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Quantitative effect of target translation on small RNA efficacy reveals a novel mode of interaction.

Lavi-Itzkovitz A, Peterman N, Jost D, Levine E - Nucleic Acids Res. (2014)

Bottom Line: Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding.Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA.Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA.

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Efficient translation increases the efficacy of gene silencing. (A, C, E) Gene expression (measured by GFP fluorescence per OD600) for (A) 10 sodB, (C) four hns and (E) seven csgD variants with and without induction of RyhB, DsrA (aTc = 0 or 8 ng/ml, IPTG = 0.5 mM in both), or OmrA (IPTG = 0 or 1.0 mM, aTc = 10 ng/ml in both). (B, D, F) sRNA efficacy (defined as the ratio of target expression in the presence and absence of the sRNA) with respect to each variant, measured for (B) RyhB-sodB, (D) DsrA-hns and (F) OmrA-csgD, plotted against expression in the absence of the sRNA.
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Figure 3: Efficient translation increases the efficacy of gene silencing. (A, C, E) Gene expression (measured by GFP fluorescence per OD600) for (A) 10 sodB, (C) four hns and (E) seven csgD variants with and without induction of RyhB, DsrA (aTc = 0 or 8 ng/ml, IPTG = 0.5 mM in both), or OmrA (IPTG = 0 or 1.0 mM, aTc = 10 ng/ml in both). (B, D, F) sRNA efficacy (defined as the ratio of target expression in the presence and absence of the sRNA) with respect to each variant, measured for (B) RyhB-sodB, (D) DsrA-hns and (F) OmrA-csgD, plotted against expression in the absence of the sRNA.

Mentions: To characterize the relative ribosomal binding strength of the variants in our library we compared GFP expression without sRNA induction (0.5-mM IPTG, no aTc added) in a microplate reader. The different strains in our library demonstrated a wide range of GFP expression levels (Figure 3A, black bars), which correlated with their predicted ribosome binding strength (Supplementary Figure S9A). To measure the effect of the ribosome binding strength on sRNA efficacy, we repeated our measurements with induction of RyhB (IPTG = 0.5 mM, aTc = 8 ng/ml) (FigureĀ 3A, red bars). In most strains, the presence of RyhB reduced GFP expression, as expected from the repression of sodB by RyhB.


Quantitative effect of target translation on small RNA efficacy reveals a novel mode of interaction.

Lavi-Itzkovitz A, Peterman N, Jost D, Levine E - Nucleic Acids Res. (2014)

Efficient translation increases the efficacy of gene silencing. (A, C, E) Gene expression (measured by GFP fluorescence per OD600) for (A) 10 sodB, (C) four hns and (E) seven csgD variants with and without induction of RyhB, DsrA (aTc = 0 or 8 ng/ml, IPTG = 0.5 mM in both), or OmrA (IPTG = 0 or 1.0 mM, aTc = 10 ng/ml in both). (B, D, F) sRNA efficacy (defined as the ratio of target expression in the presence and absence of the sRNA) with respect to each variant, measured for (B) RyhB-sodB, (D) DsrA-hns and (F) OmrA-csgD, plotted against expression in the absence of the sRNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231754&req=5

Figure 3: Efficient translation increases the efficacy of gene silencing. (A, C, E) Gene expression (measured by GFP fluorescence per OD600) for (A) 10 sodB, (C) four hns and (E) seven csgD variants with and without induction of RyhB, DsrA (aTc = 0 or 8 ng/ml, IPTG = 0.5 mM in both), or OmrA (IPTG = 0 or 1.0 mM, aTc = 10 ng/ml in both). (B, D, F) sRNA efficacy (defined as the ratio of target expression in the presence and absence of the sRNA) with respect to each variant, measured for (B) RyhB-sodB, (D) DsrA-hns and (F) OmrA-csgD, plotted against expression in the absence of the sRNA.
Mentions: To characterize the relative ribosomal binding strength of the variants in our library we compared GFP expression without sRNA induction (0.5-mM IPTG, no aTc added) in a microplate reader. The different strains in our library demonstrated a wide range of GFP expression levels (Figure 3A, black bars), which correlated with their predicted ribosome binding strength (Supplementary Figure S9A). To measure the effect of the ribosome binding strength on sRNA efficacy, we repeated our measurements with induction of RyhB (IPTG = 0.5 mM, aTc = 8 ng/ml) (FigureĀ 3A, red bars). In most strains, the presence of RyhB reduced GFP expression, as expected from the repression of sodB by RyhB.

Bottom Line: Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding.Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA.Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA.

Show MeSH
Related in: MedlinePlus