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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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Binding assays of wild-type and truncated SR1/ahrC RNA pairs. Binding experiments were performed as described in Materials and Methods section. Autoradiograms of gel-shift assays are shown. The concentration of unlabelled ahrC RNA species or SR1 species is indicated. F, labelled RNA, D duplex between SR1 and ahrC RNA. (A) Binding assays with wild-type and truncated SR1 derivatives. SR1 species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to the targets. ahrC376 comprising the 3′ part of ahrC mRNA with nt 113 to 483 was used in all cases. Above, the schematic representation of the SR1 species is shown. (B) Binding assays with wild-type and truncated ahrC species. ahrC RNA species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to SR1186. (C) Overview on the ahrC mRNA species used in this work. The sequence of the ahrC gene is shown. Regions A′ to G′ complementary to SR1 are indicated by grey boxes, the SD sequence is underlined. Start and stop codon are shown in Italics. Below, a schematic representation of the 5 ahrC-mRNA species used in this work is shown. Black rectangle, SD sequence. grey boxes, regions complementary to SR1.
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Figure 2: Binding assays of wild-type and truncated SR1/ahrC RNA pairs. Binding experiments were performed as described in Materials and Methods section. Autoradiograms of gel-shift assays are shown. The concentration of unlabelled ahrC RNA species or SR1 species is indicated. F, labelled RNA, D duplex between SR1 and ahrC RNA. (A) Binding assays with wild-type and truncated SR1 derivatives. SR1 species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to the targets. ahrC376 comprising the 3′ part of ahrC mRNA with nt 113 to 483 was used in all cases. Above, the schematic representation of the SR1 species is shown. (B) Binding assays with wild-type and truncated ahrC species. ahrC RNA species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to SR1186. (C) Overview on the ahrC mRNA species used in this work. The sequence of the ahrC gene is shown. Regions A′ to G′ complementary to SR1 are indicated by grey boxes, the SD sequence is underlined. Start and stop codon are shown in Italics. Below, a schematic representation of the 5 ahrC-mRNA species used in this work is shown. Black rectangle, SD sequence. grey boxes, regions complementary to SR1.

Mentions: So far, only for a few chromosomally encoded regulatory sRNAs, secondary structures have been determined experimentally. Examples include MicF (18), OxyS (30), RNAIII of S. aureus (31), DsrA (32), Spot42 (19), RyhB (20) and MicA (14,21). Since computer-predicted RNA structures often deviate from experimentally determined ones [e.g. RNAIII of pIP501 (33) or RNAI/RNAII of pT181 (34)], we performed limited digestions with structure-specific ribonucleases in vitro to determine the secondary structure of SR1. The wild-type SR1 (205 nt) as well as the 3′ truncated species SR1132, the 5′ truncated species SR198 and the 5′ and 3′ truncated species SR178 were 5′-end labelled, gel-purified and treated with RNases T1 (cleaves 3′ of unpaired G residues), T2 (unpaired nucleotides with a slight preference for A residues) and V1 (double-stranded or stacked regions). Figure 1A shows an analysis of SR1205 and the truncated species SR1132 whereas Figure 2B contains the schematic representation of the structure of SR1205 derived from the cleavage data. The experimentally determined structure for wild-type SR1 comprises three main stem-loops: SL1 (nt 1 to 112), SL2 (nt 138 to 154) and the terminator stem-loop SL3 (nt 173 to 203) interrupted by two single-stranded regions SSR1 (nt 113 to 137) and SSR2 (nt 155 to 172). It deviates from the structure predicted with Mfold in the 5′ as well as in the 3′ portion: The 5′ part was found to be single-stranded between nt 38 and 51, and the double-stranded stem proved to be much longer than predicted and comprises 20 paired nucleotides (nt) interrupted by three internal loops or bulged-out bases, respectively, compared to only 10 paired nt in the predicted structure. For the 3′ part, two stem-loops and the terminator stem-loop were predicted by Mfold, whereas the structure probing data support in addition to the terminator stem-loop only the second stem-loop SL2 in the centre of a long single-stranded region.Figure 1.


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)

Binding assays of wild-type and truncated SR1/ahrC RNA pairs. Binding experiments were performed as described in Materials and Methods section. Autoradiograms of gel-shift assays are shown. The concentration of unlabelled ahrC RNA species or SR1 species is indicated. F, labelled RNA, D duplex between SR1 and ahrC RNA. (A) Binding assays with wild-type and truncated SR1 derivatives. SR1 species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to the targets. ahrC376 comprising the 3′ part of ahrC mRNA with nt 113 to 483 was used in all cases. Above, the schematic representation of the SR1 species is shown. (B) Binding assays with wild-type and truncated ahrC species. ahrC RNA species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to SR1186. (C) Overview on the ahrC mRNA species used in this work. The sequence of the ahrC gene is shown. Regions A′ to G′ complementary to SR1 are indicated by grey boxes, the SD sequence is underlined. Start and stop codon are shown in Italics. Below, a schematic representation of the 5 ahrC-mRNA species used in this work is shown. Black rectangle, SD sequence. grey boxes, regions complementary to SR1.
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Figure 2: Binding assays of wild-type and truncated SR1/ahrC RNA pairs. Binding experiments were performed as described in Materials and Methods section. Autoradiograms of gel-shift assays are shown. The concentration of unlabelled ahrC RNA species or SR1 species is indicated. F, labelled RNA, D duplex between SR1 and ahrC RNA. (A) Binding assays with wild-type and truncated SR1 derivatives. SR1 species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to the targets. ahrC376 comprising the 3′ part of ahrC mRNA with nt 113 to 483 was used in all cases. Above, the schematic representation of the SR1 species is shown. (B) Binding assays with wild-type and truncated ahrC species. ahrC RNA species were 5′ end-labelled with [γ32P]-ATP and used in at least 10-fold lower equimolar amounts compared to SR1186. (C) Overview on the ahrC mRNA species used in this work. The sequence of the ahrC gene is shown. Regions A′ to G′ complementary to SR1 are indicated by grey boxes, the SD sequence is underlined. Start and stop codon are shown in Italics. Below, a schematic representation of the 5 ahrC-mRNA species used in this work is shown. Black rectangle, SD sequence. grey boxes, regions complementary to SR1.
Mentions: So far, only for a few chromosomally encoded regulatory sRNAs, secondary structures have been determined experimentally. Examples include MicF (18), OxyS (30), RNAIII of S. aureus (31), DsrA (32), Spot42 (19), RyhB (20) and MicA (14,21). Since computer-predicted RNA structures often deviate from experimentally determined ones [e.g. RNAIII of pIP501 (33) or RNAI/RNAII of pT181 (34)], we performed limited digestions with structure-specific ribonucleases in vitro to determine the secondary structure of SR1. The wild-type SR1 (205 nt) as well as the 3′ truncated species SR1132, the 5′ truncated species SR198 and the 5′ and 3′ truncated species SR178 were 5′-end labelled, gel-purified and treated with RNases T1 (cleaves 3′ of unpaired G residues), T2 (unpaired nucleotides with a slight preference for A residues) and V1 (double-stranded or stacked regions). Figure 1A shows an analysis of SR1205 and the truncated species SR1132 whereas Figure 2B contains the schematic representation of the structure of SR1205 derived from the cleavage data. The experimentally determined structure for wild-type SR1 comprises three main stem-loops: SL1 (nt 1 to 112), SL2 (nt 138 to 154) and the terminator stem-loop SL3 (nt 173 to 203) interrupted by two single-stranded regions SSR1 (nt 113 to 137) and SSR2 (nt 155 to 172). It deviates from the structure predicted with Mfold in the 5′ as well as in the 3′ portion: The 5′ part was found to be single-stranded between nt 38 and 51, and the double-stranded stem proved to be much longer than predicted and comprises 20 paired nucleotides (nt) interrupted by three internal loops or bulged-out bases, respectively, compared to only 10 paired nt in the predicted structure. For the 3′ part, two stem-loops and the terminator stem-loop were predicted by Mfold, whereas the structure probing data support in addition to the terminator stem-loop only the second stem-loop SL2 in the centre of a long single-stranded region.Figure 1.

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