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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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Secondary structure probing of the SR1/ahrC complex. (A) Alterations in the SR1 secondary structure upon complex formation with ahrC mRNA. Purified, 5′ end-labelled SR1186 (13 nM) was incubated with increasing amounts of unlabelled ahrC376 (80, 200 and 800 nM), complex allowed to form for 5 min at 37°C and subjected to limited cleavage with the RNases indicated. The digested RNAs were separated on 8% denaturing gels. Autoradiograms are shown. RNase concentrations used were: T1: 10−2 U/μl, T2: 10−1 U/μl, V1: 10−1 U/μl C, control without RNase treatment, L, alkaline ladder. Left; entire gel. Right, long run of the same samples allowing a better separation of the complementary regions B, C, D, E and F. Nucleotide positions are included. Altered T1, T2 and V cleavages are indicated by the symbols shown in the box. Right half, below: SR178: For a better resolution of the complex within complementary regions F and G, the secondary structure of the complex between SR178 (6.25 nM) and ahrC376 (80, 200, 800 and 1600 nM) was mapped, the same concentrations of T1, T2 and V were used and the products separated by a long run on an 8% gel. (B) Alterations in the ahrC secondary structure upon complex formation with SR1. Purified, 5′ end-labelled ahrC136 or ahrC376 (13 nM) was incubated with increasing amounts of unlabelled SR1186 (80, 200 and 800 nM), complex formation, cleavage and gel separation were performed as above. (C) Schematic representation of the SR1 secondary structure with indicated structural changes upon binding to ahrC RNA. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to ahrC RNA are highlighted by grey boxes. (D). Schematic representation of the secondary structure of ahrC136 and ahrC376 with indicated structural changes upon binding to SR1. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to SR1 are highlighted by grey boxes. Nucleotide numbering for both RNAs is as in Figure 2.
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Figure 3: Secondary structure probing of the SR1/ahrC complex. (A) Alterations in the SR1 secondary structure upon complex formation with ahrC mRNA. Purified, 5′ end-labelled SR1186 (13 nM) was incubated with increasing amounts of unlabelled ahrC376 (80, 200 and 800 nM), complex allowed to form for 5 min at 37°C and subjected to limited cleavage with the RNases indicated. The digested RNAs were separated on 8% denaturing gels. Autoradiograms are shown. RNase concentrations used were: T1: 10−2 U/μl, T2: 10−1 U/μl, V1: 10−1 U/μl C, control without RNase treatment, L, alkaline ladder. Left; entire gel. Right, long run of the same samples allowing a better separation of the complementary regions B, C, D, E and F. Nucleotide positions are included. Altered T1, T2 and V cleavages are indicated by the symbols shown in the box. Right half, below: SR178: For a better resolution of the complex within complementary regions F and G, the secondary structure of the complex between SR178 (6.25 nM) and ahrC376 (80, 200, 800 and 1600 nM) was mapped, the same concentrations of T1, T2 and V were used and the products separated by a long run on an 8% gel. (B) Alterations in the ahrC secondary structure upon complex formation with SR1. Purified, 5′ end-labelled ahrC136 or ahrC376 (13 nM) was incubated with increasing amounts of unlabelled SR1186 (80, 200 and 800 nM), complex formation, cleavage and gel separation were performed as above. (C) Schematic representation of the SR1 secondary structure with indicated structural changes upon binding to ahrC RNA. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to ahrC RNA are highlighted by grey boxes. (D). Schematic representation of the secondary structure of ahrC136 and ahrC376 with indicated structural changes upon binding to SR1. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to SR1 are highlighted by grey boxes. Nucleotide numbering for both RNAs is as in Figure 2.

Mentions: The results from the binding assays indicate that SR178 is sufficient for efficient complex formation with ahrC mRNA and that without opening of the 5′ half of the terminator stem-loop no efficient complex can form. To investigate the alterations in the secondary structures of SR1 and ahrC upon pairing, the secondary structure of the SR1186/ahrC376 complex was determined. To ascertain alterations in the SR1 structure, labelled SR1186 was incubated with a 6- to 60-fold excess of unlabelled ahrC RNA, the complex was allowed to form for 5 min at 37°C, and, subsequently, partially digested with RNases T1, T2 and V1. In parallel, free SR1186 was treated in the same way. Figure 3A shows the result. As expected, no significant alterations were observed within the 5′ 112 nt of SR1 that contain only region A (nt 15 to 19) complementary to ahrC. By contrast, significant alterations in the T1, T2 and V cleavage pattern were observed within the other six complementary regions B, C, D, E, F and G (Figure 3A, right half). The data are summarized in Figure 3C: Whereas in region B, only one reduced T1 cut was detected at G113, drastic alterations were observed in both regions C and G: In C, all 9 nt complementary to ahrC showed reduced T2 cleavages, G126 and G127 exhibited reduced T1 cleavage and at U123 and U125, an induction of V1 cleavage was detected indicating that this region became double-stranded upon pairing with ahrC. The same was true for region G, where the cleavage pattern at all positions was altered compared to free SR1: nt 175 to 181 showed a decreased T2 cleavage, among them G176 and G181 a reduced T1 cleavage, whereas at U180 and G181 new V cuts appeared. Fewer changes were found in regions D, E and F, where G133 (region D), U146 and A147 (region E) and G156, U157 and U158 (region F) were not single-stranded anymore and, instead, U132 and U133 (region D), A148 and A149 (region E) as well as U155 and G156 (region F) showed induced V cleavages, i.e. became double-stranded.Figure 3.


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)

Secondary structure probing of the SR1/ahrC complex. (A) Alterations in the SR1 secondary structure upon complex formation with ahrC mRNA. Purified, 5′ end-labelled SR1186 (13 nM) was incubated with increasing amounts of unlabelled ahrC376 (80, 200 and 800 nM), complex allowed to form for 5 min at 37°C and subjected to limited cleavage with the RNases indicated. The digested RNAs were separated on 8% denaturing gels. Autoradiograms are shown. RNase concentrations used were: T1: 10−2 U/μl, T2: 10−1 U/μl, V1: 10−1 U/μl C, control without RNase treatment, L, alkaline ladder. Left; entire gel. Right, long run of the same samples allowing a better separation of the complementary regions B, C, D, E and F. Nucleotide positions are included. Altered T1, T2 and V cleavages are indicated by the symbols shown in the box. Right half, below: SR178: For a better resolution of the complex within complementary regions F and G, the secondary structure of the complex between SR178 (6.25 nM) and ahrC376 (80, 200, 800 and 1600 nM) was mapped, the same concentrations of T1, T2 and V were used and the products separated by a long run on an 8% gel. (B) Alterations in the ahrC secondary structure upon complex formation with SR1. Purified, 5′ end-labelled ahrC136 or ahrC376 (13 nM) was incubated with increasing amounts of unlabelled SR1186 (80, 200 and 800 nM), complex formation, cleavage and gel separation were performed as above. (C) Schematic representation of the SR1 secondary structure with indicated structural changes upon binding to ahrC RNA. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to ahrC RNA are highlighted by grey boxes. (D). Schematic representation of the secondary structure of ahrC136 and ahrC376 with indicated structural changes upon binding to SR1. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to SR1 are highlighted by grey boxes. Nucleotide numbering for both RNAs is as in Figure 2.
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Figure 3: Secondary structure probing of the SR1/ahrC complex. (A) Alterations in the SR1 secondary structure upon complex formation with ahrC mRNA. Purified, 5′ end-labelled SR1186 (13 nM) was incubated with increasing amounts of unlabelled ahrC376 (80, 200 and 800 nM), complex allowed to form for 5 min at 37°C and subjected to limited cleavage with the RNases indicated. The digested RNAs were separated on 8% denaturing gels. Autoradiograms are shown. RNase concentrations used were: T1: 10−2 U/μl, T2: 10−1 U/μl, V1: 10−1 U/μl C, control without RNase treatment, L, alkaline ladder. Left; entire gel. Right, long run of the same samples allowing a better separation of the complementary regions B, C, D, E and F. Nucleotide positions are included. Altered T1, T2 and V cleavages are indicated by the symbols shown in the box. Right half, below: SR178: For a better resolution of the complex within complementary regions F and G, the secondary structure of the complex between SR178 (6.25 nM) and ahrC376 (80, 200, 800 and 1600 nM) was mapped, the same concentrations of T1, T2 and V were used and the products separated by a long run on an 8% gel. (B) Alterations in the ahrC secondary structure upon complex formation with SR1. Purified, 5′ end-labelled ahrC136 or ahrC376 (13 nM) was incubated with increasing amounts of unlabelled SR1186 (80, 200 and 800 nM), complex formation, cleavage and gel separation were performed as above. (C) Schematic representation of the SR1 secondary structure with indicated structural changes upon binding to ahrC RNA. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to ahrC RNA are highlighted by grey boxes. (D). Schematic representation of the secondary structure of ahrC136 and ahrC376 with indicated structural changes upon binding to SR1. Altered T1, T2 and V cleavages are denoted as shown in the box. Regions complementary to SR1 are highlighted by grey boxes. Nucleotide numbering for both RNAs is as in Figure 2.
Mentions: The results from the binding assays indicate that SR178 is sufficient for efficient complex formation with ahrC mRNA and that without opening of the 5′ half of the terminator stem-loop no efficient complex can form. To investigate the alterations in the secondary structures of SR1 and ahrC upon pairing, the secondary structure of the SR1186/ahrC376 complex was determined. To ascertain alterations in the SR1 structure, labelled SR1186 was incubated with a 6- to 60-fold excess of unlabelled ahrC RNA, the complex was allowed to form for 5 min at 37°C, and, subsequently, partially digested with RNases T1, T2 and V1. In parallel, free SR1186 was treated in the same way. Figure 3A shows the result. As expected, no significant alterations were observed within the 5′ 112 nt of SR1 that contain only region A (nt 15 to 19) complementary to ahrC. By contrast, significant alterations in the T1, T2 and V cleavage pattern were observed within the other six complementary regions B, C, D, E, F and G (Figure 3A, right half). The data are summarized in Figure 3C: Whereas in region B, only one reduced T1 cut was detected at G113, drastic alterations were observed in both regions C and G: In C, all 9 nt complementary to ahrC showed reduced T2 cleavages, G126 and G127 exhibited reduced T1 cleavage and at U123 and U125, an induction of V1 cleavage was detected indicating that this region became double-stranded upon pairing with ahrC. The same was true for region G, where the cleavage pattern at all positions was altered compared to free SR1: nt 175 to 181 showed a decreased T2 cleavage, among them G176 and G181 a reduced T1 cleavage, whereas at U180 and G181 new V cuts appeared. Fewer changes were found in regions D, E and F, where G133 (region D), U146 and A147 (region E) and G156, U157 and U158 (region F) were not single-stranded anymore and, instead, U132 and U133 (region D), A148 and A149 (region E) as well as U155 and G156 (region F) showed induced V cleavages, i.e. became double-stranded.Figure 3.

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