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In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA.

Heidrich N, Moll I, Brantl S - Nucleic Acids Res. (2007)

Bottom Line: The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down.Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS.Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA.

View Article: PubMed Central - PubMed

Affiliation: AG Bakteriengenetik, Friedrich-Schiller-Universität Jena, Philosophenweg 12, Jena D-07743, Germany.

ABSTRACT
Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5' part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

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Intracellular concentration of SR1 under different growth conditions. Bacillus subtilis strain DB104 was grown to OD560 = 2 (log phase) or OD560 = 4.5 (stationary phase), respectively, 5 ml or 1.5 ml culture, respectively, were withdrawn and used for the preparation of total RNA and subsequent northern blotting. Lanes 1 and 2, 6.6 and 33.3 fmol of in vitro synthesized, purified SR1, lanes 3 and 4, DB104 (Δsr1::cat) with 6.6 and 33.3 fmol of in vitro synthesized, purified SR1 mixed at the beginning of the RNA preparation, lanes 5, two and three parallels of RNA isolated from DB104. An autoradiogram of the northern blot is shown.
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Figure 7: Intracellular concentration of SR1 under different growth conditions. Bacillus subtilis strain DB104 was grown to OD560 = 2 (log phase) or OD560 = 4.5 (stationary phase), respectively, 5 ml or 1.5 ml culture, respectively, were withdrawn and used for the preparation of total RNA and subsequent northern blotting. Lanes 1 and 2, 6.6 and 33.3 fmol of in vitro synthesized, purified SR1, lanes 3 and 4, DB104 (Δsr1::cat) with 6.6 and 33.3 fmol of in vitro synthesized, purified SR1 mixed at the beginning of the RNA preparation, lanes 5, two and three parallels of RNA isolated from DB104. An autoradiogram of the northern blot is shown.

Mentions: To determine the intracellular concentration of SR1 in B. subtilis in logarithmic and stationary growth phase, strain DB104 was grown in complex medium, and samples were withdrawn at OD 2 (log phase) and OD 4.5 (onset of stationary phase). Cell numbers were determined upon plating of appropriate dilutions of the harvested cultures on agar plates. Total RNA was prepared, separated on a denaturing polyacrylamide gel alongside defined amounts of in vitro synthesized SR1 and subsequently, subjected to northern blotting (Figure 7). Losses during RNA preparation were calculated using in vitro synthesized SR1 mixed with the same amount of DB104::Δsr1 cells at the beginning of the RNA preparation. A comparison with the same amounts of untreated RNA yielded ∼80% loss. Loading errors were corrected by reprobing with labelled oligonucleotide C767 complementary to 5S rRNA. Using this quantification procedure, the amount of SR1 within one B. subtilis cell was calculated to be ∼20 molecules in log phase and 200–250 molecules in stationary phase, corresponding to an approximate intracellular concentration of 30 and 315 nM, respectively.Figure 7.


In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA.

Heidrich N, Moll I, Brantl S - Nucleic Acids Res. (2007)

Intracellular concentration of SR1 under different growth conditions. Bacillus subtilis strain DB104 was grown to OD560 = 2 (log phase) or OD560 = 4.5 (stationary phase), respectively, 5 ml or 1.5 ml culture, respectively, were withdrawn and used for the preparation of total RNA and subsequent northern blotting. Lanes 1 and 2, 6.6 and 33.3 fmol of in vitro synthesized, purified SR1, lanes 3 and 4, DB104 (Δsr1::cat) with 6.6 and 33.3 fmol of in vitro synthesized, purified SR1 mixed at the beginning of the RNA preparation, lanes 5, two and three parallels of RNA isolated from DB104. An autoradiogram of the northern blot is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935000&req=5

Figure 7: Intracellular concentration of SR1 under different growth conditions. Bacillus subtilis strain DB104 was grown to OD560 = 2 (log phase) or OD560 = 4.5 (stationary phase), respectively, 5 ml or 1.5 ml culture, respectively, were withdrawn and used for the preparation of total RNA and subsequent northern blotting. Lanes 1 and 2, 6.6 and 33.3 fmol of in vitro synthesized, purified SR1, lanes 3 and 4, DB104 (Δsr1::cat) with 6.6 and 33.3 fmol of in vitro synthesized, purified SR1 mixed at the beginning of the RNA preparation, lanes 5, two and three parallels of RNA isolated from DB104. An autoradiogram of the northern blot is shown.
Mentions: To determine the intracellular concentration of SR1 in B. subtilis in logarithmic and stationary growth phase, strain DB104 was grown in complex medium, and samples were withdrawn at OD 2 (log phase) and OD 4.5 (onset of stationary phase). Cell numbers were determined upon plating of appropriate dilutions of the harvested cultures on agar plates. Total RNA was prepared, separated on a denaturing polyacrylamide gel alongside defined amounts of in vitro synthesized SR1 and subsequently, subjected to northern blotting (Figure 7). Losses during RNA preparation were calculated using in vitro synthesized SR1 mixed with the same amount of DB104::Δsr1 cells at the beginning of the RNA preparation. A comparison with the same amounts of untreated RNA yielded ∼80% loss. Loading errors were corrected by reprobing with labelled oligonucleotide C767 complementary to 5S rRNA. Using this quantification procedure, the amount of SR1 within one B. subtilis cell was calculated to be ∼20 molecules in log phase and 200–250 molecules in stationary phase, corresponding to an approximate intracellular concentration of 30 and 315 nM, respectively.Figure 7.

Bottom Line: The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down.Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS.Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA.

View Article: PubMed Central - PubMed

Affiliation: AG Bakteriengenetik, Friedrich-Schiller-Universität Jena, Philosophenweg 12, Jena D-07743, Germany.

ABSTRACT
Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5' part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

Show MeSH
Related in: MedlinePlus