluate the taR*2’s in Synechocystis sp. PCC 6803. (B) The taR*2’s were evaluated in Synechocystis sp. PCC 6803. The transcription of taR*2’s was induced in Ni2+-inducible nrsB promoter. The cellular fluorescence of taR*2 in the presence of NiSO4 was normalized to 1.0. The expression-fold representing the ratio of GFPuv expression levels in the presence and absence of NiSO4 are shown. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate. (C) Northern blot analysis of taR*2’s. The transcription was stopped by adding rifampicin and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for taR*2s and 16S rRNA, respectively.

Mentions: Two scaffold-fused taRNAs, taR*2-MicF and taR*2-MicF M7.4 were evaluated in Synechocystis. This MicF M7.4 scaffold was engineered by substituting the AU and GU-base pairs present in the stem loop structures of MicF scaffold into GC-base pairs and also by replacing the loop region with an Hfq high-affinity sequence (Link et al. 2009). This engineered MicF scaffold was predicted to form a stable secondary structure from M-fold secondary structure prediction (Zuker 2003). In our previous study, a naturally occurring E. coli MicF sRNA-derived scaffold sequence was revealed to be a suitable scaffold sequence to improve the gene regulation abilities of taR*2 (Fig.1C) (Sakai et al. 2014). While the taR*2-MicF harboring an intact MicF sRNA-derived scaffold sequence enhanced the gene expression to 2.5-fold in E. coli, taR*2-MicF M7.4 harboring the engineered MicF scaffold further enhanced the gene expression to 1.5-fold, which was 4.1-fold higher than taR*2 without the scaffold sequence. Moreover, the scaffold-fused taRNAs were more stable than the taRNA without the scaffold sequence in vivo and had low function in E. coliΔhfq strain, indicating that the endogenous Hfq bound and protected the scaffold-fused taRNAs from nuclease cleavage as well as natural trans-encoded sRNAs. To evaluate taR*2-MicF and taR*2-MicF M7.4 in Synechocystis, the scaffold-fused taR*2s were inserted downstream of the Ni2+-inducible nrsB promoter (PnrsB) (Lopez-Maury et al. 2002) and the crR*2 was constitutively transcribed from the trc promoter without the lac operator sequence (PtrcΔlacO) (Fig.2A). The gfpuv gene was inserted under crR*2 as a reporter gene to construct plasmids to evaluate the riboregulators (Table1).


Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

Sakai Y, Abe K, Nakashima S, Ellinger JJ, Ferri S, Sode K, Ikebukuro K - Microbiologyopen (2015)

Scaffold-fused taR*2’s in Synechocystis sp. PCC 6803. (A) Schematic representation of the construct to evaluate the taR*2’s in Synechocystis sp. PCC 6803. (B) The taR*2’s were evaluated in Synechocystis sp. PCC 6803. The transcription of taR*2’s was induced in Ni2+-inducible nrsB promoter. The cellular fluorescence of taR*2 in the presence of NiSO4 was normalized to 1.0. The expression-fold representing the ratio of GFPuv expression levels in the presence and absence of NiSO4 are shown. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate. (C) Northern blot analysis of taR*2’s. The transcription was stopped by adding rifampicin and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for taR*2s and 16S rRNA, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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fig02: Scaffold-fused taR*2’s in Synechocystis sp. PCC 6803. (A) Schematic representation of the construct to evaluate the taR*2’s in Synechocystis sp. PCC 6803. (B) The taR*2’s were evaluated in Synechocystis sp. PCC 6803. The transcription of taR*2’s was induced in Ni2+-inducible nrsB promoter. The cellular fluorescence of taR*2 in the presence of NiSO4 was normalized to 1.0. The expression-fold representing the ratio of GFPuv expression levels in the presence and absence of NiSO4 are shown. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate. (C) Northern blot analysis of taR*2’s. The transcription was stopped by adding rifampicin and cells were harvested at the indicated time points for RNA preparation. Total RNA was analyzed using probes specific for taR*2s and 16S rRNA, respectively.
Mentions: Two scaffold-fused taRNAs, taR*2-MicF and taR*2-MicF M7.4 were evaluated in Synechocystis. This MicF M7.4 scaffold was engineered by substituting the AU and GU-base pairs present in the stem loop structures of MicF scaffold into GC-base pairs and also by replacing the loop region with an Hfq high-affinity sequence (Link et al. 2009). This engineered MicF scaffold was predicted to form a stable secondary structure from M-fold secondary structure prediction (Zuker 2003). In our previous study, a naturally occurring E. coli MicF sRNA-derived scaffold sequence was revealed to be a suitable scaffold sequence to improve the gene regulation abilities of taR*2 (Fig.1C) (Sakai et al. 2014). While the taR*2-MicF harboring an intact MicF sRNA-derived scaffold sequence enhanced the gene expression to 2.5-fold in E. coli, taR*2-MicF M7.4 harboring the engineered MicF scaffold further enhanced the gene expression to 1.5-fold, which was 4.1-fold higher than taR*2 without the scaffold sequence. Moreover, the scaffold-fused taRNAs were more stable than the taRNA without the scaffold sequence in vivo and had low function in E. coliΔhfq strain, indicating that the endogenous Hfq bound and protected the scaffold-fused taRNAs from nuclease cleavage as well as natural trans-encoded sRNAs. To evaluate taR*2-MicF and taR*2-MicF M7.4 in Synechocystis, the scaffold-fused taR*2s were inserted downstream of the Ni2+-inducible nrsB promoter (PnrsB) (Lopez-Maury et al. 2002) and the crR*2 was constitutively transcribed from the trc promoter without the lac operator sequence (PtrcΔlacO) (Fig.2A). The gfpuv gene was inserted under crR*2 as a reporter gene to construct plasmids to evaluate the riboregulators (Table1).

Bottom Line: Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp.PCC 6803.The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

No MeSH data available.


Related in: MedlinePlus